Introduction

In 2001, Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) defined probiotics as “live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host”¹. However, probiotic food consumption has been a part of different cultures around the world for many centuries. Probiotic organisms generally belong to LAB (lactic acid producing bacteria) and include Lactobacillus, Lactococcus, Bifidobacterium and many other organisms². Microorganisms of various other genera such as Bacillus, not belonging to LABs, have been identified and declared as probiotics as they exhibited probiotic characteristics³. In recent times, researchers have focussed on benefits of probiotics and many have established their health and nutritional benefits in humans. Beneficial effects of probiotics include improvement of gut health, reduction in symptoms of Irritable Bowel Syndrome (IBS)⁴, antibiotic associated diarrhoea^5,6, infectious diarrhoea caused by rotavirus^7-9, traveller’s diarrhoea¹⁰ and necrotizing enterocolitis¹¹.

Many studies have been performed on bioremediation of heavy metals by microorganisms^12-14 and have reported an interesting observations that probiotics such as Lactobacillus spp. and Bacillus spp. also have the ability to bioremediate heavy metals^15-19. This has been attributed to the structural components of their cell wall such as teichoic acid, lipoteichoic acid and peptidoglycan²⁰.

Heavy metals such as lead, chromium, nickel, cadmium, mercury and arsenic reach human gut through food and water laden with them. These heavy metals enter the food chain as a result of anthropogenic activities and cause varying levels of toxicity in humans²¹. Hence, these microorganisms are the best and most effective agents to reduce toxicity caused by heavy metals since they are safe in terms of human usage, are ingested via diet, show gut remediation and can eliminate the heavy metals by defaecation²².

In this study, B. clausii was evaluated for tolerance and survival capacity in presence of Cr (VI), Pb (II), Cd (II) and Ni (II) and the effect of exposure of these heavy metals on it.

Materials and Methods

Materials

Commercially available probiotic Bacillus clausii was procured from local chemist. It is marketed by Sanofi Synthelabo Pvt Ltd. Powai, Mumbai, India as a spore suspension under the name “Enterogermina®”. Potassium dichromate, lead nitrate, cadmium sulphate and nickel sulphate are the salts of heavy metals which have been selected for this study and were used as sources of Cr (VI), Pb (II), Cd (II) and Ni (II), respectively. Antibiotic discs were procured from HiMedia, India for kanamycin (30 μg), norfloxacin (10 μg), chloramphenicol (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), and ampicillin (10 μg).

Methods

Minimum Inhibitory Concentration (MIC)

MIC of Cr (VI), Pb (II), Cd (II) and Ni (II) for B. clausii was assessed by method of Mistry et al²³ with some modifications. Spores were germinated in nutrient broth (NB) and inoculated on nutrient agar (NA) plates supplemented with two-fold concentration of respective heavy metals ranging from 1-512 ppm. These plates were incubated at 37^o C for 24 hrs. The lowest concentration of the respective heavy metal which inhibited the growth of probiotic was considered as the MIC of that heavy metal for B. clausii.

Characterization of Heavy Metal Resistant B. Clausii

Various characterization parameters of B. clausii were evaluated before and after 24 hrs. exposure to Cr (VI), Pb (II), Cd (II) and Ni (II). This was done to assess whether the respective heavy metal exposure affects characteristics of the test organism.

Morphological Characterization

Observations on cell size, shape, colony characteristics and staining properties were performed on B. clausii before and after exposure to test heavy metals.

Biochemical Characterization

Catalase Test

Unexposed and exposed cultures of B. clausii for Cr (VI), Pb (II), Cd (II) and Ni (II) were cultured on NA slants. Few drops of H₂O₂ were added to the slant after 24 hrs. and observed for immediate bubbling²⁴.

Methyl Red and Voges Proskauer (MRVP) Test

MRVP test was performed to determine glucose fermentation products. MR test was done according to methodology of Clark and Lub²⁵ while for VP test method of Voges and Proskauer²⁶ modified by Barritt²⁷ was followed. This was done on unexposed and exposed B. clausii to Cr (VI), Pb (II), Cd (II) and Ni (II) respectively, in sets of glucose phosphate broth tubes. The tubes were inoculated and incubated for 72-96 hrs. Methyl red was added to one tube of the set to check development of acid. To the other tube, 12 drops of reagent VP-I was added followed by 2-3 drops of reagent VP-II. The tubes were exposed to air and were shaken intermittently. One tube remained uninoculated and acted as control.

Sugar Fermentation Test

Sugar fermentation test was performed according to procedure of Cappuccino and Sherman²⁸. A set of sugar fermentation tubes were prepared with different sugars (lactose, sucrose, glucose, sorbitol and mannitol). Phenol red was added to the broth as pH indicator to check for acid production and Durham’s tube were placed in the broth to check for gas production. The broth was inoculated with unexposed and exposed B. clausii to each heavy metal respectively. An uninoculated tube was kept as control.

Antibiotic Sensitivity Test

To analyse whether Cr (VI), Pb (II), Cd (II) and Ni (II) exposure caused any change in antibiotic sensitivity of B. clausii, Kirby-Bauer disc diffusion method was performed using antibiotic discs²⁹. Zone of inhibition was measured after 24 hrs. for the unexposed and exposed cultures of B. clausii.

Probiotic Characterization

Efficacy of B. clausii as a probiotic was studied by acid and bile tolerance assays after exposure to heavy metals under study. For both assays, methodology of Hassanzadazar et al³⁰ was followed with some modifications.

For acid tolerance assay, B. clausii exposed to Cr (VI), Pb (II), Cd (II) and Ni (II) respectively, was added to different broths of pH 2, 3 and 4. Growth of exposed B. clausii was monitored by viable cell count that was performed by pour plate technique for every hour for 3 hrs. Prior to plating, ten- fold serial dilution of the inoculum was prepared in 0.1% peptone water. All the experiments were done in triplicates. Cell counting was done after 24 hrs. of incubation at 37^o C.

For bile tolerance assay, NB with bile salt concentration of 0.2, 0.3 and 0.4% were prepared. B. clausii after 24 hrs. exposure to Cr (VI), Pb (II), Cd (II) and Ni (II) was added to the respective broths. Viable cell count was performed by pour plate technique. The inoculum was prepared hourly for 3 hrs. by ten- fold serial dilution in 0.1% peptone water. Colony count was done after 24 hrs. incubation at 37^o C.

Results and Discussion

Minimum Inhibitory Concentration (MIC)

In presence of nickel and chromium, B. clausii showed growth till 128 ppm and hence MIC was obtained as 256 ppm. In case of cadmium exposure, MIC was obtained at 64 ppm. However, after lead exposure, B. clausii showed good growth till 512 ppm and thus no MIC was recorded.

Earlier, environmental species of genus Bacillus have shown bioremediation of Cr (VI)³¹, Pb (II)³², Cd (II)³³ and Ni (II)³⁴. The genetic determinants for heavy metal tolerance might be borne either on plasmids, transposons or genomic DNA^35,36. In fact, these determinants are responsible for increased tolerance of microorganisms for heavy metals and are reported more in heavy metal polluted sites rather than in unpolluted sites³⁷.

The results of the assay for MIC indicate combined resistance of B. clausii and high MIC values for the heavy metals tested. The earlier studies conducted by Silver³⁸ and Alam and Imran³⁹ also have reported similar combined resistance of B. clausii. Further studies have reported that bacteria which are multi-heavy metal resistant have greater MIC values as compared to bacteria showing resistance to a single heavy metal^39-41.

According to WHO, the guideline values for Cr (VI), Pb (II), Cd (II) and Ni (II) in drinking water are 0.05 mg/L, 0.01 mg/L, 0.003mg/L and 0.07mg/L, respectively⁴². B. clausii has shown MIC values much above the permissible values of these heavy metals in drinking water, indicating that it has the potential for bioremediation of the above mentioned heavy metals.

Morphological and Biochemical Characterization

Vegetative cells of B. clausii exposed to heavy metal stress did not exhibit any change in their various morphological characteristics like cell shape, cell size, colony characters etc. Similar observations were made when heavy metal exposure did not affect the biochemical properties test organism. These results indicate that 24 hrs. exposure of B. clausii to Cr (VI), Pb (II), Cd (II) and Ni (II) could not modify or transform the organism’s basic characteristics as depicted in Table 1.

Table 1: Comparison of morphological and biochemical characteristics of B. clausii before and after exposure to Cr (VI), Pb (II), Cd (II) and Ni (II)

Antibiotic Sensitivity Test

An organism can be either tolerant or resistant to antibiotics. For an organism to be resistant, it must grow in constant presence of low concentration of antibiotic. If an organism is categorized as tolerant, it means that it can survive for short duration of high concentration of antibiotic⁴³. This helps to determine the successful clinical use of antibiotics. In this study, upon exposure to heavy metal stress, a marked increase was observed in antibiotic sensitivity of B. clausii for the antibiotics used (Fig 1). Against amoxicillin, Pb (II) exposed B. clausii showed maximum change (from 9 mm to 33 mm) (Fig 2, Fig 4) while minimum change was recorded for Ni (II) (Fig 2, Fig 5) exposed and Cd (II) exposed culture (from 9 mm to 17 mm) (Fig 2, Fig 6b). Lead exposed culture recorded maximum (from 15 mm to 34 mm) (Fig 2, Fig 4) and cadmium exposed culture recorded minimum (from 15 mm to 21 mm) change against ciprofloxacin (Fig 2, Fig 6a). After exposure to lead, B. clausii showed doubling of antibiotic sensitivity (from 11 mm to 22 mm) against norfloxacin (Fig 2, Fig 4) while minimum was for chromium exposed culture (from 11 mm to 17 mm) (Fig 2, Fig 3). The zone of inhibition increased from 8 mm to 28 mm after lead exposure which was maximum for ampicillin (Fig 2, Fig 4) while minimum increase was for Cr (VI) (Fig 2, Fig 3) and Cd (II) (Fig 2, Fig 6c) exposed B. clausii (from 8 mm to 17 mm respectively). Pb (II) exposed test organism recorded maximum change against kanamycin (13 mm to 31 mm) (Fig 2, Fig 4) while Cd (II) exposed B. clausii showed negligible change (13 mm to 15 mm) (Fig 2, Fig 6b). For chloramphenicol, after exposure to nickel, B. clausii showed a tremendous increase in the zone of inhibition from 7 mm to 32 mm (Fig 2, Fig 5) while negligible change was found in Cd (II) exposed test probiotic (7 mm to 10 mm) (Fig 2, Fig 6a). This behaviour may be related to presence of proteins involved in chloramphenicol and kanamycin resistance have also been identified in proteome of B. clausii⁴⁴. Thus, lead exposure caused maximum change in antibiotic sensitivity while cadmium exposure led to minimum change. According to above definition, B. clausii can be considered as resistant to the antibiotics tested against and can therefore be used to replenish the microflora of the gut after antibiotic treatment, even after heavy metal exposure. The results of above assay indicate that the antibiotic resistance of B. clausii decreases upon exposure to Cr (VI), Pb (II), Cd (II) and Ni (II). Similar results obtained by Tuckfield and McArthur⁴⁵, Habi and Daba⁴⁶ and Belapurkar et al¹⁹, which also support our findings.  Various researchers have postulated multiple reasons for this observation. The reasons can be inactivation of enzymes and proteins by heavy metals involved in antibiotic resistance⁴⁷, irreversible inhibition of ribosome function by heavy metals⁴⁷ or chelation of antibiotics by heavy metals thus reducing their concentration⁴⁸.

Figure 1: Diameter of zone of inhibition (mm) for B. Click here to View Figure

Figure 2: Zone of inhibition of B. clausii before exposure to heavy metals Click here to View Figure

Figure 3: Zone of inhibition of B. clausii after exposure to Cr (VI) Click here to View Figure

Figure 4: Zone of inhibition of B. clausii after exposure to Pb (II) Click here to View Figure

Figure 5: Zone of inhibition of B. clausii after exposure to Ni (II) Click here to View Figure

Figure 6: (a, b, c) Zone of inhibition of B. clausii after exposure to Cd (II) Click here to View Figure

From the above results, it can also been concluded that B. clausii has potential to resist and tolerate both heavy metals and antibiotics. In bacteria, antibiotic resistance and heavy metal tolerance are genetically linked through R- plasmid^49-51. According to the study of Alam and Imran³⁹ selective pressure of the heavy metals causes co-selection of antibiotic resistance, albeit indirectly.

Probiotic Characterization

As B. clausii is a probiotic microorganism, it can survive low pH conditions of stomach as well as in presence of bile salts in the intestinal tract. To determine the effect of heavy metal stress on its probiotic efficacy of B. clausii, acid and bile tolerance assays were performed.

Acid Tolerance Assay

When Cr (VI) exposed B. clausii was inoculated in pH 2, 3 and 4 respectively and growth was monitored for 3 consecutive hours, it was observed that maximum CFU (colony forming units) were obtained at pH 3 after 3 hrs. (Fig. 7). Similar result was obtained for Pb (II), Cd (II) and Ni (II) exposed B. clausii (Fig. 8-10). This is in corroboration with a study conducted by Sahadeva et al⁵² which says that for an organism to be acid tolerant it must show maximum growth at pH 3 after 3 hrs.

Figure 7: Acid tolerance of B. clausii after exposure to chromium Click here to View Figure

Figure 8: Acid tolerance of B. clausii after exposure to lead Click here to View Figure

Figure 9: Acid tolerance of B. clausii after exposure to cadmium Click here to View Figure

Figure 10: Acid tolerance of B. clausii after exposure to nickel Click here to View Figure

Mechanisms of acid tolerance are more prominent in Gram positive bacteria like Bacillus spp. as they attain higher cell densities which enhance biofilm formation and communication amongst cells⁵³. These mechanisms are (i) change in types of fatty acids of cell membrane⁵⁴ (ii) exchange of proton for an amino acid in presence of amino acid carboxylase^55,56 (iii) conserving structures of macromolecules like proteins with the help of chaperones^54,57,58  and (iv) counteracting the low pH with production of alkaline molecules like ammonia⁵⁹. Since the test organism belongs to genus Bacillus, it probably suggests use one or more of these mechanisms. Interestingly, despite high heavy metal stress, acid tolerance of B. clausii was not affected as concluded by the results of this assay.

Bile Tolerance Assay

For a probiotic to be categorized as bile tolerant, it must have maximal growth at 0.3% bile salt concentration after 3 hrs.^60,61 When B. clausii exposed to heavy metals under study was inoculated in bile salt concentrations of 0.2%, 0.3% and 0.4%, highest CFUs were observed at 0.3% bile after 3 hrs. Hence, subjection of B. clausii to heavy metal stress did not change its bile tolerance property (Fig. 11-14).

Figure 11: Bile tolerance of B. clausii after exposure to chromium Click here to View Figure

Figure 12: Bile tolerance of B. clausii after exposure to lead Click here to View Figure

Figure 13: Bile tolerance of B. clausii after exposure to cadmium Click here to View Figure

Figure 14: Bile tolerance of B. clausii after exposure to nickel Click here to View Figure

In contrast to defined mechanisms of acid tolerance, the methods by which bacterial cell counters bile stress is not well defined. One of the mechanism is with the use of enzyme bile salt hydrolase⁶². Apart from this, complex gene expression is also known to be responsible for bile tolerant property of bacteria^63-66.

It may be noted that even under heavy metal stress, B. clausii is able to retain its probiotic efficacy by probable use of different mechanisms and can be applicable for bioremediation of heavy metal, in vivo.

Conclusion

Anthropogenic and natural activities are the causes of Cr (VI), Pb (II), Cd (II) and Ni (II) contamination of soil and water and air. These are toxic heavy metals which reach humans through the food chain and cause detrimental effects on their health. Chromium and lead are systemic toxicants while nickel and cadmium are carcinogens. When the effects of these heavy metals were tested on a probiotic microorganism, B. clausii, no change was observed in its morphological and biochemical properties and its probiotic efficacy. Hence, it can be concluded that B. clausii, a commercial probiotic has the potential for in vivo bioremediation of Cr (VI), Pb (II), Cd (II) and Ni (II) and can therefore benefit the society at large by reducing the toxicity of these heavy metals in the human gut.

Acknowledgements

The authors wish to thank Ar. Achal Choudhary, President and Dr. Sanjay Nagar, Head, Department of Biotechnology, IPS Academy, Indore for their valuable assistance regarding infrastructural and laboratory facilities.

Financial Support and Sponsorship/ Funding

Not applicable

Conflicts of Interest

There are no conflicts of interest among authors

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Introduction

Infertility affects relatively a large number of couples of about 186 million globally.¹ Many reports have indicated that the fertilizing potential of semen in healthy men is declining over a period.^{2- 4} Epidemiological studies also show that the sperm concentration in United States declines by 1.5% per year and by 3% in Australia and Europe.⁵ Significant declines in sperm count, motility, and morphology have also been reported in Denmark,⁶ Sweden,⁷ France,⁸ Austria⁹ and Finland.¹⁰ Similarly, mean concentration of spermatozoa in Italy has also fallen down.¹¹

At present, semen analysis performed based on the WHO laboratory manual for the examination and processing of human semen is considered to be the standard method for semen examination, where the semen parameters such as semen volume, pH, sperm concentration, motility, morphology, etc. are evaluated.¹² Modern life-style changes, high stress levels, smoking, alcohol consumption, lack of exercise, exposure to radiations could be the possible causes of decline in the seminal parameters.¹³ Apart from all these factors, the genital tract infection plays a vital role in declining the seminal parameters and has a direct impact on male infertility.¹⁴ A number of studies report that 8-32% of male infertility cases are associated with infections and inflammation of the male genitourinary tract by microorganisms such as bacteria, virus, fungi, and protozoa.^15-17 Infection with these microorganisms lead to  infertility problems such as sperm damage, pyospermia, asthenospermia, teratospermia, etc by adversely affecting the spermatogenesis, causing inflammatory disorder, anatomical obstruction, scaring and initiating leukocyte response with its concomitant oxidative stress.¹⁸

Bacteriospermia, the infection with bacteria is one of the major and significant factors in male infertility that result in abnormal semen parameters and even lead to impairment of sperm functions and seminal tract obstruction.^{19, 20} However, in most of the occasions, this bacteriospermic condition among men does not considered as one of the obstacles in fertility treatment when compared to all other investigative factors such as hormone levels and irregularities in menstruation cycles among women.   Even though numerous studies independently report the effect of bacteriospermia on seminal parameters, only a few studies highlighted the importance in sperm DNA fragmentation and Reactive Oxygen Intermediates (ROI).  This review discusses on various literature that describe about the different bacteria that involve in seminal tract infection, their influence on semen parameters such as count, motility, DNA fragmentation and the role of certain radicals (Reactive Oxygen Intermediates) that are produced as a result of bacterial infection.

Effects of Bacteriospermia

Acute and chronic genital tract infections are well known causes for male infertility.²¹  Escherichia coli, is one of the main bacteria isolated from human semen has the most negative influence on sperm morphology^{22, 23}.Semen infected with E. coli has also shown abnormal parameters such as low sperm concentration and reduced motility.^{24, 25}

Klebsiellae are opportunistic gram negative bacterial pathogens which cause seminal tract infections¹⁵.It has been reported that semen sample infected by Klebsiella appeared yellowish in colour with offensive odour with many pus cells. The presence of Klebsiella negatively influenced the semen parameters and possibly could be a cause for death of all spermatozoa in semen leading to a condition called necrozoospermia.²⁶ 

Staphylococcus aureus is an ubiquitous gram positive bacteria found in semen samples of both fertile and infertile males. Studies reported that S. aureus was the most dominant microorganism in semen culture of infertile men with high prevalence rate than other bacteria.^{27, 28} Various reports signify that there is decrease in sperm motility and agglutination of sperms when spermatozoa were co-incubated with S. aureus.^29-31 It is evident that presence of S. aureus has negatively influenced the semen parameters causing spermatozoa immobilization, agglutination of spermatozoa and has had a main role in deterioration of spermatogenesis and disability of sperm function.^{32, 33} It is evident that Staphylococcal infection, can decrease the sperm count thereby leading to oligozoospermia^{34, 35} and in some cases azoospermia.³⁶

Pseudomonas aeruginosa, a gram-negative, opportunistic pathogenic bacterium is found to be associated with the seminal tract infection that produces a quorum signaling molecule, 3-oxododecanoyl-L-homoserine lactone which has detrimental effects on human spermatozoa. It affects the seminal parameters such as sperm cell death- necrosis and premature acrosomal loss.³⁷

Enterococci are gram-positive cocci and reported as the common organism isolated from semen that affect semen quality.³⁸

Chlamydia trachomatis, an obligate intracellular gram negative bacterium and the most prevalent cause of bacterial sexually transmitted infections^{21, 39} has been reported that infection with this bacterium can alter the pH of semen and reduced ejaculate volume and when C. trachomatis and human spermatozoa were co-incubated in vitro, the motility of the spermatozoa has been affected thereby leading to premature death.⁴⁰

Ureaplasma urealyticum is considered to be a potentially pathogenic species that causes both genital infections and infertility in men.⁴¹  This U. urealyticum, was divided into two biotypes;  biovar 1 and biovar 2.⁴² Infection with biovar 2 can cause increased seminal viscosity with decreased pH and also affect most of the important parameters such as sperm concentration, motility and morphology of spermatozoa.⁴³ Interestingly, this U. urealyticum causes damage to the development and vitality of human embryos generated by in vitro fertilization (IVF) and less pregnancy rate after Embryo transfer procedures.^{44, 45}

Mycoplasma genitalium and M. hominis are also found to be associated with decrease in sperm concentration and abnormality in sperm morphology respectively.⁴¹

Bacteriospermia and Sperm DNA fragmentation

DNA fragmentation in sperms may affect fertility by hindering fertilization, early embryo development, implantation, and pregnancy.⁴⁶ This could be possibly because of defects in the sperm chromatin structure that can be associated with abnormal nucleoprotein content and DNA strand breaks.⁴⁷ It was reported that increased sperm DNA fragmentation was a reason for recurrent pregnancy loss and also had negative influence on sperm morphology.⁴⁸ It was also reported that DNA fragmentation in sperms has declined the fertilization rate and pregnancy rate in IVF procedures.⁴⁹ Various factors influence fragmentation of DNA in sperms such as errors in spermiogenesis, oxidative stress, chemotherapeutic agents, radiations, poor chromatin compaction, endogenous caspases, endonucleases and infection.^50-52 It was suggested that the semen samples infected with S.  aureus, S.  epidermis, S.  haemolyticus, E.  coli, Enterococcus faecalis and agalactiae had shown high sperm DNA fragmentation and also poor sperm concentration, motility and chromatin condensation⁵³. There is another report that emphasizes the increase in DNA fragmentation index in semen samples infected with U. urealyticumand M. genitalium⁵⁴ which in turn affected the embryonic development.^{45, 55} 

Bacteriospermia and Reactive Oxygen Intermediates

Apart from directly affecting the seminal parameters such as sperm motility and morphology bacteriospermia also affects indirectly by producing oxidative stress through the release of Reactive Oxygen Intermediates.^56-58 These Reactive Oxygen intermediates include superoxide anion radical, hydrogen peroxide and hydroxyl radicals.⁵⁹ The presence of bacteria leads to the recruitment of white blood cells to the inflammatory site as a result of host defense and later the activated macrophages and neutrophils produce reactive oxygen intermediates which affect the spermatozoa.^{60, 61} It was reported that bacteria themselves or the bacterial products stimulate ROI production in leukocytes.⁶² Studies carried out to reveal the significance of ROI in human semen reveal that there was higher ROI generation in bacterial infected semen samples than the uninfected semen samples.^{63, 64} Studies show that the attack of free radicals on the sperm membrane causes reducing the potency of spermatozoa in fertilization process.^65-69 An invitro study reported that there was an increase in the ROI generated by leukocytes incubated with E. coli and S. haemolyticus. In addition, this increase in hydrogen peroxide in the presence of B. ureolyticus is believed to be associated with the superoxide dismutase (SOD) produced by the bacteria.  This Hydrogen peroxide is highly toxic to the sperms.  Moreover, the insufficiency in the levels of catalase and gluthione peroxidase leads to the increase in intensity of the oxidative stress.⁷⁰ Another study also suggests that the hydrogen peroxide and hydroxide ion produced by U. urealyticum  are highly toxic to the sperms.⁷¹ Similarly the presence of U. urealyticum in semen has caused sperm DNA damage as well as elevated seminal reactive oxygen species.⁷²

Conclusion

The infertility rates in developing countries like India is in an increasing rate as the number of IVF procedures increased at a rate of 18% and is expected to increase upto 20% by this year 2020.⁷³ This increase in infertility can be clinically correlated with the bacteriospermic condition which is common among couples undergoing IVF treatment.⁷⁴ The problem here is, these infections are asymptomatic in most of the cases that lead to a dilemma in treatment procedures.²⁶

Bacteriological investigations for semen are generally carried out only when microscopic observation reveals significant pus cells.  But it is suggested to investigate for bacterial infection regardless the number of pus cells being observed.⁷⁵ Performing microbiological testing especially for the presence of bacterial infection before any assisted reproductive  procedure has a high significance as these genital bacteria can attach to the spermatozoa which can’t be expelled even by sperm washing techniques for intra uterine insemination and in vitro fertilization procedures. Hence there is a high possibility of microbial contamination of IVF medium which can result in fertilization failure and or poor embryonic development.⁷⁶

The present review highlights the correlation of Bacteriospermia on vital sperm parameters through various published literature andsuggests to include screening for the presence of bacterial infection even if they are asymptomatic to get better fertility rate in ART procedures to overcome infertility.

Acknowledgement

The authors thank the management and staffs of Ponnaiyah Ramajayam Institute of Science and Technology (Institute Deemed to be University – U/S 3 of UG C act, 1956), Dr. Ramesh Cardiac and Multispeciality Hospital  (JCI accredited) and Institute of Bio-Medical Research (DSIR recognized In- House R&D Unit of Kanmani Fertility Centre) for their help and support. Our Sincere thanks to Dr.S.R.Suresh, Director, PRIST-Chennai ECR campus and Dr. Yaramareddy Swapna, Clinical Head, Komali Fertility Centre for their motivation to draft this paper.

Conflicts of interest

There are no conflicts of interest.

Funding Source

Nil.

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Introduction

Diabetes mellitus is a set of disorder that is mainly caused by impairment of insulin production.¹ The second cause is a complication that involves poor insulin secretion due to the low action of the β-cell.²Based on the contemporary records about the shifting age structure of the global population of these omnipresent persistent debilitating menace among adults as 8.5% which doubled the 4.7% rate during 1980, albeit in retrospect, the world wide diabetes record during 1980 was documented as 108 million which reached 382 million during the year 2013 which flared and increased to 422 million during the year 2016.³ The vital cell’s failure to detect high blood sugar keeps insulin levels low.²DM derails metabolism in several organs such as the eyes and the kidneys.⁴ Insulin resistance in the human body also results in diabetes; this is a case where cells continue to absorb sugar because they have become insensitive to the pancreatic secreted hormone. When the liver absorbs sugar even when the levels are above normal, it causes liver and diabetes complications.⁵

One of the complications that result from insufficient insulin is lipid impairment, whereby lipoproteins develop insensitivity to insulin,^6,7 A variety of complications emanating from variegated symptoms including high blood sugar, frequent urination, increased hunger which, if left untreated, diabetes can cause many deleterious damage.⁸ Moreover, serious long term implications embodying diabetic ketoacidosis (DKA), hyperosmolar hyperglycemic state, or unavoidable tragic death. Severe long term implications could result in devastating ailments embodying cardiovascular disease, stroke, kidney disease (CKD), foot ulcers, and damage to the eyes. Kidney and nervous system complications emanate from mainly high effects of blood sugar rather than hypoglycemia.⁹ The permanency of elevated glucose gradually erodes the functionality of the kidneys and induces poor responsiveness of the nerves. This complication has the capability to cause inefficiency of the organs and their complete dysfunction.⁹

For centuries, many parts of the world have documented the use of plants as direct sources of medicine. In South Asia, over 100 traditional communities each with its own set of herbs have relied on herbal extracts as multiple-disease remedies.¹⁰ Avicennia marina (Forssk Vierh) is an important mangrove species and one of the most widely distributed genera.¹¹ The genus Avicennia named after Islamic physician Abdallah Ibn Sina,¹² Owing to their breathing aerial roots (pneumatophores), the species like A. marina have been traditionally classified in the family of Verbenaceae,¹³ but their real family is Acanthaceae.¹⁴ Because of existence in very harsh conditions, in dry areas with high salinity, such as on the eastern and western coasts of the Red Sea, the leaves of A. marina have acquired adaptational features that change with each environment.¹³

Avicennia marina is the most widely distributed mangrove species across all genera in the world. It extends from the shoreline of Egypt, along East Africa, through the southern parts of the continent, along with the Red Sea and in the Oceania and Pacific regions including Australasian coasts and the Japanese shoreline.¹¹ This wide geographical reach owes to the plant’s highly regarded adaptational abilities, such as affinity for heat levels above 35 °C and advanced saltiness, for example in the Arabian Peninsula.¹⁵ The objectives of the present investigation were to assess the antihypoglycemic ramifications of aqueous extracts of A. marian against experimentally STZ-induced kidney damage.

Materials and Methods

Experimental Animals

The experiment was performed on 60 adult male Wistar albino rats (200 to 250g average body weight). The animals were housed in well-aerated individual cages in an animal room and maintained at constant temperature (24 ± 1°C) and humidity (55 ± 10%) with alternating 12 h light/ dark cycle. The rats were fed with normal commercial chow and water ad libitum. The animals were maintained in accordance with the international ethical guidelines for the care of laboratory animals and all experimental procedures were approved by the Animal Care and Use Committee of the King Abdulaziz University.

Plants Extraction

The collected leaves of A. marina plants were scientifically identified and authenticated by a plant taxonomist at the Department of Arid Land Agriculture, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia. Aqueous extracts of plant leaves were prepared according to previous reporters.^16,17

Preparation of STZ and Induction of Diabetes

Overnight-fasted adult male rats (6 weeks old) were intraperitoneally injected with a single dose (60 mg/kg) of freshly prepared STZ.¹⁸ Three days after STZ injection, the fasting blood glucose levels were measured in blood samples taken from rats’ tail by using a One Touch Ultra Glucometer (Lifescan, Johnson and Johnson, Milpitas, CA, USA). Animals with blood glucose levels ≥ 250 mg/dl were considered diabetic and used for the experiment¹⁹. This day was considered the first day of the experiment.

Experimental Design

The experimental rats were randomly divided into 4 equal groups, each of 15 rats. Group 1: Normal rats received water and fed ad libitum .Group 2: STZ-induced diabetic rats. Groups 3: diabetic rats treated orally with aqueous leaf extracts of A. marina (400 mg/kg BW/day), Group 4: Non-diabetic rats received an aqueous leaf extract of A. marina at a dose of 400 mg/kg BW/day. The treatments started on the 4^th day after STZ injection, which considered as the 1^st day of treatment, and continued daily for six weeks.

Measurement of Blood Glucose Levels

To measure the glucose levels, fresh fasting blood samples were collected from the rat’s tail vein, and glucose levels were then determined using a One Touch Ultra Glucometer (Lifescan, Johnson and Johnson, Milpitas, CA, USA).

Measurement of Serum Glucose and Insulin

Blood was collected from retro-orbital venous plexus of rats at the 6^th week, left for clotting at room temperature and serum was separated by centrifugation at 3000 rpm for 20 min. Serum glucose and insulin levels were determined at the 6^th week post- treatments by commercial kits (Roche cobas Diagnostics USA) using cobas 6000 analyzer series. Serum insulin levels were measured using insulin ELISA kits which includes an enzyme immunoassay for the quantitative determination of insulin in sera of rats (Cat. no. ezrmi-13kelisa, Billerica, MA, USA) according to a method.¹⁹

Measurement of Serum Creatinine, Urea and Uric Acid

Assessment of Serum Creatinine

The serum creatinine level was determined according to Henry et al.²⁰

Principle of the Test

Creatinine reacts with picric acid in alkaline condition to form a yellow-orange complex which is measured at 492 nm. The rate of formation of color is proportional to creatinine quantity in the sample.

Calculation    

Where

Absorbance of the sample.

Absorbance of the standard.

To convert the result (mg/dL) to umol/L divide by 88.4

Assessment of Serum Blood Urea Nitrogen (BUN).

The serum BUN level was determined according to Henry et al.²⁰

Principle of the Test

Urea is synthesized in the liver from the ammonia produced mostly by the catabolism of amino acids. Kinetic enzymatic estimation of urea uses these reactions.

Urease hydrolyses urea to ammonia. Glutamate dehydrogenase (GLDH) combines the ammonia with 2-oxoglutarate to form glutamate. In this reaction, the NADH is oxidized to NAD+ and this change is detected photometrically as a decrease in absorbance at 340 nm (Warburg’s optical test).

Calculation

Where:        n = 50.0 mg/dL (8.33 mmol/L).

Assessment of Serum Uric Acid

The method of Young²¹ was used to determine the level of uric acid.

Principle of the Test

Uric Acid is oxidized by uricase to produce allantoin and H2O2. 2-Hydroxy-2, 4, 6-tribromobenzoic acid (TBHBA) + 4-aminophenazone (4-AAP) + H2O2, in the presence of POD, produces a colored chromagen that is measured at 520 nm. The color intensity at 520 nm is proportional to the concentration of uric acid in the sample.

Calculation

Where:

A is absorbance of the sample or standard.

To convert the result (mg/dL) to mmol/L divide

 Histopathological Examinations

The kidney tissue samples were collected at the 6^th week post-treatments after sacrifice and fixed in 10% neutral formalin. After fixation, the samples were embedded in paraffin, 5-μm sections were cut, stained with hematoxylin and eosin (H&E), and then examined for pathological studies using light microscopy.²²

Immunohistochemical Study

The standard immunohistochemical methods were adopted for detection of apoptotic caspase3 and diabetic insulin biomarkers in pancreatic tissue.²³ The tissue sections were routinely microwave-treated to unmistaken the epitopes of antigen.²⁴ The Biotin-Streptavidin (BSA) system was used to visualize the apoptotic and insulin markers.²⁵ Diaminobenzidine (DAB) was used as chromogen since it allows a permanent preparation. Hematoxylin counterstain was done.

Statistical Analysis

The obtained data in this study were expressed as mean ± standard error (SE). Statistical significance of the difference between groups, with more than two categories, was determined by one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) post-hoc test. The statistical software package used for analysis was Statistical Package for So

The kidney tissue samples were collected at the 6^th week post-treatments after sacrifice and fixed in 10% neutral formalin. After fixation, the samples were embedded in paraffin, 5-μm sections were cut, stained with hematoxylin and eosin (H&E), and then examined for pathological studies using light microscopy.²²

Immunohistochemical Study

The standard immunohistochemical methods were adopted for detection of apoptotic caspase3 and diabetic insulin biomarkers in pancreatic tissue.²³ The tissue sections were routinely microwave-treated to unmistaken the epitopes of antigen.²⁴ The Biotin-Streptavidin (BSA) system was used to visualize the apoptotic and insulin markers.²⁵ Diaminobenzidine (DAB) was used as chromogen since it allows a permanent preparation. Hematoxylin counterstain was done.

Statistical Analysis

The obtained data in this study were expressed as mean ± standard error (SE). Statistical significance of the difference between groups, with more than two categories, was determined by one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) post-hoc test. The statistical software package used for analysis was Statistical Package for Social Sciences (SPSS 24). The values were considered to be significantly different when the P value was < 0.05.²⁶

Results

Fasting Blood Glucose

Our final results confirmed that while fasting, the level of blood glucose (mg/dL) of STZ-induced diabetic animals (G2) and STZ-induced diabetic rats administered A.marina extract (G3) remained significantly elevated (p ≤ 0.001) compared to normal control group (G1). Daily oral treatment of STZ-induced diabetic rats (G3) with A.marina leaf extract induced non-significant changes in blood glucose levels as compare to diabetic control group (G2) (Figure 1)

Results

Fasting Blood Glucose

Our final results confirmed that while fasting, the level of blood glucose (mg/dL) of STZ-induced diabetic animals (G2) and STZ-induced diabetic rats administered A.marina extract (G3) remained significantly elevated (p ≤ 0.001) compared to normal control group (G1). Daily oral treatment of STZ-induced diabetic rats (G3) with A.marina leaf extract induced non-significant changes in blood glucose levels as compare to diabetic control group (G2) (Figure 1)

Figure 1: Effect of extract of A. marina on blood glucose (mg/dL) levels in normal Click here to View Figure

Serum Glucose

A comparison of serum glucose differences are illustrated in Figure 2 The serum glucose level (mmol/L) of STZ-induced diabetic rats (G2) and STZ-induced diabetic rats treated with A.marina extract (G3) showed highly significant increase (p ≤ 0.001) as compared to the normal control group (G1). However, the treatment of diabetic rats (G3) with A.marina leaf extracts revealed non-significant changes in the serum glucose levels in comparison to STZ-induced diabetic rats (G2).

Figure 2: Effect of extract of A. marina on serum glucose (mmol/L) levels in normal  Click here to View Figure

Serum Insulin

Figure 3 exhibited a highly significant decrease (p ≤ 0.001) in serum insulin level (mlU/L) of STZ-induced diabetic rats (G2) and treated diabetic animals (G3) compared to the normal control group (G1). The treatment of diabetic groups (G3) with A.marina exhibited non- significant changes in the serum insulin levels compared to STZ-induced diabetic rats (G2).

Figure 3: Effect of extract of A. marina on serum insulin Click here to View Figure

Serum Creatinine

The levels of serum creatinine (μmol/L) in STZ-induced diabetic rats (G2) revealed a significant increase (P ≤ 0.01) compared to the normal control group (G1).  However, the treatment of diabetic rats (G3) with A.marina leaf extracts revealed non-significant change in the serum creatinine levels in comparison to normal control group (G1). Nevertheless, the treatment of the STZ-induced diabetic rats (G3) with A.marina exhibited non-significant change in the serum creatinine levels when comparison was made to the diabetic control group (G2) (Figure 4).

Figure 4: Effect of extract of A. marina on serum creatinine Click here to View Figure

Serum Blood Urea Nitrogen (BUN)

The serum BUN levels (mmol/L) of the STZ-induced diabetic rats (G2) showed a highly significant increase (P ≤ 0.001) compared to the normal control rats (G1). Similarly, the STZ-induced diabetic rats treated with A. marina (G3) demonstrated a highly significant increase (P ≤ 0.001) in the levels of serum BUN when compared with the normal control rats (G1). However, administration of A. marina to diabetic rats (G3) showed a non-significant difference in the serum BUN level compared to diabetic control group (G2) (Figure 5).

Figure 5: Effect of extract of A. marina on serum BUN Click here to View Figure

Serum Uric Acid

A comparison of serum uric acid levels (umol/L) was illustrated in Figure 6. Data recovered from the serum uric acid levels of STZ-induced diabetic rats (G2) showed a significant increase (P ≤ 0.01) as compared with the normal control group (G1). However, the treatment of diabetic rats (G3) with A.marina leaf extracts revealed non-significant change in the serum uric acid levels in comparison to normal control group (G1). On the other hand, daily oral administration of A.marina leaf extracts to STZ-induced diabetic rats (G3) resulted in a significant decrease (P ≤ 0.05) in serum uric acid levels as compared with the diabetic control group (G2).

The daily oral administration of A. marina to non-diabetic rats (G4) induced non-significant changes in the levels of blood glucose, serum glucose, creatinine, blood nitrogen urea and uric acid compared to normal control group (G1) (Figure 1-6).

Figure 6: Effect of extract of A. marina on serum uric acid Click here to View Figure

Histopathological Examinations of Kidney Tissue

Renal parenchyma and stroma of normal rats (G1) were normal with keeping features of the nephron units, collecting tubules, papillary and pelvic structures (Figure 7). But renal serial sections of STZ-induced in diabetic rats (60 mg STZ/kg BW, Single ip) (G2) revealed that moderate an array of multifocal necrotic areas (coagulative necrosis) with pyknotic or karryoretic nuclei and deep eosinophilic cytoplasm with keeping a ghost of the tubular and glomerular architecture and distinct inflammatory reaction (Figure 8). Additionally, variable degrees of degenerative changes including cloudy swelling, vacuolar and hydropic degeneration were noticed. Besides this, mild dilatation of some distal convoluted tubules and collecting tubules with partial atrophy of their lining epithelium was recorded. Moreover, a few apoptotic cells were seen in the tubular epithelium together with peculiar perivascular edema and mild to moderate congestion of intertubular and glomerular blood vessels and capillaries, sometimes with erythrocytic extravasations (Figure 8).

Figure 7: Photo-micrograph for rat’s kidney G1 Click here to View Figure

Figure 8: Photo-micrograph of rat’s kidney G2 Click here to View Figure

Renal lesions of STZ-induced diabetic rats (60 mg STZ/kg BW, single ip) treated with A. marina 400 mg/kg BW (daily oral dose for 6weeks) (G3) were represented by characteristic perivascular edema with vacuolation of the vascular walls. Some of the nephron units (glomeruli and tubules) were apparently normal, but others showed degenerative and early necrotic and apoptotic changes. The renal papillae and pelvis were apparently normal in most parts; however, a few of them showed mild vascular and tubular dilatations of the surrounding tissues, besides focal exfoliative change in the lining epithelium (Figure 9)

Figure 9: Photo-micrograph of rat’s kidney G3, Click here to View Figure

Sections from kidneys of non-diabetic rats, treated with A. marina, 400 mg/kg BW (daily oral dose for 6weeks) (G4), revealed apparently normal nephron units including glomeruli and different types of tubules. The renal papillae, pelvis, medullary rays and blood vessels were also apparently normal. However, a few renal tubular epithelia, especially in the cortex, showed mild degenerative changes, mainly hydropic degeneration (Figure 10).

Figure 10: Photo-micrograph of rat’s kidney G4, Click here to View Figure

Immunohistochemical Kidney Findings

Kidney sections of normal rats (G1) denoted normal parenchyma free from any apoptotic changes except for a very few cells in the medullary collecting tubules (0.5-1%) /HPF (Figure 11). Meanwhile, renal tissue of STZ-induced diabetic (60 mg STZ/kg BW, Single ip dose) (G2) revealed about 3-5% positive apoptotic reaction of the tubular epithelium, particularly in the medulla (Figure 12). However, kidney sections of STZ-induced diabetic rats (60 mg STZ/kg BW, single ip dose) treated with A. marina 400 mg/kg BW (daily oral dose for 6 weeks) (G3) revealed about 6-8% /HPF of the tubular epithelial cells and 3-5% /HPF of the glomerular cells with positive cytoplasmic apoptotic reaction (Figure 13). Concomitantly, Some of the renal sections of non-diabetic rats treated with A. marina, 400 mg/kg BW (daily oral dose for 6 weeks) (G4) were free of apoptosis, but other sections showed 4-6% cells/HPF of both tubular and glomerular cells with early apoptotic changes as proved by weak brownish cytoplasmic reactivity to caspase-3 (Figure 14).

Figure 11: Photo-micrographs of rat’s kidney Click here to View Figure

Figure 12: Photo-micrographs of rat’s kidney, G2, Click here to View Figure

Figure 13: Photo-micrographs of rat’s kidney, G3 Click here to View Figure

Figure 14: Photo-micrographs of rat’s kidney,G4 Click here to View Figure

Discussion

Diabetes mellitus is a common metabolic disorder, characterized by hypoinsulinemia, hyperglycaemia, hyperlipidemia, hyperaminoacidemia, and disturbances of acute impact on carbohydrates, proteins and lipid metabolisms resulting from serious deleterious defects in either insulin secretion, insulin action, or both.^27,28 Remedies developed on the principles of chemical and conventional medications are usually of short affectivity, with a risk of adverse side effects and are usually of high prices, particularly for third world populations. Consequently, treatment of DM with plant derived phytochemicals that are easily obtained and are relatively cheap appears extremely likable.²⁹ Several investigations were applied to review the effectiveness of the plant extracts on diabetes evoked by STZ in many experimental animal tissues.^30,31 Many natural products can suppress the enzyme activities responsible for the production of glucose, its absorption, and insulin effectiveness.^32,33Other studies have clarified those positively effective components in plant extracts that can modify apoptosis of β-cell and promote insulin action.^34-36 The anti-diabetic effects of A. marina have been explored in many studies.^37,38

In our current investigation, diabetes was induced by the administration of a single dose of STZ (60 mg/kg B.W), tested animals with blood glucose levels above 250 mg/dl on the 3^rd day post treatment of the STZ injection were regarded as diabetic rats.¹⁹ In this study, the dramatic elevation of the fasting blood glucose and serum glucose levels (at 6^th week) after induction of diabetes were encountered in the diabetic rats as compared with the normal control rats.  The treatment of STZ-induced diabetic rats with A. marina revealed non-significant changes in blood and serum glucose levels in comparison with the diabetic control group. Streptozotocin is a cytotoxic compound especially toxic to the pancreatic β-cells, which are responsible for the production of insulin in mammals. It enters pancreatic β-cells through GLUT2 channels in the cellular plasma membrane, which evidently leads to cellular toxicity and local immune reactions leading finally to hypoinsulinemia and hyperglycemia in animals.³⁹ The harmful effect of STZ on β-cells leads to the development of deficient production of insulin and thus, the elevation of blood glucose level occurs²³. Moreover, a significant increase of serum/plasma glucose could provoke additional destruction of β-cells of the pancreas.⁴⁰

Insulin is the playmaker in diabetes of all types and has a crucial function in glucose homeostasis.   All patients with type-1 diabetes would like to have hormone treatment for good  health (insulin-dependent) unless they receive islet cells or whole organ transplant; several patients with type-2 diabetes could need this hormone once their β-cell function declines over time⁴¹. The findings of this study indicated that, at the end of the experimental period, the serum insulin levels were significantly decreased in STZ-induced diabetic rats as compared with the normal control ones. Administration of aqueous extracts of A. marina to diabetic rats resulted in a non- significant change in serum insulin levels when compared with the diabetic control group.

Moreover, creatinine is a well-authenticated as a metabolic derivative of muscle creatine and phosphocreatine and its concentration in serum is proportional to the body muscle mass. Therefore, the amount of creatinine is usually stable and its elevated levels indicate diminished renal function, as it is easily excreted by the nephrons.⁴² Low serum creatinine levels are positively associated with an increased risk of incident dysglycemia.^43,44 Likewise, other studies recorded a positive association between serum creatinine and diabetes prevalence.⁴⁵ In the current study, there was a significant increase in serum creatinine level of STZ-induced diabetic rats as compared with the normal control rats. The daily administration of aqueous extract of the investigated plants has a no impact on serum creatinine of diabetic animals as compared with STZ-induced diabetic rats. In contrast, Gandomani and Malati⁴⁶ indicated that A. marina at a dose of 400 mg/kg significantly decreases serum pro-inflammatory cytokines in rats with a consequent reno-protective effect.

It was formally documented that urea is the end product of protein catabolism in the living system and hence, it is synthesized in the liver from ammonia which is produced consequently as a result of the deamination of amino acids. Diabetic nephropathy is one of the major causes of chronic renal failure which is associated with an increased level of BUN and creatinine.⁴⁵ Another urgent factor is uric acid which is the metabolic end product of purine catabolism and its elevation levels in serum signifies renal functional impairment. Serum uric acid level has been suggested to be associated with the risk of type-2 diabetes. Albeit, biologically, uric acid plays an important role in weakening of insulin resistance in animal models by suppressing the bioavailability of nitric oxide, which is essential for insulin-stimulated glucose uptake.⁴⁷

In the current study, our evaluation documented a significant increase in a cascade manner embodying increase in serum blood urea nitrogen and uric acid levels of STZ-induced diabetic rats as compared with normal rats. Treatment of STZ-induced diabetic rats with A. marina revealed non-significant change in serum blood urea nitrogen in comparison to the diabetic control group. The results of the current study are contrary to that obtained by Mirazi  et al.⁴⁸ The authors concluded that hydro-alcoholic extract of A. marina leaves at doses of 400mg or 800mg/kg. B.W could be able to treat renal toxicity induced by CCl₄ in male rats with a significant reduction of urea. On the other hand, daily oral administration of A. marina extract to STZ-induced diabetic rats resulted in a significant decrease in serum uric acid levels in comparison to the diabetic control group

Diabetic nephropathy is a major complication of DM and a leading instigator of end-stage renal failure, as a worldwide calamity. The severity of diabetic nephropathy is one of the major factors determining the prognosis of diabetic patients.⁴⁹ The current investigation on the histo-morphology of the kidney of normal rats showed normal structures of nephron units in almost all the examined sections with keeping features of normal glomeruli, proximal and distal convoluted tubules, loops of Henle, collecting tubules, renal papillae, and renal pelvis. Moreover, the renal blood vessels and intertubular capillaries were in good condition. Renal sections of STZ-induced diabetic rats revealed multifocal coagulative necrotic areas with a pyknotic or karyopyknotic nuclei, deep eosinophilic cytoplasm, and distinct inflammatory reaction. Variable degrees of degenerative changes with mild dilatation of some distal convoluted and collecting tubules along with the partial atrophy of their lining epitheliums were also recorded. Peculiar perivascular edema and mild to moderate congestion of intertubular and glomerular blood vessels and capillaries with erythrocytic extravasations were also seen.

The results of the current study are partially similar to that obtained by Zafar et al.⁵⁰ who found large aggregates of lymphocytes in the interstitium of rat’s renal tissue following single IP injection of STZ (45 mg/kg B.W). The authors added that an inconstant number of proximal convoluted tubules showed signs of renal-tubular necrosis; their epithelial lining was disrupted and discontinued with pyknotic nuclei, vacuolated cytoplasm, broken cellular membranes and disappearance of brush borders. Nephropathic lesions of the current investigation coincide with that of  Balamash et al.⁵¹ This battery of astute scientists have observed that the renal tissue of normal rats showed normal cortex and medulla were intact; renal corpuscles being the main feature of the cortex. Moreover, the outer layer of Bowman’s capsule was lined by simple squamous epithelium and the glomerulus looked like cluster of capillaries covered by the inner layer of Bowman’s capsule. Furthermore, renal tubules in the cortex mainly include the proximal and distal convoluted tubules and these tubules were lined by the cuboidal epithelium.

Kidney lesions in STZ-induced diabetic rats treated with A. marina were represented by characteristic perivascular edema with vacuolation of the vascular walls. Some of the nephron units (glomeruli and tubules) were normal; meanwhile, others showed degenerative and early necrotic changes. The renal papillae and pelvis were normal in most parts; however, a few of them showed mild vascular and tubular dilatations of the surrounding tissue, besides, focal exfoliative changes in the lining epithelium were also seen. With such lesions, a speculated mild decrease in the renal filtration threshold along with a consequent insignificant increase in the creatinine, urea, and uric acids could be expected.

The characteristic renal lesions in the current investigation could lead to induction of dramatic changes in the renal biochemical markers. Such changes could be attributed to the direct nephrotoxic effect of STZ compound and the hyperglycemic secondary toxic metabolites. This information is in agreement with what was revealed earlier that diabetic nephropathy is a major complication of DM and a leading cause of end-stage renal failure worldwide.⁵² A fact that led to the severity of diabetic nephropathy is one of the major factors determining the prognosis of diabetic patients with the subsequent results that consider it a major determinant of morbidity and mortality in patients with DM. Therefore, previous scientific reports showed that diabetic nephropathy was induced by hyperglycemia via several mechanisms such as oxidative stress along with induction and increase of glycation reaction. Henceforth, the production of highly reactive oxygen radicals was attributed to the hyperglycemic oxidative stress which could lead to prominent cellular cyto-toxicity in different tissues including kidney.⁵³ Moreover, various investigations showed that the level of lipid peroxides as MDA was elevated both in serum and kidney tissues and the levels of the anti-oxidant enzyme were decreased in renal homogenates of diabetic rats.⁵⁴

Diabetic renal pathologic lesions are contributed to the angio-bio-physiologic complication of DM, which leads to chronic renal problems.⁵⁵ Apoptosis probably contributes to provoke diabetic nephropathy.⁵⁶ Apoptotic protein p53 and active caspase-3 are major biomarkers of apoptosis.⁵⁷ Oxidative stress can activate p53 leading to its nuclear accumulation resulting in cell cycle arrest, apoptosis, or cell removal from the proliferative pool.⁵⁷ Apoptotic p53 up-regulation is reported in several different tissues in DM, including kidney.^59,60 Apoptotic changes were induced by p53 through stimulation of caspases.⁶¹ Caspases constitute a group of cysteine proteases which exist in the cell as an inactive zymogen.⁶² Caspase-3 is the executioner caspase of apoptosis that becomes activated upon cleavage in the apoptotic process leading to the morphological features of apoptosis.⁶³ Elevated levels of active caspase-3 are reported in many renal diseases.^64,65

Kidney sections from different experimental groups of the present study revealed 6-8%/HPF in the tubular epithelial cells together with 3-5%/HPF in the glomerular cells of STZ-induced diabetic rats treated with A. marina. The observations of the current study are generally similar to that obtained by Al-Rasheed et al.⁶⁶ The authors reported that apoptotic markers (BAX and caspase-3) were significantly increased and the anti-apoptotic marker (BCL-2) was decreased in kidneys of STZ-induced diabetic rats. The authors added that topographical localization and double immunohistochemical analysis suggested that podocytes were the main apoptotic cells under these conditions, although endothelial and mesangial cells were also affected. This confirms and extends the previous observations of Susztak et al.⁵⁶ The authors identified topographically cell type(s) undergoing apoptosis and it was consistent with findings of Meyer et al.⁶⁷ and Steffes et al.⁶⁸ where the authors reported that significant podocyte apoptosis could be observed in diabetic patients, parallel with the progression of renal disease. The findings of the present study are in partial agreement with the results obtained by Ahmed et al.⁶⁹ The authors declared that the pro-inflammatory cytokine TNF-α and the apoptotic mediators (p53 and caspase-3) were remarkably decreased in the kidney of STZ-induced diabetic rats as a result of treatment with VDR activator (paricalcitol), while the expression of anti-apoptotic protein BCL-2 was increased. Furthermore, it should be mentioned consequently that in the aforementioned study, vitamin D was used as a synthetic chemical compound, but it can be naturally obtained from many phytochemical plant sources.⁷⁰

Finally, the apoptotic p53 protein together with pro-apoptotic proteins were consequently transported into the mitochondria as they encourage an increase in the mitochondrial membrane permeability and lead to energize of cytochrome c, which adheres to the apoptotic protease activating factor 1 (APAF-1) and with the caspase-9 proenzyme, to form a complex called the “apoptosome”. The latter, consecutively, synergized caspase-9 with consequent stimulation of caspase-3 proenzyme to the protease stage, which then sticks to the effector caspase group. Effector caspase-3, -6, and -7 do not need an adaptor protein for dimerization-induced activation. Rather, effector caspases spontaneously dimerize but are only activated upon cleavage between the small and large subunits by an active initiator caspase. Activated effector caspases then cleave a number of protein substrates to initiate apoptosis leading to the subsequent dismantling of cellular components,^71-74 evidently the effector caspases induce intracellular protein lysis and the morphological distinctive changes of apoptosis.⁷⁵ It was reported that apoptosis could aggravate the pathomechanisms of diabetic nephropathy and nephrotoxicity through caspase-3 expression.⁷⁶ The mitochondrial oncogene product BCL-2, prevented caspase-3 activation during a variety of proapoptotic conditions.⁷⁷ 

Conclusion 

The findings of this study evidently showed that the aqueous leaf extract of A. marina exerted protective effect against streptozotocin-induced nephrotoxicity in male rats as demonstrated by amelioration of the aforementioned biochemical parameters, histopathological and immunohistochemical changes in the kidney sections and restored renal tissue architecture near normal levels. The plant extract showed antihyperglycemic, insulinotropic, and renoprotective actions. Moreover, the findings of this investigation may lead to the development of novel anti-diabetic drugs through the use of up to date technologies which is described as a highly needed imperative

Funding source

There is no funding source

Conflict of Interest

The author of this study did not have any type of conflict of interest.

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Introduction

Algal flora has been studied by several workers throughout the world. Rindi and Guiry (2003)¹ studied algae from the walls of Galway city, western Ireland. Alghanmi and Jawad (2017)² studied the biodiversity of cyanobacteria from agricultural fields from Al-Diwaniyah city and a total of (26) species were recorded by them, out of which Oscillatoria was dominant. Bernstein et al (2011)³ performed a study to show cyanobacterial allergenicity.

The algal flora of India was studied by several workers. Sethi et al. (2012)⁴ collected different samples from the biological crust and subaerial habitat from the eastern region of India and reported (24) species of cyanobacteria and (6) species of microalgae. Satpati et al. (2013)⁵ studied mangrove forest at Sundarban and recorded (32) species of algae. Datta and Keshri (2014)⁶ investigated soil and subaerial algae from village Burdwan, West Bengal, India and recorded (22) taxa of blue-green algae. Kharkongor and Ramanujam (2014)⁷ reported (85) taxa of algae collected from tree barks from forested areas of Meghalaya. Satpati and Pal (2016)⁸ recorded Trentepohlia rigidula from two very distinct habitats like tree bark and cemented wall from West Bengal, India. Adhikary and Keshri (2015)⁹ studied cyanobacterial biofilms on the stone temple from Bhubaneshwar and reported (17) species of cyanobacteria while in monsoon additional 25 species of cyanobacteria were observed in the biofilms of these temples. Palanivel and Uma Rani (2016)¹⁰ studied two temple tanks from the suburb of Chennai were they found chlorophyceae was the dominant group at both the temple tanks. Dirborne and Ramanujam (2017)¹¹ studied algal flora from the pine forest and subtropical broadleaf forest from East Khasi Hills Dist. of Meghalaya with a comparative study on cyanobacteria and diatoms. Das and keshri (2017)^12,13 studied algal diversity from Koch-Bihar a district from West Bengal situated at foothills of Eastern Himalayas from where they reported (11) taxa of coccoid cyanoprokaryotes belonging to (5) genera and (24) taxa of Oscillatoriales under cyanoprokaryotes.

Similar work was carried out in different parts of Maharashtra by several workers. Pandkar et al. (2010, 2012)^14,15 reported algal diversity from Fergusson College with a very rare algal species named Oedocladium for the first time from Pune. Pandkar (2011)¹⁶ performed daily air sampling for 11 months at Nagpur city and identified (16) algal genera out of which Phormidium, Microcoleus, Scytonema, Anabaena, Lyngbya were known to be allergenic. Nikam et al. (2013)¹⁷ studied cyanobacterial diversity from Ahmednagar, Pune and Satara district of Maharashtra and reported (94) cyanobacterial species belonging to (38) genera, (14) families and (5) orders from 627 soil samples collected. Mahadik and Jadhav (2013)¹⁸ studied the Ujani reservoir from Indapur tehsil under the Pune area and reported (75) species under (42) genera of algae. Nimbhore and Jadhav (2014)¹⁹ studied algal flora of the Brinjal field of Aurangabad tehsil and reported (21) species under (14) genera belonging to cyanophyceae, chlorophyceae and bacillariophyceae. Wadhave (2014)²⁰ studied rice fields from Bhadrawati tehsil from Chandrapur dist. Maharashtra and reported (74) algal taxa.

Only two reports were there on studies carried out at Fergusson College as Pandkar (2010, 2012)^14,15. Apart from these reports on the Fergusson campus, there is not a single report from Fergusson hill. Hence these particular sites were chosen for present taxonomic study which will give an insight on changes taking place in algal composition over the past few years.

In the current paper, results are acquired by collecting algal samples from various terrestrial and subaerial sources and cultures obtained from them are reported. The study represents a comparative account of algal flora from the Fergusson campus and Fergusson hill as well as reports allergic algae from these two places.

Material and Method

Fergusson college campus is located in Pune City, Maharashtra, India (18⁰31’17.75”N & 73⁰50’20.17”E). It is divided into two sites as FC Campus and FC Hill. Both the sites are always flooded with students. These sites were also utilized by the elderly for recreational activities.

Samples from FC hill and FC campus were collected in July (2019). A total of 34 samples were collected out of which 17 were from FC campus and the remaining 17 were from FC hill respectively. Samples were collected from different sites like tree barks, stones, cemented walls and water tanks. The dry samples were collected using a scalpel and was stored in a clean zip lock bag while water samples were collected using centrifuge tubes. Collected samples were inoculated in the B.G.11 medium under natural conditions. Growth was observed after three weeks of inoculation. Upon optimum growth, semi-permanent slides were prepared using glycerin as a mounting medium. The identification of algae was done by using standard available literature (Desikacharya, 1959; Prescott, 1954)^23,24.

B.G. 11 broth with minerals, M1958-500G (HIMEDIA, Lot # 0000314677) was used to make media while LM5209 binocular microscope with LM1918 5MP CMOS camera was used for identification and microphotography. All other reagents used were of lab grade.

Figure 1:  Divisional Diversity at FC Campus Click here to View Figure

Figure 2: Generic Diversity at FC Campus Click here to View Figure

Figure 3: Total Forms per Spot at FC Campus Click here to View Figure

Table 1: Comparative Account of Algal Diversity Observed at Various Spots from FC Campus

Sampling spots from FC Campus- CS I and CS II- Soil from FC Campus; CW I to CW VIII -Wall scraping FC Campus; CT I to CT VII-Tree scraping from FC Campus.

Result and Discussion

Total of 116 cyanophyta members were observed from 17 different spots from the FC campus. Fig. 1 clearly showed a dominance of cyanophyta (90%) with a scarce presence of chlorophyta (8%) and bacillariophyta (2%) respectively. Cyanophyta was further divided into coccoid and filamentous forms. Filamentous (62) forms were little more than coccoid forms (54). In filamentous forms genus, Phormidium (20) was dominant with P. fragile reported from (8) samples. The second dominant group among filamentous cyanophyte was Oscillatoria (13) were O. limosa was reported from (5) samples. Lyngbya was reported from (9) samples out of which L. lutea was reported from (4) samples. Scytonema was reported from (7) samples with species like S. hofmanni and S. burmanicum from (2) samples each. Apart from these filamentous forms, Plectonema tomasinianum was reported from (2) samples followed by Nostoc, Dasygloea, Microcoleus, and Hapalosiphon were reported from (1) sample only. (Table 1)

In coccoid forms, Microcystis (19) was dominant followed by Chroococcus (18). From Chroococcus, C. minutus (8) was reported most of the time. Gloeocapsa were reported from (6) samples out of which G. nigrescens was identified up to species level from (5) samples. Merismopedia was reported from (4) samples with M. punctata reported from (2) samples. Other coccoid forms recorded were Aphanothece stagina, Aphanocapsa biformis, Synechocystis aquatilis and Synechococcus aeruginosus from one sample each. Desmid (8) and diatoms (3) were scarce belonging to chlorophyta and bacillariophyta respectively. (Table 1)

Total of 98 cyanophyta members were observed from 17 different spots from FC hill. Fig. 4 clearly showed the dominance of cyanophyta (78%) followed by chlorophyta (16%) while bacillariophyta (5%) and euglenophyta (1%) were scarcely recorded. Coccoid (52) forms were little more than filamentous (46) forms. Coccoid forms recorded showed the dominance of Chroococcus (26) with C. minutus (12) recorded from most of the samples. A second largest genus recorded from coccoid forms was Microcystis (18) were M. aeruginosa was identified up to species level from (1) sample only. Genus Aphanocapsa was recorded from (4) samples which were further identified up to species level as A. grevillei, A. elachista, A. biformis and A. roeseana. Other coccoid forms reported were Gloeocapsa (2) and Gloeothece rupesrtris (1). (Table 2)

Figure 4: Divisional Diversity at FC Hill Click here to View Figure

Figure 5: Generic Diversity at FC Hill Click here to View Figure

Figure 6: Total Algal Forms per Spot at FC Hill Click here to View Figure

In filamentous forms, Phormidium (23) was dominant with P. fragile (7) recorded most of the time. Scytonema was reported from (5) samples with S. julianum and S. hofmanni recorded from (1) sample each. Oscillatoria was reported from (4) samples with species like O. princeps, O. vizagapatensis and O. acuta reported from (1) sample each. Lyngbya (3), Arthospira (2) and Microcoleus were identified up to generic level while Plectonema wollei and Dasygloea amorpha were identified up to species level from (1) sample only. Desmid (18) and diatoms were reported from chlorophyta and bacillariophyta respectively. (Table 2)

Table 2: Comparative Account of Algal Diversity Observed at Various Spots from FC Hill

Sampling spots from FC Hill- HS I and HS II- Soil from FC hill; HST I to HST VIII- Stone scrapping from FC hill; HW 1 and HW II –Wall scrapping from FC  hill; HT I to HT IV- Tree scrapping from FC hill; HTA I and HTA II- Tank water from FC  hill.

Total (116) and (98) cyanophytic members from FC campus and FC hill respectively showed species richness which also points towards various substratum are adding to its algal diversity. Upon comparison of Fig.3 and Fig.6, it has been observed that tree scraping, stone scraping, and wall scraping shows a majority of cyanophytic algal forms compare to soil and water samples. It can be due to undisturbed crevices of trees, stones, and walls while soil samples and tank water sample shows less algal forms which may due to destructive human activity.

From Fig.2 and Fig.5, it’s quite clear that genera like Chroococcus, Microcystis, Gloeocapsa, Phormidium, Oscillatoria were present abundantly while Merismopedia, Lyngbya and Scytonema were present optimally at both the sites. All these cyanophytic members possess well-developed sheath around there cell/trichome which might help them withstand adverse environmental conditions. This finding correlates with Pandkar (2010, 2012)^14,15. Similar results were obtained by Sethi (2012)⁴, Karande (2012)²³. According to Sethi cyanobacteria were prominent in soil, building facades as well as on tree bark while green alga flourished only if sufficient moisture was available in the substratum while Karande stated that higher numbers of microalgae were reported from biofilms collected from the higher altitude. According to results obtained by Roy (2015)²⁴ on studies carried on East Kolkata Wetlands of West Bengal showed the presence of chlorophyte being dominant over cyanophyte but species like Chroococcus, Merismopedia and Synechococcus flourish throughout the year.

Microphotograph from FC Campus

Cyanophytic Coccoid Forms

Cyanophytic Filamentous Forms

Microphotograph from FC Hill

Cyanophytic Coccoid Forms

Cyanophytic Filamentous Forms

Conclusion

Total 129 algal forms were observed from FC Campus and 125 algal forms were observed from FC Hill out of which 116 and 98 were cyanophyta members from FC Campus and FC hill respectively. Cyanophyta members were found to be dominant at both the places with the presence of chlorophyta, bacillariophyta and euglenophyta respectively. The dominance of cyanophyta members was due to the presence of well-developed sheath around them which helps them to withstand adverse environmental conditions.

The present algal taxonomic study will provide insight into how algal diversity changed over the past few years. Forms such as Phormidium, Lyngbya, Scytonema, Microcystis reported to be allergenic has been encountered. Presences of allergenic algae manifest medical threat to humans were this study will act as baseline data.

Acknowledgment

The authors are thankful to Principal Fergusson College (Autonomous) Pune, SPPU, BARTI and Head Department of Botany Fergusson College for granting permission for completion of this work. We are also thankful to Bhagyashree U. Kute (S.Y. B.Sc. Student) for providing help during sampling and culturing work.

Conflict of Interest

We confirm that we do not have any conflict of interest.

Funding

Under STAR and MAST program of UGC-CE (2019-2020).

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Introduction

Tuberculosis (TB) remains a main public health problem in India, accounting for a quarter of the eight million instances of TB that arise worldwide. Despite the fact that tablets are available for remedy, TB stays nevertheless as a massive burden, in aid negative settings and the arena’s most vital motive of death specifically in India. When clinical knowledge is used to manual coverage and practices, evidences are ranked in line with the relative merits of various data.¹ Over the decade, numerous new interventions in TB manipulate had been advanced and recommended in WHO hints and carried out into India’s TB manipulate programmed. In the northeastern states of India, TB and PRG are found to be endemic. While guidelines for brand spanking new interventions are normally based on proof from standard population, little is thought from tribal regions. According to Indian state TB statistics 2017 and 2018 it is found that Nagaland a North Eastern state of India inhibited by tribal population accounts to 5826 TB Patients Notified Public Sector out of 2,050,220 population. The Revised National Tuberculosis Control Programme (RNTCP) has adopted multiple strategies so as to reach overall coverage of tuberculosis diagnosis and treatment. However, rural populations such as those living in the hilly terrain of Nagaland have limited access to health structures due to “poor road conditions and lack of basic communication services”, which may also hinder completion of TB treatment²⁰. Due to limited evidence, at the side of other factors, this could have hindered wide-scale use of tribal unique proof based interventions. One of the approaches is proof from it as a minimum one desirable systematic assessment. Its purpose is to reduce large portions of statistics to usable dimensions. Some declare that doing so is an effective medical approach; one of this is much less time ingesting and greater dependable then engaging in new research. The use and usefulness of systematic critiques is one effective mechanism for improving the proof available to tell population fitness decision making.¹

Moreover they contend that the various occasions where in man or woman research is executed allow reviews consequences to be generalized throughout distinct contexts and emerge more extra tremendous than person research. There are currently rare systematically accumulated statistics on the availability of proof for scale-up of newly encouraged interventions for TB control in tribal areas.¹

The polymorphism in the ABO blood group remains important in population genetic studies, estimating the availability of compatible blood, evaluating the probability of hemolytic disease in the new born, resolving disputes in paternity/ maternity and for forensic purposes². The frequencies of ABO and Rh phenotypes in different populations have been extensively studied. Rh system emerged as second most important blood group system due to hemolytic disease of newborn and its importance in RhD negative individuals in subsequent transfusions once they develop Rh antibodies. The D antigen, after A and B, is the most important red cell antigen in transfusion practice^3.

Several physical, emotional and mental problems appear to be associated with procrastination. It may create embarrassment and inferiority complex among students of which the found negative relationship between level of ego identity and procrastination; it lessens confidence among students and their expectancy of completing a task⁴; resulting in unhealthy sleep, diet and exercise habits ⁵ yields to higher rates of smoking, drinking, digestive ailments, insomnia and cold and& flu symptoms⁶; increases a lot of stress, worry, and fear leading a miserable life with shame and self-doubt creating and raising anxiety and deteriorates self-esteem⁷ affects achievement of goals creating anxiety⁸ and causes higher stress, low self-esteem, depression, cheating, plagiarism, higher use of alcohol, cigarette and caffeine and decreased ability to maintain healthy self-care habits like exercise and eating ⁹

The study was conducted with the objectives to the context of disease epidemiology and procrastination, upon various Indian tribal communities, who are highly isolated both demographically and topographically, from the mainstream Indian populations. Blood bank usually has a problem of ever-changing stock position and it being very difficult to predict the prevalence of a particular blood group at a particular time. The present study was done to assess the prevalence of blood groups in different categories of Northeast India with the following specific objectives;       (i) To evaluate the most common Blood group among tuberculosis patients (ii) To compare and study the controlled groups with those of tuberculosis patients.

Research Methodology

The present study was descriptive in nature. Therefore survey approach was considered and adopted to collect the data from respective respondents. 

Population and Sampling

The present study was delimited to the TB patients of the olive Christian Hospital, Dimapur, Nagaland. The population of the study is tuberculosis patients of the above said hospital with the ABO blood groups. Among the population 50 TB patients were selected as a sample through the random sampling technique Further, 50 control group respondents were selected from the faculty members of St. Joseph University to evaluate and compare the result. Overall 100 respondents were considered for the study including patients and control group.

Data Collection

The study was descriptive in nature; therefore, the researchers considering the survey approach was appropriate to adopt for data collection. The researchers has developed two different set of questionnaire for patient group and control group respectively based on 5 point Likert scale technique. And the developed an questionnaire has been validated through the pilot study with the 50 respondents. Some corrections were identified and rectified in the pilot study. Finally, the questionnaires were administered on TB Patients of olive Christian Hospital, Dimapur, Nagaland and Control group peoples. The collected data were coded and analyzed in terms of percentage and mean score through Ms-Excel.

Laboratory Analysis

Once the samples were collected the ABO and Rh blood group testing was done from the laboratory using the standard protocol of AB D Antisera typing Kit.

Statistical Analysis

The gene and allele frequencies of blood group, are calculated by Hardy-Weinberg model using S2 ABO estimator software.¹⁰ Allele Frequencies are calculated under the assumption of Hardy–Weinberg equilibrium and expressed as percentages. The chi – square test is used to compare observed allelic and genotypic frequency distributions of the blood group and Rh antigens to that of under the Hardy–Weinberg.¹¹

Results and Discussion

Association studies between ABO blood groups with Procrastination among tuberculosis patient’s diseases are the hallmarks for unravelling the genetic pattern of complex diseases. Studying the relationship between allelic and genotype frequencies of candidate genes among both affected and healthy subjects, to understand the genetic etiology of complex human traits, is an efficient method to elucidate their disease pathogenesis. It is found that female occurred in the order O> A >B. The allele frequency of blood group O is the highest, 𝜒2 The goodness of fit test was resulted in value was = 5.1 and p value was 0.02. This only significant value(Table.2) There is the high proportion of Rh (D) +ve individuals than the Rh – ve in the study populations. (Table.1)

Table 1: Frequency of ABO blood groups and Rh factor in Patients and Controls

Table 2: Shows the overall allele frequencies for the ABO in Patients and Controls

* Statistically significant.

Hence forth, we tried to elucidate the possible association of the ABO blood groups with Procrastination among tuberculosis disease patients. The ABO blood groups with Procrastination were observed to be more prevalent among cases than controls, as shown in Table. 2 and 3. Genotype distributions, allelic frequencies and the corresponding odds ratios (OR) were calculated for each variant as shown in Table: 4.

Table 3: Distribution of ABO blood frequencies in Cases and Controls stratified according to gender

χ2 : Chi-square with 1 degree of freedom; OR: odds ratio

No significant association was observed in the ungrouped data. This occurred in the order ABM (Dominant model). The allele frequency of blood group A +B Vs O is the highest, 𝜒2 The goodness of fit test was resulted in value was = 4.6 and p value was 0.05 this is significant value (Table. 4) However, when segregated the subjects into male and female, we found that the O Blood group homozygous genotype has a significant prevalence upon females than in males.

Table 4: Distribution of ABO blood frequencies (dominant and recessive model) in Patients and Controls

After the discovery of blood groups, numerous studies on associations of blood groups and various diseases were performed. Identifying the prognostic and associating factors, which predict the condition of the disease and its response to the treatment, can play an important role in determining the therapeutic strategies.

This study demonstrated that blood group O+ve was commonest and O-ve was least frequent among blood donors. This is in agreement with the studies that performed on blood donors¹² and population of Tehran Province.¹³

Previous studies of the ABO pattern among patients with pulmonary tuberculosis were made at a time when the incidence was much higher than at present in Copenhagen. If a weak correlation exists between tuberculosis and some of the blood groups, it could easily have been obscured when the disease was more frequent. This is likely especially if recurrences, sequelae or non-bacillary patients were included. The ABO pattern of these patients is closer to or identical with the ABO pattern of normal persons. In this context it should be recalled that the diagnosis of pulmonary tuberculosis is less exact among abacillary patients. This fact may well explain the discrepancies in the ABO pattern of bacillary and abacillary patients, since the latter group may contain a number of patients not suffering from tuberculosis.

The present study showed that there was an association between tuberculosis and the blood groups B and AB in this region of the Dimapur City. The deviations were however not significant¹⁴.Many studies with similar intent were conducted earlier.

It is Suggested blood Groups O and AB individuals are more susceptible to TB¹⁵. However, a study by Rao et al., concluded that blood Groups O and A were the most common blood groups associated with PTB.¹⁶ A study in Gujarat, a significant association was discovered between blood Group AB and pulmonary TB.¹⁷ Similarly, Jain¹⁷ had similar observations for AB blood group and pulmonary TB. People with blood Group O showed protection from TB in a Chinese population.¹⁸This could be one of the most important for the observed deviations from the expected ABO pattern, but it remains speculative so far. Studies suggests that self-administered TB treatment is feasible for patients living in areas with limited or no access to health services.²⁰

Conclusion

The study concluded that procrastination effects on the O +ve blood groups patients genotype is significantly associated more with the female patients (p = 0.02) than male patients (p = 3.8). The allele frequency of blood group O is the highest, 𝜒2 The goodness of fit test resulted in value was  5.1 and p value was 0.02  among tuberculosis patients in the tribal populations of  Nagaland. The significant association of the O blood genotype with Procrastination among tuberculosis patients, especially, with females of the tribal populations was observed.

The ABO blood groups with Procrastination were observed to be more prevalent among tuberculosis patients than the controls. When separated the subjects into male and female, it is found that the O Blood group homozygous genotype has a significant prevalence upon females than in males. With the observed association of ABO genotype, Procrastination with tuberculosis, this study anticipates more studies with larger cohorts to extend and elucidate. The in progress study be responsible for spirited information on the Procrastination of TB among the tribal population of  Nagaland which jerry can be situated used as a standard data for future epidemiological studies.

References

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Introduction

The clinical signs of COVID-19 in children less than 16, identified by acute respiratory syndrome year, were fever, dry cough and abnormal laboratory findings summarized as increase level of creatine kinase MB and procalcitonin, decrease lymphocytes counts (Xu et al., 2020b). While the clinical signs of COVID-19 in older shown same symptoms with high virus load firstly in the upper respiratory tract and in also detected in stool (Zhou et al., 2020).  Also, digestive symptoms are common in patients suffering from COVID-19 in a recently submitted article (Gu et al., 2020; Mao et al., 2020).

The intestinal microbiome has a role in development and differentiation of the immune system as the gut microbiota provides signals pathways to stimulate the normal development  as well as the maturation of immune cells (Lazar et al., 2018).

Also, the lung microbiota population differed from mild and moderate in the chronic obstructive pulmonary disease. Streptococcus was the most predominant genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways (Pragman et al., 2018).

Human gut microbiota has a role in drug effectiveness and metabolism (Jourova et al., 2016). Also, Murine microorganisms provide chemicals that lead to the potential development of new therapeutics against multiple disease categories (Mayer and Lehmann, 2000).

In china many patients admitted firstly to hospital suffer GIT manifestation, so we want to explore the effect of modulation of Gut Microbiota on complications of COVID-19 which could be the cause of death.

Drugs and Gut Microbiota

Antiprotozoal drug pentamidine treat ulcerative colitis indirectly through immune modulation (Esposito et al., 2012), Immune cells release cytokines, interleukins and pro-inflammatory signaling molecules (Shanahan, 2001).  Also, The anti-atherosclerotic effect of Berberine (BBR) is related to alterations in gut microbiota compositions, indicating the potential therapeutic value of pharmacological approaches that may modulate the gut microbiota in treating atherosclerosis (Shi et al., 2018). A larger cohort study suggested that the medication can alter gut microbiome composition and so the correlation between species abundance in the human microbiome and drug sensitivity evaluation consider very important for development of new successful and beneficial therapy (Maier et al., 2018).

Indeed, in HIV‐infected patients, the changes in gut microbial flora resulted in a generalized immune activation (Deeks et al., 2013).  Notably, the antimicrobial effect of Chloroquine could potentially explained  to the changes in the gut flora, resulting in stimulation of DCs to produce IFN‐α2, and this effect is more than the inhibitory effect of Chloroquine on IFN production (Routy et al., 2015). As a huge number of cells were exhibited in the gut-associated lymphoid tissue (GALT), and according to the microbial translocation theory, the damage of intestinal mucosa by the inflammation may allow passage of products of the gut microbiome which further enhance HIV-related immune hyperactivation (Lederman et al., 2013; Marchetti et al., 2013).

Zinc, Coronavirus and Gut Microbiota

The high levels of dietary Zn reduced  plasma cortisol level in LPS-challenged pigs at days 9 and 19 and the dietary Zn improved the growth performance of the newly weaned piglets through the modulation in gut microbiota as well as reduction of cortisol response following an immune challenge (Namkung et al., 2006). Notably, Zn ion was reported to stop the initiation step of  EAV-RNA synthesis and  elongation and template binding was reduced in the case

Paracetamol Safe in Coronavirus Infection

The implication of TNF‐α in virus‐induced hepatitis failure considered a features of coronavirus in the form of fulminant hepatic necrosis while the  paracetamol poisoning does not cause an increase in the  TNF‐α activity in the circulation (Devictor et al., 1992). So, the paracetamol could be safe as antipyretic drug in case of viral infection.

Corticosteroids and Coronavirus

It was noticed that the plasma SARS-CoV RNA concentrations in the second and third week of illness were significantly more abundant in patients who received early hydrocortisone treatment as compared to those who given the placebo. So, the early corticosteroid treatment was accompanied by a higher subsequent plasma viral load (Lee et al., 2004).

The Hypnotics and Coronavirus

The Abnormalities of sleep are common in hospitalized patients suffer diseases, but the mechanisms and consequences are still not well understood. for example, In one pilot study of patients  suffer from insomnia comorbid with RA, eszopiclone 3 mg, sleep aid,  improved sleep and daytime function measures over the treatment period, as well as some measures of disability, and quality of life and RA-associated pain (Roth et al., 2009) . Additionally, Night calm sodium is a composition of eszopiclone 3mg that could help in reducing the virus count in the upper respiratory tract but need more investigation. Also, there is a clinical studies (ClinicalTrials.gov Identifier: NCT00822679) on effect of eszopiclone on Inflammatory Mediators in Patients suffer from Acute Coronary Syndrome (https://clinicaltrials.gov/ct2/show/NCT00822679)

Why are Children Less Susceptible to Corona Infection ?

Why children not affected by corona could be explained by a recent study demonstrated that neonatal colonization of the gut microbiota is essential for the reduction of the numbers of iNKT cells in the intestine, and iNKT cells have been enhanced the mediating allergic responses in the lungs (Mazmanian et al., 2008). In addition, the microbial compounds stimulate the peripheral B cells through B cell-intrinsic MYD88 signaling and inhibit IgE production. Also, the decrease in the levels of peripheral IgE resulted in a reduction in the numbers of basophils, and reduced the risk of allergic airway inflammation. EAE, experimental autoimmune encephalomyelitis (Kamada et al., 2013).

Dietary Fermentable Fiber Content, Gut Microbiota and Allergic Inflammation in the Lung.

Host-microbe crosstalk affects the inflammation in peripheral tissues especially in the lung tissue, which is poorly understood. Also, it was found that dietary fermentable fiber modulates the microbiota of gut and lung, by imbalance the ratio of Bacteroidetes to Firmicutes (Trompette et al., 2014).  Also, the short-chain fatty acids (SCFAs), metabolites of gut microbiota metabolism of the fiber, had a beneficial role for protection against the inflammation.  Notably, the circulating levels of SCFAs were increased and protected against the allergic inflammation in the lung tissue in mice fed a high-fiber diet increased whereas a low-fiber diet decreased the levels of SCFAs and enhanced the allergic airway disease. Additionally, the treatment of mice with the SCFA propionate resulted in the modulation of the bone marrow hematopoiesis that was characterized by the enhanced generation of dendritic cell (DC) precursors and macrophage and subsequent seeding of the lungs by the DCs with high phagocytic capacity. The effects of propionate on allergic inflammation were based on the G protein-coupled receptor 4. Moreover, the  dietary fermentable fiber and SCFAs can build the immunological environment in the lung and influence the severity of allergic inflammation (Cummings et al., 1987; Hill et al., 2010). As recent evidence has indicated that the microbiota could influence the immune cell homeostasis and the susceptibility to allergic inflammation (Herbst et al., 2011; Olszak et al., 2012).

Cytokines Storm and Cause of Death Due to Coronavirus

Cytokine storm syndrome is active immune response that leads to frequently fatal multi-organ dysfunction syndrome. The first screening tool for the possibility of a cytokine storm syndrome diagnosis in hospitalized patients with Covid-19 was the elevated serum ferritin values (Mehta et al., 2020). The death in patients suffering Cytokine storm syndromes is impossible as corticosteroids are contraindicated here due it increases the virus load in the body as described above. Although some cases have recovered as the artificial liver blood purification system could enhance the removal of inflammatory mediators and block cytokine storm (Xu et al., 2020a).

Vitamins and Coronavirus

Vitamins like A, D, and C are very important keys for immune system improvement while its deficiency results in increased risk of mortality. Notably children with vitamin A deficiency have a greater risk of illness and death due to respiratory tract infections (Ross, 1996). While the Pre-existing deficiency of vitamin A seem to worsen the infection and vitamin A supplementation has been reported to reduce the risk of death in 6–59 month old children by about 23–30% In the case of pneumonia that is associated with measles. Similarly, the raising of serum 25(OH)D concentrations through vitamin D supplementation, or exposure to sunlight, could reduce the incidence, severity, and risk of death from influenza, pneumonia, and the current COVID-19 epidemic (Grant et al., 2020). Also vitamin C could reduce signs and complications of covid-19 through reduce the cytokine storm in acute respiratory tract infection (Banerjee and Kaul, 2010; Boretti and Banik, 2020) and improvement of immune system status.

Discussion

The inflammation of the lung occurs later as the corona virus firstly detected in upper respiratory tract and the stools (Mao Et Al., 2020; Xu Et Al., 2020b; Zhou Et Al., 2020). Notably, many patients suffered firstly from gastrointestinal manifestations and could alter the gut microflora population (Gu Et Al., 2020; Mao Et Al., 2020).Intriguingly, the nutritional support and application of the prebiotics or probiotics could be  helpful to regulate the balance of intestinal microbiota and decreased the risk of secondary infection due to bacterial translocation after coronavirus infection (Xu Et Al., 2020a).  Also, the unique gut microbiota profile perform many specific regulations in host nutrient metabolism, the maintenance of integrity of the gut mucosal barrier constructions, modulation of immune system, and protection against pathogens (Restrepo). Moreover, the dietary fermentable fibers could modulate the population of the gut microbiota through the imbalance of the ratio of firmicutes to bacteroidetes and releasing of circulating short-chain fatty acids (scfas) that could have ameliorative affect to allergic inflammation in the lung (Cummings Et Al., 1987; Hill Et Al., 2010). ). In contrast, the disturbance in gut microbiota has been implicated in different lung diseases, including allergy, asthma and cystic fibrosis (Anand And Mande, 2018).

Notably, drug inducing its pharmacological effects through modulation of gut microbiota like the antimicrobial effect of Chloroquine could potentially explained  to the changes in the gut flora (Routy et al., 2015).

Regarding different incidence, mortality of coronavirus in different countries all over the word could be that a certain population had partially solid immunity like BCG vaccination (Aaron et al., 2020) and also may be the nutritional habitats and composition of food could reduce the mortality rate like in Germany.

Finally, the Eating of organic whole plant foods supported with good amounts of leafy greens and enriched with fiber-rich foods (legumes, whole grains, beans, vegetables) , avoiding animal derived products including (poultry, fish, pork, beef, and dairy), and use a lactobacillus probiotic containing products to the everyday routine are key factors for health gut microbiota (Rinninella et al., 2019).

In conclusion, the modulation of gut microbiota by drugs and/or vitamins are very important for improvement of immune system and may enhance the protection against viral infection and reduction of clinical signs but further investigation is needed to study clinical available data from different global countries.

Conflict of Interest

All authors have no conflict of interest

Acknowledgments

We thank prof dr. Mehvish Taqi Editorial lead and Social Media Manager at Enviro Research Publishers. Bhopal, Madhya Pradesh, India who support and help us. We also thank our vice dean of postgraduate and research prof dr. Sabry El Khoudry, faculty of veterinary medicine, Mansoura university, Egypt. For his support

Financial Support

There is no financial support to this mini-review article

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Introduction

The human papillomavirus (HPVs) infection is implicated in the development of cervical cancer. Continuing infection by the high-risk HPV (specifically, type 16) can initiate cancer of the vulva, cervix, vagina, anus, oropharynx, and penis (Crosbie et al. 2013; Bernard et al. 2010). The connection between cervical cancer and HPVs infection is well recognized (Sundstrom et al. 2019; Chen et al. 2019; Almazrou et al. 2020; Jradi and Bawazir, 2019). In 2018, a total of 570,000 incidences of HPVs infection-related cancer appeared, whereas 311,000 mortalities were acknowledged. It has also been recognized that cervical cancer ranks the second most common form of cancer in females of 15-44 years group (https://gco.iarc.fr/today). Cervical cancer is the ninth most frequently diagnosed cancer among the Saudi women of 15-44 years group (Jradi and Bawazir, 2019; https://gco.iarc.fr/today; Bruni et al. 2019). In 2018, 316 Saudi women were diagnosed with cervical cancer, wherein 158 resulted in death (World Health Organization, 2017). According to literature (Turki et al. 2013), the prevalence of the HPV infection is escalating in a worrying proportion in Saudi Arabia. Three preventive vaccines (Table 1) for young girls (9-13 years), and young adults (13-126 years) have been introduced (Sundstrom et al. 2019; Markowitz et al. 2007; Rashid et al. 2016; Harper et al. 2004; Villa et al. 2005). It also noted that the available vaccines could protect more than 2/3^rd of the population against HPV-16 and HPV-18 genotype infections (Alsbeih, 2014; Gosadi, 2019; Hussain et al. 2016; Doorbar, 2006). An increase in the degree of perception and the elementary familiarity of the disease, along with its available vaccines, helps to control and prevent the disease. Similarly, the outcome of the present study of the subject matter can help control the prevalence of cervical cancer caused by HPV in Saudi Arabia. Accordingly, the author aimed to perform the titled survey in the Northern Region of Saudi Arabia.

Table 1: Types of HPV vaccines

Methodology

This study was undertaken from September 2016 to November 2017 at the Northern Border University (NBU, Saudi Arabia), which is located in the Northern Region of Saudi Arabia. Four health colleges, namely, The College of Medicine, The College of Nursing, The College of Pharmacy, and the College of Applied Medical Sciences, located in Arar and Rafha cities were included in the present study. The affiliated students of the university were from the urban areas as well as the rural areas, but the quasi-totalities were belonging to urban areas. This cross-sectional study was performed by a pre-tested questionnaire (Table 2), which was shared with the students after their consent. A total of 434 students participated in this study, wherein 258 girl students and 144 boy students (18-26 years) contributed to the study. The collected records from the survey were analyzed anonymously by allocating arbitrary codes.

Diagram 1: Diagram of the General Methodology Click here to View Diagram

Results

Of the 402 students interviewed, the number of girls was 258 and 144 boys (64.18% and 35.82 % respectively). The results are summarized as the number of answers given by the respondents for each question (Table 2). A total of 8040 answers were presented by the interviewed students (20 x 402 = 8040), wherein 2625 (32.64%) answered as ‘‘Don’t know’’. Accordingly, it can be assumed that every third participant student was unaware of HPV and cervical cancer. Moreover, 46.26 % (186 students) of the total number of students never heard about cervical cancer. Human papillomavirus (HPV) as a correct answer for the possible causes of cervical cancer is known by 133 students (33.08 %), and 24.62% of the students answered that HIV causes cervical cancer. The knowledge about prevention and vaccine for cervical cancer showed similar values among the students.

Table 2: The pre-test questionnaire and the obtained results (N = 402)

Furthermore, only 167 students (41.54%) were sure that cervical cancer is preventable, whereas 235 students (58.45%) did not know or supposed that cervical cancer is not avoidable. The majority of the participants (61.19%) were unaware of the available vaccine for HPV, while 38.80% were aware that this type of cancer is preventable by vaccine. It has also been observed that only 75 students (18.66%) knew that the vaccine for HPV is available in Saudi Arabia. Question 12 was related to the effect of available vaccines against HPV and associated illness. The data collected from the questionnaire showed that 31.09% of students knew that vaccines could treat HPV infection and HPV-associated disease, whereas 68.90% did not know about it.

According to the World Health Organization (WHO), Merck’s Gardasil received approval from the US Food and Drug Administration (FDA) in 2006. Shortly afterward, it was provisionally recommended for girls and women aged nine to 26 years. In the present study, only 49 students (12.19%) were able to reveal the correct age group for which the HPV vaccines have been approved, whereas 38.30% did not know the ideal age for such vaccines. About 39.8% of respondents answered that both males and females could get the HPV vaccines, 3.23% supposed that only a male can get vaccines, and 32.09% revealed that only a female could get these vaccines. It is well known that ‘‘HPV vaccination combined with regular screening is the best strategy to avoid developing cervical cancer (World Health Organization, 2017). However, no health checkup is needed before getting the vaccine. The percentage of students who were aware of it was 14.18%, whereas others did not know the answer (44.53%) or gave the wrong answer (41.29%).

The cervical cancer screening test is recommended in women aged > 30 years every five years, including a combined check of cytology and HPV testing (co-testing). The standard cervical cytology test is recommended alone every three years (Saslow et al. 2012;

Moyer and U.S. Preventive Services Task Force, 2012; Domgue et al. 2019). One query focused on the awareness of the interviewed students about the cervical cancer screening test. The results were varied from don’t’ know (41.29%) to Pap test (14.93%) and HPV test (12.19%). Once infected by the HPV virus, will it be possible getting the vaccine? The majority of participants (46.02) were not aware of it, 24.13% responded that it is possible, whereas 29.85% did not believe in HPV vaccine efficacy in HPV post-infected women. The WHO recommends that two doses of an HPV vaccine be given to 9-14 years old girls as a priority (World Health Organization, 2017). It was surprising that only 8.71% of students gave the correct answer, whereas others either provided incorrect answers or did not know about vaccine doses. The most critical obstacle preventing the participants from receiving/advising HPV vaccination was inadequate information (22.14%). This was followed by the worry about the complications (18.66%), above the age (18.41), vaccine availability (16.17%), high cost, and the concern about the efficacy of the vaccine. It has also been identified that 38.06% of students consider medical school teaching as the primary source of information about HPV associated diseases and prevention. This was followed by the internet (20.15), books & friends (6.72%), newspaper (2.24), and television (1.74%). A large part of the students (24.38%) did not hear about it.

Discussion

Cervical cancer is the ninth most frequently diagnosed cancer among Saudi women of the 15-44 age group (Jradi and Bawazir, 2019; Bruni et al. 2019). About 316 Saudi women had cervical cancer in 2018, wherein 158 died of this disease (World Health Organization, 2017). Cervical cancer is preventable as well as curable if detected early (Rashid et al. 2016; Alsbeih, 2014). Mostly advanced-stage cases are reported in Saudi Arabia due to inaccessible screening tests (Alsbeih, 2014; Dosoky et al. 1995; Manji, 2000). The age-standardized incidence rate of cervical cancer cases attributable to HPV (per 100,0000 women) in Saudi Arabia is relatively low as compared to other countries (2.5 in Saudi Arabia, 1.9 for Yemen, 6.4 for UAE, 8.4 for UK and 75.3 in Swaziland) (Bruni et al. 2019). In 2013, the WHO had reported that 6.51 million Saudi women older than 15 years are at risk of developing cervical cancer (World Health Organization, 2017), and HPV-16 and HPV-18 cause 70% of the worldwide cervical cancers. The HPV-16 (30%), HPV-18 (8.0%), and HPV-45 (5%) are the most prevalent genotypes reported in Saudi Arabia (Turki et al. 2013).

A combination of knowledge, awareness, educational programs, HPV screening, and HPV vaccination can reduce and control cervical cancer cases (Rashid et al. 2016). Accordingly, the present study aimed to get information about the level of awareness and knowledge toward cervical cancer, HPV, and attitude to HPV vaccination among health college students. We observed a persistent answer ”don’t know” and the present cross-sectional study and about half of the interviewed students did not hear at all about cervical cancer.

Globally, the awareness level among students about cervical cancer and HPV is weak. The knowledge regarding the prevention and vaccine is also found to be very poor. This is alarming because the concept of the protection against this type of cancer is primarily built upon the procurement of necessary and basic understanding and awareness toward HPV and cervical cancer. In the present study, 61.19% of the interviewed students assumed that there is no available vaccine for HPVs; 18.66% knew that there is an available vaccine against HPV in Saudi Arabia; 39.8% answered that both sexes could get the vaccine. This is the first report undertaken to understand the awareness about cervical cancer and HPV in the Northern Region of Saudi Arabia. However, previous studies have been conducted in other regions of the country like Riyadh (Almazrou et al. 2020; Jradi and Bawazir, 2019). A lack of awareness about HPV, cervical cancer, and the HPV vaccine among the studied women groups in Riyadh has been reported (Jradi and Bawazir, 2019). This study further elaborated that the health care providers (other than physicians) (30%) were not aware of the preventive measures, wherein 63.3% did not exercise any screening procedure for cervical cancer because of the lack of screening facilities. This study corroborates our finding, and the reason for the reduced level of the HPV, cervical cancer, and the HPV vaccine are well harmonized (Jradi and Bawazir, 2019). Among other reasons, misconception, non-availability of the educational programs at the academic and community level, and the socio-cultural factors (modesty and decency) may explain the reduced level of awareness among participants about the HPV, cervical cancer and its vaccine. The lack of information about HPV linked to the lack of knowledge of the vaccines is one of the numerous barriers that prevent vaccination recommendation.

Conclusion

This study exposed a reduced level of knowledge about HPV and cervical cancer among students of the Health Colleges in the Northern Border University. It is imperative to improve awareness about this subject among the participant students. Further, it is believed that there exists no national HPV immunization program in Saudi Arabia. However, such programs are running successfully in many countries, for example, in the USA (National Breast and Cervical Cancer Early Detection Program, NBCCEDP), in UK (Cervical screening), in Australia (The National Cervical Screening Program). It is suggested that the National Transformation Program to ensure the realization of Kingdom’s Vision 2030 in the domain of Public Health System and Health Disasters Management will be the adequate framework for incorporating The ‘‘Saudi Arabian Cervical Screening Program: SACSP’’.

Acknowledgment

The author, thanks to all the students of the Northern Border University, who participated in this study.

Conflict of Interest

The author declares that there is no conflict of interest.

Funding Source

There is no funding source.

References

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2. Bernard H. U., Burk R. D., Chen Z., van Doorslaer K., zur Hausen H., de Villiers E. M. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology. 2010; 401(1): 70‐79.
3. Sundstrom B., Smith E., Delay C., Luque J. S., Davila C., Feder B., Paddock V., Poudrier J., Pierce J. Y., Brandt H. M. A reproductive justice approach to understanding women’s experiences with HPV and cervical cancer prevention. Soc. Sci. Med. 2019; 232: 289-297.
4. Chen J., Deng Y., Ao L., Song Y., Xu Y., Wang C. C., Choy K. W., Tony Chung K. H., Du Q., Sui Y., Yang T., Yang J., Li H., Zou C., Tang, T. The high-risk HPV oncogene E7 upregulates miR-182 expression through the TGF-β/Smad pathway in cervical cancer. Cancer lett. 2019; 460: 75-85.
5. Almazrou S., Saddik B., Jradi H. Knowledge, attitudes, and practices of Saudi physicians regarding cervical cancer and the human papilloma virus vaccine. J. Infec. Public Health. 2020; 13(4): 584-590.
6. Jradi H., Bawazir, A. Knowledge, attitudes, and practices among Saudi women regarding cervical cancer, human papillomavirus (HPV) and corresponding vaccine. Vaccine. 2019; 37(3): 530-537.
7. https://gco.iarc.fr/today (Accessed on March 1, 2020)
8. Bruni L., Albero G., Serrano B., Mena M., Gómez D., Muñoz J., Bosch F. X., de Sanjosé S. ICO/IARC Information Centre on HPV and Cancer (HPV Information Centre). Human Papillomavirus and Related Diseases in the World. Summary Report 17 June 2019. 2019 [https://www.hpvcentre.net/statistics/reports/XWX.pdf; Accessed on March 1, 2020].
9. World Health Organization. Questions and answers about HPV vaccination: Information for parents and caregiver. 2017: 1-17. (http://www.euro.who.int/__data/assets/pdf_file/0009/356841/Q-and-A_HPV_Parents_EN.pdf; Accessed on March 1, 2020)
10. Turki R., Sait K., Anfinan N., Sohrab S., Abuzenadah, A. Prevalence of Human Papillomavirus in Women from Saudi Arabia. Asian Pac. J. Cancer Prev. 2013; 14(5): 3177-3181.
11. Markowitz L. E., Dunne E. F., Saraiya M., Lawson H. W., Chesson H., Unger E. R., Centers for Disease Control and Prevention (CDC), & Advisory Committee on Immunization Practices (ACIP). Quadrivalent Human Papillomavirus Vaccine: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommend. Reports. 2007; 56: 1-24.
12. Rashid S., Labani S., Das B. C. Knowledge, Awareness and Attitude on HPV, HPV Vaccine and Cervical Cancer among the College Students in India. PLoS One. 2016; 11(11): e0166713.
13. Harper D. M., Franco E. L., Wheeler C., Ferris D. G., Jenkins D., Schuind A., Zahaf T., Innis B., Naud P., De Carvalho N. S., Roteli-Martins C. M., Teixeira J., Blatter M. M., Korn A. P., Quint W., Dubin G., GlaxoSmithKline HPV Vaccine Study Group. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet. 2004; 364(9447): 1757-1765.
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Introduction

⁴Factors to be Considered in The Design of Colon-Targeted Drug Delivery Systems

Anatomy and Physiology of Colon

Gastrointestinal Transit

The GI tract is divided into stomach, small intestine and large intestine. The large intestine extending from the ileocaecal junction to the anus is divided into three main parts. These are the⁶ colon, the rectum and the anal canal. The colon itself is made up of the caecum,the ascending colon, the hepatic flexure, the transverse colon, the splenic flexure, the descending colon and the sigmoidal colon.

pH in the Colon: Average⁸ pH in the GI tract

The⁹ transit time of dosage forms in GI tract

¹²Colonic Microflora

A large number of anaerobic and aerobic bacteria are present throughout the entire length of the human GI tract. The upper region of the GIT has a very small number of bacteria and predominantly consists of gram-positive facultative bacteria .The concentration of bacteria in the stomach is usually less then 10³ colony-forming units/ml (CFU/ml) and most commomaly isolated species are streptococci, staphylococci, lactobacilli and various fungi. In the distal part of the small intestine, a higher concentration of anaerobic bacteria is found .The lower ileum has a bacteria concentration of 10⁷-10⁸ CFU/ml.The concentration of bacteria in the human colon is 10¹¹-11¹² CFU/ml.

Role of Absorption Enhancer

Absorption enhancers used in colonic drug delivery are

⁵Pharmaceutical approaches to colon targeted drug drug delavery

Evaluation of Colon –Targeted Drug Delivery System

Different in vitro and in vivo methods are used to evaluate the colonic drug delivery systems.

of colon-specific drug delivery system including rat (Van den Mooter et al., 1995; Leopold and Friend, 1995) and the pig (Harboe et al., 1989).

A-In Vitro Methods

The ability of the coats/carriers to remain for intact in the physiological environment of the stomach and small intestine is generally assessed by conducting drug release studies in 0.1N HCL for 2 hrs. (Mean gastric emptying time) and in pH 7.4 Sorensen’s phosphate buffer for 3 hrs.(mean small intestine transit time) using USP ¹dissolution rate test apparatus or flow through dissolution apparatus.

B- In Vivo Methods

Different animal models are used for evaluating in vivo performance of colon-specific drug delivery systems. Guinea pigs were used to evaluate colon-specific drug delivery from a glycoside prodrug of dexamethasone (Friend et al., 1991). Other animal models used for the in vivo evaluation of colon-specific drug delivery system including rat (Van den Mooter et al., 1995; Leopold and Friend, 1995) and the pig (Harboe et al., 1989).

In vivo Technique

The in vivo techniques are

String Technique

Endoscope Technique

Radio telemetry

Roentgenography

Gamma scintigraphy

Conclusions

A considerable amount of research work has been carried out on the development of colon-specific drug delivery system for the last two decades. The advantages of targeting drug specifically to the diseased colon are reduced incidence of systemic side effects, lower dose of drug, supply of the drug to the biophase only when it is required and maintenance of the drug in its intact from as close as possible to target site.

References

1. The United State Pharmacopeia, Asian ed.; (2003).
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4. Jain, N.K.; Advances in controlled & Novel Drug Delivery, CBS Publishers, 89 (2005).
5. Chourasia, M.K. and Jain, S.K., Pharmaceutical approaches to colon targeted drug delivery system; Dr.Hari Singh Gour University, Sagar.
6. Colon-specific drug delivery system. J. Pharma. Sci, 62: 1-8 (2000).
7. Colonic transit of different sized tablets in healthy subjects. Control Rel., 26:

Introduction

The primary goal of research based pharmaceutical organizations around the globe is to discover, develop and procure regulatory approvals for marketing new drugs for the treatment or prevention of diseases in humans and animals. A clinical trial is a research study in human volunteers to determine safety and efficacy of a treatment i.e. an investigational new drug, method of diagnosis and screening¹. Chemicals and biologics that do show promise in preclinical studies face extensive, strict and thorough clinical trials in healthy volunteers and in high-risk population. Preclinical research allows evaluation of the pharmacokinetics, pharmacodynamics, dose-response profiles and toxicological potential of a drug. Preclinical studies also determine the optimal formulation and dose for phase I clinical trials, analyze physicochemical characteristics of the testing compound and provide the rationale for the proposed therapeutic indication. Approval to initiate human clinical trials is based on the results from preclinical pharmacological and animal toxicity studies. The design of the clinical trials is based on the chemical nature of the product being tested, results of preclinical animal studies and intended clinical uses. This is particularly relevant for biopharmaceuticals that frequently demonstrate complex and multiple pharmacological activities.^2-10

Types of Clinical Trials¹¹

Treatment trial for new combination of drugs and new approaches to therapy.

Prevention trial to search more effective ways to prevent disease in new patients and to prevent disease from returning.

Diagnostic trial to find better tests to diagnose a particular disease.

Screening trial to examine the best way to detect certain diseases.

Quality of life trials to explore way to improve comfort and the quality of life for patients with a chronic disease.

Good Clinical Practices (GCP)

Good clinical practice is a set of guidelines for biomedical studies that encompasses the design, conduct, termination, audit, analysis, reporting and documentation of the studies involving human subjects.

The guidelines Seek to Establish Following Cardinal principles^12-15

Protection of the rights of human subjects involving informed consent procedures and ethics committee consultation.

Standardization of activities with in the sponsor company achieved by the use of good management system and written standard operating procedures (SOPs).

Documentation and archiving to a standard that provides an intact paper trial which will confirm all events occurring during the study.

Monitoring and audit of investigation procedures and documentation by the sponsor.

Authenticity of biomedical data generated i.e. verification of trial documentation by regulatory authority audit, though these procedures are developed to a different extent in the various systems.

Sponsor and investigator¹⁶

The sponsor takes responsibility for and conducts the clinical trials and may be an individual or pharmaceutical manufacturer, research institute or private organization. Sponsors are responsible for selecting qualified investigators and monitors, informing investigators, reviewing and monitoring studies and maintaining trial record.

Investigators, such as physicians and other professionals qualified by training and experience, conduct or supervise the clinical study. They prepare a general outline of the planned studies that specifies the duration of the study, number of subjects that will be involved, and clinical observations and laboratory tests to be performed.

Monitor

The monitors are responsible for ensuring the quality and integrity of the data obtained from clinical trials, as well as the rights and safety of human subjects participating in the study. The monitor must visit the clinical site frequently to ensure that clinical trials are carried out according to the scheduled plan and that investigators have followed the specified regulations. The monitor also reviews the reports that are prepared by the investigator and submitted to the sponsor.

The objectives of the monitoring are to verify that^17,18

The rights and well being of human subjects are protected.

The reported trial data are accurate, complete and verifiable from source documents, hospital case records, laboratory reports, X-rays scan etc.

The conduct of the trial is in compliance with the protocol/amendment(s) approved by regulatory authorities and ethics committee with GCP and with the applicable regulatory requirement(s).

The monitor’s role is to be¹⁹

Messenger between the investigator and the sponsor.

Organizer of investigator meetings, key documents and trial logistic.

Negotiator of agreements and budget.

Inspector of case record forms, source data, ethics and consent documents.

Trainer of investigator and his team.

Observer trial conduct at the hospital site through monitoring.

Reporter of trial status protocol deviations, site issues.

Monitors, to function effectively, have to be thoroughly familiar with the investigational product(s), the protocol, written informed consent form and any other written information to be provided to subjects, the sponsor’s SOPs, GCP and the applicable regulatory requirement(s).

Protocol

A document that states the background, objectives, rationale, design, methodology and statistical considerations for study. It also states the condition under which the study shall be performed and managed. The content and format of the protocol should take into consideration the adopted SOPs, the regulatory requirements and the guiding principles of GCP. The prerequisites for the study include¹⁶

Investigational Pharmaceutical Product.

Preclinical supporting data.

Protocol.

Relevant components of protocol

General information.

Objectives and justification.

Study design.

Inclusion, exclusion and withdrawal of subjects.

Handling of the product(s).

Assessment of efficacy.

Assessment of safety.

Statistics.

Data handling and management.

Quality control and quality assurance.

Ethical and safety consideration.

Clinical Trials from Phase-I to Phase-IV

There are four stages of clinical trials and they are summarized as follow:

Phase-I Trial (Human/Clinical Pharmacology trial)^20,21

A Phase-I study represents the initial evaluation of an investigational new drug in people. The primary objective is to assess the safety of the new drug by testing a range of doses or dosing intervals in a sequential manner. The total number of subjects included in Phase-I studies is usually in the range of 20-80 and these studies require fewer than 12 months to complete. These studies are often carried out in healthy adult volunteers using clinical, physiological and biochemical observations. At least 2 subjects should be used on each dose. Investigators trained in clinical pharmacology and having the necessary facilities to closely observe and monitor the subjects usually carry out phase-I trials. These may be carried out at one or two centers.

Other Objectives in Phase-I Studies May also Include;

Determining the extent of drug metabolism.

Identifying interactions with other medications.

The mechanism of action.

Identifying preferred route of administration.

Gaining early evidence on drug effectiveness.

Conducting thorough dose ranging and dose response studies early in the product development reduce the possibility of later failed phase-II or III studies.

The Major Functions of Phase-I Trial Unit May be Grouped as Follows

Maintenance of an Ethics Committee

Secretarial organization e.g. checklists of members, documents sent and received, taking and typing minutes.

Recruitment of committee members ensuring a balance of medical and lay members and that they are independently minded.

Help with elections of chairman or other officers.

Contact with other departments e.g. legal department on questions of indemnity.

Contact  with  other  organizations  for guidelines.

Recruitment and Care of Volunteers

Pretrial tests for hepatitis antigens and in some cases HIV.

Records of participation in trials, including blood taken.

Records of honoraria paid.

Contact with general practitioners.

Contact with pathology laboratories re sample.

Pretrial (group) discussions for each trial.

Pre and post trial medical examinations.

Instructions during trial e.g. declaration of all concomitant treatments, avoidance of alcohol, strenuous exercise and driving a car.

Organization of Trials

Protocol development and circulation.

Drug supplies, labeling and dose allocation schedule.

Instructions to nurses and technicians recording data.

Dipstick pathology tests on blood and urine samples.

Storage of samples for assay, etc.

Maintenance of equipment and quality control.

Records of results, especially adverse reactions.

Practice of emergency procedures.

Organization, analysis and presentation of data.

Report writing.

Dissemination and interpretation of results

Ethics Review Board (ERB)

Clinical research in healthy subjects can result in benefits for society. Ethical aspects of research include reasoned analyses of moral obligations including²²:

The moral duty of justice requires the investigators to be fair in the treatment of research subjects. Thus, the treatment of participants in clinical studies should be compensated adequately for inconvenience, discomfort and loss of time when participating in studies.

Beneficence requires that the research activities benefit study subjects and other people.

Nonmalfeasance, not doing harm.

Respect for autonomy i.e. allowing the individual to determine what happens to him/ her.

It is an independent body constituted of medical/scientific professionals and non-medical/ non-scientific members whose responsibility is to ensure the protection of rights, safety and well being of human subjects involved in clinical trial.

An ideal ERB should include²³;

At least five members (quorum).

At least one member whose primary area of interest is in non-scientific area.

At least one member who is independent of the institution/trial site.

The main responsibility of ERB is²⁴;

To safeguard the dignity, rights, safety and well being of the potential research participants.

To ensure and verify that universal ethical values and international scientific standards are followed with a view on local community values and customs.

To help in the development and the education of a research community responsive to local health care requirements.

Phase-II Trial (Exploratory trials)

In Phase-II trials a limited number of patients are studied carefully to determine possible therapeutic uses, final formulation, dose and dosing regimens and further evaluation of safety and pharmacokinetics. Normally10-12 patients should be studied at each level. These studies are usually limited to 3-4 centers and carried out by clinicians specialized on the concerned therapeutic areas and having adequate facilities to perform the necessary investigations for efficacy and safety.²⁵

Element of Phase-II protocol include front page, introduction, objectives, ethical aspects including payments to patients, drug supply, labeling and randomization code, patient selection, methods, statistical analysis, dropouts, protocol amendments, adverse events, timetable, resources and personnel, financial indemnity, publication of results and record form.²⁶

Phase-III (Confirmatory Trials)

The purpose of these trials is to obtain sufficient evidence about efficacy and safety of the drug in a larger number of patients, generally in comparison with a standard drug and/or a placebo as appropriate. Clinicians in the concerned therapeutic areas, having facilities appropriate to the protocol, may carry out these trials. If the drug is already approved/marketed in other countries, Phase-III data should generally be obtained on at least 100 patients distributed over 3-4 centers primarily to confirm the efficacy and safety of the drug, in Indian patients when used as recommended in the product monograph for the claims made.^27,28 Phase-IV (Studies performed after marketing of the pharmaceutical product)

Trials in Phase-IV are carried out on the basis of the product characteristics on which the marketing authorization was granted and are normally in the form of post marketing surveillance, assessment of therapeutic value, treatment strategies used and safety profile. The major objectives of phase IV studies are to obtain additional data of the drug’s safety and effectiveness and determine new uses for or abuse of the drug²⁹. The FDA requires that the manufacturer maintain and establish post marketing records and reports. This is necessary for two reasons³⁰;

An investigational drug is tested in a relatively small number of patients compared with the number of patients who may use the drug after it is marketed.

Long term adverse effects may not be discoverable before approval.

As a result of post marketing information, the FDA may withdraw its approval of an NDA.

Under this provision, the manufacturer must submit to the FDA reports of any serious adverse drug reactions and any new information relating to the drug’s safety and efficacy, including information about;

Current clinical studies.

The quantity of drug distributed.

Labeling and advertising.

Informed Consent Process

Informed consent involves the process whereby explicit information is provided to participants regarding the treatment in clinical trials. It also describes the obligation of the investigator to inform the subject about the personal benefits and risks the individual faces in the study as well as the significance of the research for the advancement of medical knowledge and social welfare.

The informed consent is designed so patients participating in a clinical trial can obtain an adequate understanding of the objective and procedures of the clinical trial. Patients participate in such trials voluntarily. The information given to the subjects must be written in lay terms and include the following key components³¹:

A statement explaining the objective of the study, duration of the subject’s participation and procedures of clinical trial.

Description of any possible risk, adverse reactions, discomforts and benefits to the subject.

A statement that the confidentiality of the subject’s records will be maintained.

An explanation of whether compensation and medical treatment are available if injury occurs during the clinical trial.

A statement that participation is voluntary and refusal to participate will not involve penalty or loss of benefits to the subject.

A list of persons to contact for questions related to the clinical trial and trial related injury.

The law requires the investigator to secure the informed consent of the patient or a representative for the administration of an experimental drug. The patient consent must be in written in phase 1 and 2. In phase 3 consent may be oral if the physician decides it is necessary or it is preferable to written consent and this decision is recorded in the patient’s medical record. Patient consent may not be necessary when it is not feasible to obtain the consent of the patient or a representative or when in the professional judgment of the physician, informed consent is not in the best interest of the patient.³²

Clinical data management

Clinical Data Management (CDM) is the entire process involved with taking original raw data from the clinical sites with compiling and validating it, so that it is suitable for reporting purpose. In CDM process, the clinical trial data are collected and organized by using a computerized database that completely and accurately reflects the findings and events of the trial. CDM is key component of the multi-disciplinary team involved in setting up, running and reporting clinical trials. A data manager defines how these data are collected, tracks the data and checks their completeness, accuracy and consistency. Data management is involved in all aspects of processing clinical trial data, working with a range of computer applications and database system to support collection, cleaning and management of subject or patient data. CDM preferably includes³³:

Input into the design of protocol, which defines what data are to be collected and at what times.

Design and approval of case report forms, on which subject’s data are collected.

Database design for the study, ensuring it meets requirements for data entry and reporting.

The CDM process starts with design of protocol and it continues till the submission of clinical study report to regulatory authority for marketing approval. The CDM process is complicated, but if all team members perform their role specifically with team spirit then it runs smoothly. The team members involved in CDM process are database designers, data entry operators, data loaders, data managers and statistical programmers and medical writers. Each member involved in CDM process has a unique role and responsibilities.

Softwares used for clinical trial management, patient recruitment process, financial aspects of trials etc. and there are some specialized clinical data management softwares. Some of the softwares which are used for managing clinical research activities are listed below³⁴:

Clintrial

Clinpharma

Teleform

SyMetric

Adventrial

Study Manager

SAS Software

Tanangra-statistical analysis

These software help to achieve better relationships and interactions with regulators, investigators, clinicians and consumers through pre-emptive handling of AE problems based on earlier detection of signals and trends. Costs are reduced while data quality and analytical capability improve. Time to submission and to market is accelerated by controlled and validated reconciliation of data, while the risk of regulatory scrutiny is reduced.

Schedule Y

Schedule Y provides exhaustive information as well as lays down regulatory requirements for conduct of clinical trials. It also consists of information on animal pharmacology, details on types of fixed dose combinations (FDCs) of drugs, conduct of animal toxicity studies, stability testing of drugs (accelerated and real time), contents of proposed clinical trial protocol and report, data elements for reporting serious adverse drug events occurred during the trial, checklist for informed consent document and different phases of clinical trials.^4,35

Clinical Research Organisation (CRO)

An organization to which the sponsor may transfer or delegate some or all of the tasks, duties and / or obligations regarding a clinical study. All such contractual transfers of obligations should be defined in writing. A CRO is a scientific body-commercial, academic or other. Following are some Clinical Research Companies in India^36,37;

Accutest Research Laboratories (I) Pvt. Ltd., Ace Biomed Pvt. Ltd., Actimus Biosciences Pvt. Ltd., Apothecaries Ltd., Asian Clinical Trials, Aurigene Discovery Technologies, Avra Laboratories, Bioserve Biotechnologies (I) Pvt. Ltd., Chembiotek Research International, Clinigene International, Clininvent Research Pvt. Ltd., Clin Tec India International Pvt. Ltd., Clintrac International Pvt. Ltd., D & O CRO, Dr. Reddy’s Laboratories Limited, Eli Lilly and Company (India) Pvt. Ltd., Glaxo Smith Kline Pharmaceutical Limited, Gokula Metro Polis Clinical Laboratories Pvt. Ltd., GVK Biosciences Pvt. Ltd., iGATE Clinical Research International Private Limited, Intas Pharmaceuticals, International Tech Park Ltd., INTOX Private, Jubilant Clinsys, Johnson & Johnson Ltd., Kendle India, Lambda Therapeutic Research Pvt. Ltd., Lotus Labs Pvt. Ltd., Lupin Limited, Magene Life Sciences, Manipal Acunova, Matrix Laboratories Limited, Metropolis Clinical Laboratories, Novo Nordisk India Private Ltd., Omnicare Clinical Research, Pharma-Olam International, Pharmanet, PPD Pharmaceutical Development India, Quintiles Technologies (India) Pvt. Ltd., Ranbaxy Laboratories Ltd., Reliance Clinical Research Services Pvt. Ltd., Reametrix India, Roche Scientific Company (I) Pvt. Ltd., Sanofi-Aventis (Aventis Pharma Limited), Sipra Labs Pvt. Ltd., SIRO Clinpharm Pvt. Ltd., SRL Ranbaxy Limited, Sterling Synergy Systems, Suven Life Sciences Limited, Synchron Research Pvt. Ltd., Triesta Sciences, Torrent Pharmaceutical Limited, Veeda Clinical Research, Vimta Labs Limited, Zydus Cadila.

Acknowledgements

The authors are thankful to Mr. J.P. Singh, librarian, K.I.E.T. School of Pharmacy, for his help to collect the required literature.

References

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14. Lee, C.J., Managing Biotechnology In Drug Development, CRC Press, Boca Raton, FL (1996).
15. Soul-Lawton, J. and Kroon, R., Good Clinical Practice, In: A Guide To Clinical Drug Research, 2^nd Edition, Cohen, A. and Posner, J. (Eds.), Kluwer Academics, Boston, MA, 145 (2000).
16. Gupta, A. and Godinho, M.H.S., Pharma Times, 37(7), 20 (2005).
17. Gilhotra, N., Pharma Review, 41 (October 2006).
18. Bhatt, A., Pharma Times, 37(7), 18 (2005).
19. Bhatt, A., Pharma Bioworld, 56 (March-Apr 2005).
20. Fidler, K. and Koch, I., Handbbok Of Clinical Drug Research, Glenny, H. and Nelmes, P. (Ed.), Blackwell Scientific, Oxford (1986).
21. Spiker, B., Guide To Clinical Studies and Developing Protocols, Raven Press, New York (1984).
22. Gupta, A. and Godinho, M.H.S., Pharma Times, 37(7), 17 (2005).
23. Royle, J.M. and Snell, E.S., J. Clin. Pharmacol., 21, 548 (1986).
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25. Armitage, P., Sequential Clinical Trials, 1^st Edition, Blackwell Scientific, Oxford (1975).
26. Pocock, S.J., Clinical Trials: A Practical Approach, 1^st Edition, John Wiley, Chichester, 125 (1983).
27. Pocock, S.J., Clinical Trials: A Practical Approach, 1^st Edition, John Wiley, Chichester, 148 (1983).
28. Spriet, A. and Simon, P., Methodology of Clinical Drug Trials, Translated by Edelstein, and Weintraub, M., Kerger, Basle, 127 (1985).
29. Rawson, N.S.B., Post Marketing Surveillance, In: Comprehensive Medicinal Chemistry, Vol-I, 1^st Indian Edition, Kennewell, P.D. (Ed.), Pergamon Press, Oxford, 625 (2005).
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37. Clinical Research Companies in India, Pharma Buzz, 1(6): 58 (2006).

Introduction

Ticks are obligate hematophagous ectoparasites of terrestrial vertebrate hosts, including cattle, goats and wild animals. They utilize blood for growth, general metabolism and reproduction. Vitellogenesis and oviposition in females, for instance, cannot occur unless females have taken a blood meal (Akov, 1982). Ticks take several milliliters of blood depending on whether they are “hard” or “soft” tick, and on their developmental stage. Blood digestion is slow and intracellular (Arthur, 1965; Balashov, 1972; Araman, 1979; Akov, 1982). Adult female ixodids may ingest ten times their body weight of blood (Alan Young, Pers. com.).

Theileriosis is the name given to infections caused by several species of Theileria (Theiler, 1911). Theileria parasites are of great economic importance in many parts of the world where they cause disease to domestic animals (Purnell, 1977) -the two most important in Africa being Theileria parva and Theileria annulata. These diseases have been given several names in different countries (Norval et al., 1992). Theileria parva infections are referred to as East Coast Fever, Corridor disease or January disease, while T. annulata infections are called tropical theileriosis. Theileria parva is transmitted mainly by the brown ear tick Rhipicephalus appendiculatus Neumann, 1901 (Norval et al., 1992). During feeding, ticks inject T. parva sporozoites into the blood of cattle, resulting in progressive lymphoproliferative disease initiated by the transformation of parasitized mononuclear cells in the lymph nodes. Once inside the lymphocytes, the sporozoites develop into multinucleate macroschizonts.

Despite several in vitro tick studies (Stiller & Coan, 1995; Kuhnert, 1996; Kuhnert et al., 1998; Neese et al., 2000; Rechav et al., 2000) and transmission experiments (Voigt, 1993; Humphrey-Smith, 1993; Waladde et al., 1995; Inokuma & Kemp, 1998; Kimbita & Silayo, 1997; Musyoki et al. 2004), little is known about the role of the different blood fractions ingested by the tick on the development of the parasite Theileria parva. In this study, the role of the blood fractions in the development of the parasite T. parva, and the subsequent invasion of bovine host lymphocytes was investigated. Through this study a technique that can be used to investigate infectivity of sporozoites in transmission experiments in vitro was also developed.

Materials and Methods

Ticks and T. parva parasites: Theileria parva (Muguga stock) and the tick R. appendiculatus maintained in the laboratory at the International Livestock Research Institute (ILRI) in Nairobi, Kenya, were used in these studies.

Tick attachment, preparation of ear wash, and ear-wash composition

Two ear wash extracts (methanol and diethyl ether) were compared in their composition and enhancement of tick attachment. Rhipicephalus appendiculatus adults which normally attach to the ears to feed, hence the name brown ear tick, were left to feed for 4 days. The ears were washed using two solvents- methanol and diethyl separately, in order to extract tick attractants. A large volume of the diethyl ether (1 litre) was used to wash the two cattle ears and the mixture was concentrated by evaporating off the ether in a rotor evaporator. Several precautions were taken, since ether is volatile and inflammable. The evaporator (Rotarvapor RE) was particularly selected for this purpose and a warm water bath was used to heat the ether. The apparatus had a Liebig condenser for condensation of the ether. The excess vapor was cooled down in an “ice cold trap” dry ice. The system was put in a fume hood that created a vacuum, by blowing air from the room out through a vent. The concentrate was divided into aliquot parts and kept at -20^oC until needed. The methanol concentration was carried out in a 55^oC water bath.

An attempt was made to analyze the possible components of the ear wash extract using thin liquid chromatography (TLC) with a solvent system of the ratio 1 ethyl acetate: 4 hexane. Under ultraviolet light, three bands were observed, one at methanol and at each diethyl ether (color pink). The diethyl ether had several compounds extracted compared to methanol. These were later found to be phenols, salicylaldehyde, 4-cresol, 2,6-dichlorophenol, 2,6-dibromophenol and 2-bromo-6-chlorophenol, responsible for the attraction of ticks. The extract was utilized as attractants in the in vitro feeding chambers. The extracts from the two solvents were compared for tick attachment. Diethyl ether proved to be the most effective solvent in that more than 90% nymphal attachment was observed compared to methanol of 40-50%.

Semiochemicals

The membrane was made attractive for tick attachment by providing a combination of sui chemicals (semiochemicals of hosts and tick fecal material), a temperature of 37^oC and relative humidity (Rh) of 80-85% were provided as physical stimuli. The external surface of the membrane was therefore treated with cattle/tick ear wash and then sprinkled with tick fecal material collected from the tick incubation tubes similar to Walladde et al. (1996).

The in vitro Tick Feeding System

The in vitro tick feeding system, similar to that described by Walladde et al., (1993, 1995, 1996), with minor modifications (Fig. -1), was used to study the role of the blood meal fractions on infectivity of blood lymphocytes by T. parva and on the maturation of the sporozoites.

Detoxification of the Membranes

The toxic substances in the chambers (mainly the glue dissolved in chloroform) were removed by rinsing each of them in a water bath at least 3 times at 80^oC for at least 8 hr at a time, after which the chambers were then dried under a hood for 12 hr. The interiors of the chambers were sterilized with a 1:1:1 mixture of gentamycin, penicillin/streptomycin and fungizone. The external aperture was covered with zinc oxide plaster, which kept it sealed except when blood was introduced or changed.

Tick Application

Following the chamber preparation, the ear wash concentrate was carefully daubed onto the membrane and a cluster of 500-1000 nymphs was applied onto the treated membrane surface. Adult ticks were first sexed in an electronic tick trough heated at the sides to prevent them from escaping before they were applied to the membrane at a 1:1 sex ratio. To avoid overcrowding and piercing the membrane, which could lead to chamber leakage, only 60-100 adults were used.

Maintenance of Chambers

During the experimental period, blood was changed twice daily at 08:00 and 17:00 hrs, under sterile conditions obtained by wiping the hood with 70% ethanol. The chambers were rinsed with normal saline (9.0 g/l NaCl) before fresh blood was put into them. Any chambers with very dark red or black blood suspected of contamination were rinsed with a mixture of penicillin/streptomycin, gentamycine and fungizone. The blood was introduced into the chambers by opening the zinc oxide plaster on top of the blood chamber (Fig. – 1) under a sterile hood, previously swabbed with 70% ethanol. The aseptically – collected heparinized cattle blood (10-12ml) was pipeted into the chamber using a pipette aid (Drummon, UK) fitted with a 10 ml disposable plastic pipette (Costar, Cambridge, Massachusetts, USA) and the aperture was then immediately sealed with the plaster. The pipette tip was placed at the side of the chamber when dispensing blood to avoid piercing through the membrane. The whole feeding system was placed in an incubator at 37^oC with a 5% CO₂ flow. The incubator had a Rh of 80-85%, obtained by placing an open tray containing saturated potassium chloride solution inside (Alan Young, Per. com).

Separation of Blood Components for in vitro System

Blood samples from cattle (6 – month – old Borans) were collected in heparin (1:5000 units) by jugular venipuncture and divided into samples of whole blood, plasma and red blood cells. The plasma was obtained by centrifugation of whole blood (1000 x g; 4oC) in a heraeus sepatech megafuse. After removing the clear plasma, the cells were cleared of the buffy coat layers by sucking with Pasteur pipettes. Some of the blood was left uncentrifuged. The cells, whole blood, as well as the plasma, were all warmed to 37^oC in a water bath for 15 min before they were transferred into the feeding chambers. Three replicas comprising 2 chambers each were set up for 3 different tick batches. The chambers were maintained at 37^oC, 5% carbondioxide and 80-85% Rh. The relative humidity was obtained by keeping a tray of saturated potassium chloride in the incubator throughout the experiment. The blood samples were changed twice daily and the chambers were rinsed with normal saline between the changes.

In vivo Feeding of Ticks

For comparison with the in vitro system, ticks were also fed in vivo on rabbits. In short, about 100 males and females in ear bags were fed on rabbit ears for 4 days. The rabbits were kept in cages and a leather collar was put around their necks to prevent them from detaching the ear bags. To ensure homogeneity in the samples, the ticks put on the rabbits were from the same batches as those applied in vitro.

Selection of Tick Batches

Ticks were infected as nymphs by letting them feed on artificially-infected cattle when the erythrocyte piroplasm counts were high (at least >10%). Engorged nymphs were maintained at 23-25⁰ C and 80 % Rh and allowed to molt to adult stage. The adult ticks used in determining the sporozoite infectivity of the peripheral blood lymphocytes (PBLs), were first screened for their infection rates (percentage of infected ticks per number examined) by Fuelgen staining of whole salivary glands after the ticks had fed on rabbits for 3 days. This was necessary in order to standardize the number of infected acini used in the interaction of the sporozoites with the PBLs. Tick batches that showed at least 80% infection rates were used. The abundance (the mean number of infected acini per tick) was calculated from the infection rates. Since the required number of infected acini was 1000 and the abundance was 94.6, the number of ticks that were to be dissected was obtained by dividing the required number of infected acini by the abundance (1000/94.6). Thus, only 11 ticks were required for dissection.

Synchronization of in vitro and in vivo feeding systems

In order to compare sporozoite maturation and their ability to infect lymphocytes, the two feeding systems, the in vitro and the in vivo, were synchronized so that fresh sporozoites were obtained on the same days. This was carried out by applying at least 60-100 adults (both sexes) in the feeding chambers at least 24-48 hr prior to the application of ticks on the rabbit ears (Table 1). This time period allowed the in vitro-fed ticks to attach, since the ticks put on the rabbits attached faster. In the feeding chambers, plasma, whole blood or erythrocytes were separately added and the ticks were allowed to feed for 4 days.

Tick Dissections for Sporozoites

Ticks were removed with a pair of forceps, washed in detergent for a few minutes, followed by a rinse in distilled water. They were then sterilized by rinsing in 70% ethanol and finally in RPMI 1640 cell culture medium and N-(2-Hydroxyethyl) piperazine N’-2-ethane-sulfonic acid (HEPES) medium. The salivary glands were dissected out under sterile conditions in a Laminar Flow Hood as described in Sebitosi et al., (1998).

Handling of T. parva Sporozoites for in vitro Infection

Viable sporozoites (Muguga stock) were prepared from the salivary glands of R. appendiculatus adults fed for 4 days on different blood fractions or on rabbits, as described above. They were dissected in a special medium consisting of RPMI 1640 + HEPES , L-glutamine, fetal calf serum (FCS)(5%), gentamycin and penstrep (antibiotics) 5 ml to keep the parasites alive. The sporozoite containing salivary glands from the three groups were separately homogenized in the above medium in a 0.1ml homogenizer and the volume was made up to 0.1ml. All these processes were carried out under a hood. The homogenate was centrifuged at 150 x g for 5 min to remove salivary gland debris, and the supernatant was used without further purification in the infection of lymphocytes.

Preparation of PBLs for in vitro Infection

Cattle blood was obtained from uninfected 6-9 months old Boran cattle (Bos indicus). It was drawn from the jugular vein with a 16-gauge needle into a syringe containing an equal volume of Alsevers solution and heparin. The cattle were kept on a farm free from East Coast fever, and brought to the ILRI farm at the age of 1-2 weeks, where they were maintained. They were screened for antibodies to T. parva using the indirect immunofluorescent antibody test (IFAT), immunoblotting and indirect enzyme linked immuno sorbent assay (ELISA) (Goddeeris et. al., 1982; Katende et. al., 1990) before the experiment started.

Processing of PBLs

The preparation of PBLs was carried out according to the technique of Goddeeris et al., (1982) and Katende et al. (1995), with minor modifications, in a horizontal laminar – airflow hood. Using a pipette, an aliquot of 30ml of blood was carefully layered on 20ml Ficoll Hypaque gradients contained in a 50ml polypropylene conical tube and centrifuged at 900 x g for 30 min in a Beckman centrifuge without brakes at room temperature (24^oC). The centrifugation, without brakes, ensured that the cells settled properly.

Table 1: Synchronization of the in vivo and in vitro feeding systems for Rhipicephalus appendiculatus

Figure 1: In vitro feeding system for the hard tick Rhipicephalus appendiculatus Click here to View Figure

Figure 2: Light microscope photograph of uninfected bovine lymphocytes obtained from in vitro infection of lymphocytes with sporozoites derived from R. Click here to View Figure

Figure 3: Theileria parva schizonts obtained from in vitro infection of lymphocytes with sporozoites derived from R. Click here to View Figure

The PBLs were aspirated from the interface using a pipette and mixed with an equal volume of Alsevers solution in a 50ml polypropylene conical tube. The cells were pelleted by centrifugation at 450 x g at room temperature for 10 min. The PBLs were then washed three times in 50 ml Alsevers solution for resuspension and centrifugation at 180 x g for 10 min, to remove the platelets. The final wash was carried out in an L-15 culture medium. Finally, the pellet was resuspended in the L-15 medium, the composition of which was as follows: L-15 medium, FCS (20%), tryptophosphate broth (10%), 20mm HEPES, L-glutamine (200mM), penistrep and gentamycin (50mg/ml).

Standardization of Lymphocytes

Twenty microliters (20µl) of the cell suspension was added to 180µl trypan blue mixed on a vortex mixer (Type 167000), and the cells counted in a hemocytometer.

In vitro infection of lymphocytes with T. parva sporozoites

The infection of lymphocytes with T. parva sporozoites was carried out in a 96-well cell culture plate (Costar). A 100 µl sporozoite suspension at a concentration of 100 acini/well, for maximum infection, was mixed with a similar amount of PBLs and incubated at 37^oC in a 5% CO₂/air mixture for 1 hr to interact freely. The plates were then removed from the incubator and 200µl of the mixture was transferred into a 24-well cell culture plate (Costar) containing 500 µl feeder cells in RPMI + 10 % FCS. The plates were set up in duplicates and incubated at 37^oC in a 5% CO₂/air mixture.

The sporozoites and the lymphocytes were left to interact for 10 days with constant microscopic examination to check for schizonts. From the start of the neutralization assay (day 0) up to day 4, the mixture was left alone without disturbance. On day 5, 1 ml of the medium was removed and replaced with fresh medium (L-15 plus 20% FCS) containing 1 % mycostatin and 0.1 % gentamycin. The cells were left undisturbed for day 6 and day 7, and on day 8 cytospins were made.

Checking for Sporozoite Infectivity in Lymphocyte

Cytospins were necessary to produce flat cells on a glass slide for easy counting. Using a 1 ml pipette, the contents of each well were mixed and aliquots from 2 wells were transferred into a glass tube (Röhren Tubes, Sarstedi, Germany) and centrifuged at 150 x g for 5 min at 0^oC to settle the cells without pelleting them. Fifty microliters of the supernatant was resuspended in a similar amount of PBS and vortexed (Super-mixer). In the cytospin, a volume of 50µl of the cell suspension was centrifuged at 70 x 10 rpm using low acceleration for 6 min. This was done on glass slides, which were lined with filter- paper cards. The slides were removed from the cytospin, air – dried for 2 min, fixed in methanol (5 min) on a rack and then Giemsa-stained (30 min). They were then washed in distilled water and examined under the microscope for the presence of intralymphocytic schizonts.

Schizont scores – All the slides were viewed carefully for schizonts. Both the number of cells in the field and the infected ones were counted and the percentage of infected cells (schizonts) was calculated out of a total of 400 cells.

Results

The use of aggregation pheromones and semiochemicals made from ear wash extraction and tick fecal material enhanced tick attachment to the membranes. Freshly prepared ear wash gave the best attachment results. Compared to methanol, diethyl ether proved to be the most effective solvent in that more than 90% nymphal attachment was observed compared to methanol of 40-50% tick attachement.

About 70-80% of ticks feeding on whole blood attached within 24-48 hr. In the erythrocyte or cell – feeding chambers, there was about 90% attachment of ticks within 24 hr. The ticks fed well and took on a characteristic reddish color, which distinguished them from all the other adults. The cells produced the best attachment, seen as “cones” on the membrane in the first 24-48 hrs. The in vivo (rabbit) feeding groups also had very good attachment (about 90-100 %) and fed well. The plasma however produced fewer attachments within the first 24-48 hrs.

The infection rates of T. parva sporozoites in the ticks used to infect the cattle were at least 80%. The abundance was 94.6 and intensity 118.25. The total acini examined were 2,838 and total acini used were 1000 extracted from a mean of 11 ticks per replicate. The total ticks per treatment were equal to the total acini required divided by abundance (a value of 10.57) or 11 ticks.

The lymphocytes (PBLs) that were not infected by the T. parva sporozoites were oval in shape (Figure 2). Those that were parasitized had an expanded nucleus and cytoplasm, and the schizonts were visible as pink dots in a blue cytoplasm (Figure 3). The sporozoites from the batch were viable, as predicted. The sporozoite infectivity was highest in the ticks fed on rabbits (67.9%), followed by whole cattle blood given in vitro 20%, erythrocytes (1%), and almost no infectivity was perceived in the plasma-fed ticks (Table 2).

Challenges of in vitro Feeding

The in vitro feeding chambers containing plasma had frequent leakages and self-sealing occurred. They also showed slow and poor tick attachment and over 20% adult mortality. The Baudruche membrane appeared bleached. The feeding chambers were also problematic in that there was frequent fungal infection and leakages. The in vitro system can be optimized to use blood from slaughterhouses thereby reducing the use of laboratory animals.

Discussion

The entry of sporozoites into the bovine lymphocytes during tick feeding is an important stage in the transmission of theileriosis. Once the tick feeds, it takes in a lot of piroplasms (about 1 million; Allan Young personal communication) whose development depends upon several midgut factors that the parasite encounters during its development-such as lectins and enzymes among others. Since the midgut factors are blood-meal-induced, the resultant infections would depend upon the type of host blood meal taken at the time of infection. Using the in vitro system and tissue culture techniques to study the various fractions of the blood meal and subsequent infectivity, some insight into the blood meal components influencing parasite development was gained.

The use of odour baits, aggregation attachment pheromones and /or semiochemicals in insects and ticks seems to be a natural phenomenon. The combination of host hair, tick fecal material, carbon dioxide and ear washings extract used in this study, enhanced the nymph attachment and is in agreement with other reports (Voigt et al., 1993; Waladde et al., 1995; 1996; Kuhnet, 1996).

Table – 2: Influence of blood meal fractions on the sporozoite infectivity of T. parva in peripheral blood bovine lymphocytes (PBLs)

In some species of Amblyomma, the males produce the aggregation attachment pheromone (Rechav et al., 2000). Odour and tactile stimuli therefore seem to play an important role in tick attachment to artificial feeding chambers, as in the natural habitants. Whole blood from cattle supplied in vitro resulted in the highest level of sporozoite infectivity, followed by the erythrocytes, whereas the plasma resulted in almost no parasite infectivity.

Furthermore, ticks that were fed plasma did not attach effectively and detached frequently. This may have been caused by the lack of a vital factor for attachment in the plasma. There was, however, a good attachment in those ticks fed on a whole-blood meal. The erythrocyte-fed ticks, on the other hand, displayed a high number of attachments, implying that it is perhaps the erythrocytes that attract the ticks to attachments. However, the erythrocytes alone, despite attracting large numbers of ticks to attach, were unable to support a high number of mature, viable sporozoites ( 1% compared to 20% in the case of a whole blood meal) to infect the lymphocytes and eventually transform into schizonts.

The presence of erythrocytes therefore seems to be necessary in T. parva maturation. The mechanism that limited the sporozoite entry into the lymphocytes, especially in the case of plasma – fed ticks (as evidenced by the lack of schizont parasitosis) therefore needs further study. It is likely that vital ingredients in the plasma that are important in the sporozoite maturation process and establishment were lacking. This could have prevented the sporozoite in the vector from maturing to a crucial stage necessary for penetrating the lymphocytes, or the receptor-ligand interaction between the parasite and the lymphocytes might have been inhibited.

In this study, the in vivo system has been demonstrated to far supersede the in vitro system in sporozoite infectivity, as is evident in the high parasitosis among the rabbit-fed ticks compared to the whole-blood-fed ticks in vitro. This is the first time that sporozoites have been obtained from ticks in vitro and used to infect lymphocytes in vitro. Unlike the in vitro systems developed by others (Stiller and Coan 1995; Kuhnert, 1996; Kuhnert et al., 1998; Musyoki et al., 2004), this particular system was made from baudruche membranes with pheromones from tick feaces and ear wash. It is an important new avenue opened for the study of pathogen transmission, and is significant from the standpoint of evaluating potential immunogens in controlling the disease by vaccination against the parasite. It is concluded that the cells together with the plasma constitute the favorable environment in which sporozoites mature and are able to infect. If sporozoites do not mature to a certain critical stage, then they lack the necessary receptors to bind and penetrate into the lymphocytes.

Acknowledgements

We wish to acknowledge the assistance of the members of the tick Unit at the International Livestock Research Institute (ILRI), Nairobi, Kenya and those of Lab 5 in the preparation of the Theileria parva infections in cattle and ticks. Special thanks go to Dr. Antony Musoke, Prof. R.I.S.Agbede, Mr. Steven Mwaura and Mr. John Tangus. For the cell culture techniques, we acknowledge the assistance of Dr. Paul Spooner, Dr. Subash Morzaria, Mr. James Gachanja and Juma.

References

1. Akov, S. Blood digestion in ticks. Physiology of ticks (ed. by F.D. Obenchain and R. Galun), 197-211. Pergamon Press, Oxford (1982).
2. Araman, S.E. Protein digestion and synthesis in ixodid females. Recent advances in acarology (ed.by J.G. Rodriguez). 1. 385-395. Academic Press, New York (1979).
3. Arthur, D.R. Feeding in ectoparasitic Acari with special reference to ticks.Advances in Parasitology 3, 249-298 (1965).
4. Balashov, Y.S. Blood sucking ticks (Ixodidae).Vectors of disease of man and animals. Miscellaneous Publications of Entomological Society of America, 8, 161-362 (1972).
5. Goddeeris, B.M., Katende, J., Irvin, M A.D. & Chumo, R.S.C. Indirect fluorescent antibody test for experimental and epizootiological studies on East coast fever (Theileria parva) infection in cattle. Evaluation of a cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science, 33, 360-365 (1982).
6. Humphrey-Smith, I., G. Donker., A. Turzo., C.Chastel & Schmidt- Mayerova, H. Evaluation of mechanical transmission of HIV by the African soft tick, Ornithodoros moubata. AIDS, 7, 341-347 (1993).
7. Inokuma, H. & Kemp, D.H. Establishment of Boophilus microplus infected with Babesia bigemina by using in vitro feeding technique. Journal of Veterinary Science, 60, 509-512 (1998).
8. Katende, J.M., Goddeeris, B.M., Morzaria, S.P., Nkonge, C.G. & Musoke, A.J. Identification of a Theileria mutans-specific antigen for use in an antibody and antigendetection ELISA. Parasite Immunology, 12, 419-433 (1990).
9. Kimbita, E.N & Silayo, R.S. Use of an in vitro infectivity assay in comparison with histological techniques in the study of Theileria parva sporozoites maturation. Veterinary Parasitology, 70, 83-97 (1997).
10. Kuhnert, F. Feeding of hard ticks in vitro: New perspectives for rearing and for the identification of systemis Acaricides. Altex, 13, 76-87 (1996).
11. Kuhnert, F., Issmer, A.E. & Grunewald, J. Partly automated in vitro feeding of adult Amblyomma hebraeum. Alte, 15, 67-72 (1998).
12. Musyoki, J.M., Kiara, H.K. & Kokwaro, E.D. Comparative studies on the infectivity of Theileria parva in ticks fed in vitro and those fed on cattle. Experimental and Applied Acarology, 32, 51-67 (2004).
13. Neese, P.A., Soneshine, D., Kallapur, V.L., Apperson, C.S & Roe, R.M. Absence of insect juvenile hormone in the American dog tick, Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae). Journal of Insect Physiology, 46, 477-490 (2000).
14. Norval, R.A.I., Perry, B.D. & Young, A.S. The epidemiology of Theileriosis in Africa. 481, Academic Press (1992).
15. Purnell, R.E. East coast fever: some recent research in East Africa. Advances in Parasitology, 15, 83-132 (1977).
16. Rechav, Y., Drey, C., Fielden, L.J. & Goldberg, M. Production of pheromones by artificially fed males of the tick Amblyomma maculate (Acari:Ixodidae). Journal of Medical Entomology, 37, 761-765 (2000).
17. Sebitosi, E.N.K., Kaaya, G.P., Young, A.S. & Agbede, R.I.S Lectins in the brownear tick Rhipicephalus appendiculatus. International Journal of Acarology, 24, 159-163 (1998).
18. Stiller D. & Coan, M.E. Recent developments in elucidating tick vector relationships for anaplasmosis and equi (1995).
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Introduction

The study of the physiological adaptations of an organism to various environmental conditions, one of which is the hemostatic system, represents a current scientific problem. Thus, the main goal of this work was to assess the effect of stress reactions caused by hypoxia on carp coagulogram indices.

In view of the fragmentation of the sources, the differences in methodology and facilities,^1,2 as well as the lack of standardization of the study of hemostasis in various fish species under various stressful environments, this problem remains unclear.

The internal, external, and general pathways of the blood coagulation system of bony fish were first demonstrated in the works of other researchers ^3,4,5. Studies conducted on bony fish indicate that the coagulation process is fundamentally similar to that of other vertebrates, in particular to mammals ^1,6,7. Recently, Russian researchers obtained data on the functionality of primary and secondary hemostasis in various fish species with low activity of plasma factors, such as fibrinogen level, TP, and PP compared with mammals.⁸ A comparative analysis of these data revealed that there are both similarities and differences in the coagulogram indices in different classes of fish.⁹

Fish handling can result in a significant stress response, and it is safe to assume that interspecies differences in blood coagulation in fish may well be the result of different responses of these fish to stress.

Stress resulting from fish-farming operations is mainly associated with hypoxia, which is exacerbated by increased activity during the pursuit, attempts to break free of the net, etc. Stressful reactions are caused mainly by catecholamines and cortisol,^15,16,17 which act for two different but overlapping time periods.

Since the content of catecholamines has not been measured in most studies on fish stress, some authors identify them by indirect signs.^18,19,20 However, the adrenal response to stress in fish remains unclear. Smith claims that adrenaline secretion increases under conditions of acute hypoxia.¹⁸ Some studies show that catecholamines, primarily adrenaline, play a significant role in all types of reproduced stress.²¹

There is an extensive database of studies on the effect of various types of stress of commercial and wild fish. In most of these studies an increase of cortisol (and glucose) concentrations in plasma were experimentally recorded. ^{22,23,24,25,26,27,28,29,30,31,32} In other experiments, the levels of cortisol, glucose, and some other hematological parameters did not change significantly.³³ The conclusions of some studies emphasize the importance of cortisol in fish as a stress hormone,^34,35 as well as an indicator of various disturbances in the aquarium environment.³⁶ Other researchers also noted that the level of cortisol in the organs and tissues of fish can be used in biomonitoring their wintering conditions.¹⁴ Wedemeyer and Yasutake suggested using cortisol and glucose of plasma for fish health evaluation.¹⁸

The literature indicates that coagulation time in fish is a rather unstable indicator, which depends not only on the method of blood drawing, but also on environmental factors and the physiological state of fish.³⁷ The same source suggested that the stress factors obviously increase the rate of blood coagulation in fish, although the author advises that a caught fish is a fish subjected to a severe stress, and interspecific differences in blood coagulation in fish may well be the result of differences resistance of these fish to stress.

It is also worth noting a review of studies where the activation of hemostatic mechanisms in fish under stress conditions was identified.¹ According to a number of the researches coagulation time is a good indicator of stress, and its reduction can be the result of an increase in the level of catecholamines and cortisol that are released during stress.

Material and Methods

The research was conducted in strict accordance with ethical principles established by the European Convention on protection of the vertebrata used for experimental and other scientific purposes (adopted in Strasbourg in March 18, 1986 and confirmed in Strasbourg in June 15, 2006) and approved by the local ethic committee of the Federal State Budgetary Educational Institution of Higher Education the Vereshchagin State Dairy Farming Academy of Vologda (Record 12, dated December 3, 2015).

The studies were carried out in aquaria conditions on 8 carps (Cyprinus carpio), grown under production-line conditions in a fish farm. The fish were kept in a tank with continuous water circulation and forced aeration at a temperature of 18–20 ° C; the feeding regime was one time per day with a specialized food for this fish species.

Blood drawing from animals was carried out after twenty-four-hours acclimatization and also 24, 48, and 72 hours after the influence of the stress factor what meets the method of conducting an acute experiment for fish. The complex stress factor was the actual fish capture for blood sampling and the total hypoxia, as well as the further deprivation of dissolved oxygen to the environment by turning off the oxygen compressor for all the next days of the experiment.

Fish were anesthetized with clove oil at a dose of 0.033 ml / l.³⁸ Blood drawing was carried out with a syringe from the tail hemal canal in a volume of 0.77% of the fish body weight (1.2 kg on average) for examination of the coagulogram —  in plastic test-tubes (3.8% sodium citrate solution in a ratio of 1: 9), for the analysis of stress markers — in plastic test-tubes without anticoagulant. All studies were performed during the first two hours after blood drawing.

The parameters of plasma-coagulation hemostasis were determined on the coagulometer “Thrombostat” manufactured by Behnk Elektronik, Germany.³⁹ To assess the state of plasma-coagulation hemostasis the following indicators were determined: APTT (activated partial thromboplastin time), PT (prothrombin time), TT (thrombin time) using human thrombin, quantitative analysis of fibrinogen. The plasma fibrinolytic activity was measured by detecting soluble fibrin-monomer complexes (SFMCs) in an o-phenanthroline sample (tablet version). The concentration of cortisol in blood plasma was determined by means of solid-phase chemiluminescent immunoassay. Plasma glucose concentration was determined by biochemical method. Blood hemoglobin was determined by the cyanmethemoglobin photometric method using KFK-2 (ZOMZ, USSR). The level of dissolved oxygen in water was determined by means of O2-test-systems (Tetra, Germany).

The values of the results obtained are presented in the form of the average value and the standard error of mean (M ± m). The significance of differences in the coagulogram indices of carps was evaluated using the Mann-Whitney test for independent samples. The significance of differences in the coagulogram indices and the carp stress markers obtained during the experiment was evaluated using the Wilcoxon test for dependent samples. A qualitative interpretation of the strength of the connection between stress reactions and coagulogram indices was performed according to the obtained value of R based on the Cheddock scale ⁴⁰.

Results and Discussion

As a result of the experiment, the following data on the dynamics of oxygen in the aquarium were obtained: on the 1st day — 5.0 mg / l, on the 2nd day — 4.8 mg / l, on the 3rd day — 3.2 mg / l, and on 4th day – 2.0 mg /l.

It was noted that the plasma cortisol level on the 1^st and 3^rd days after the combined exposure to stress factors was significantly different from the 2^nd day, and by the 4^th day the cortisol level decreased significantly. This can be explained according to classical concepts on the process occurrence in stages, where the 1^st and 2^nd days of the experiment correspond to the first stage (anxiety), the resistance stage appears on the 3^rd day with a significant increase of plasma cortisol level, and its sharp decrease by the ^4th day corresponds to the stage of exhaustion [graph. 1].

Graph 1: Dynamics of Fish Stress Markers during the Experiment Click here to View Figure

The plasma glucose level in the experiment on the 1^st day was significantly different from the 3^rd and 4^th days, while the 2^nd one had significant differences with only the 4^th day. Analyzing the dynamics of glucose level, one can draw the same parallel with the occurrence in stages of the stress process as with cortisol [tabl.1].

Table 1: Dynamics of Carp Stress Markers

The concentration of hemoglobin on the ^1st, 2^nd and 4^th days of studies was determined under the conditions of hypoxia. It was on the 1^st day significantly different from the 2^nd and 4^th days. An obvious decrease can be noticed in the chromoprotein in the blood of experimental animals during the experiment. Most likely, this effect is caused by blood loss associated with daily blood drawing for tests. Along with this, the effect of corticosteroids is not ruled out.

APTT on the 2^nd and 3^rd day of the experiment was significantly different from that on the 4^th day. It can be noted that by the fourth day this indicator underwent a sharp understatement of time according to which the rate of the first phase of hemocoagulation increased.

PT on the 3^rd day has significant differences with the indicators of the 2^nd and 4^th days. Studying PT in dynamics it should be noted that it practically did not change during the experiment.

The amount of fibrinogen in the tested animals on the 3^rd day of the experiment had significant differences from that on the 1^st and 4^th days. It can be seen that by the 4^th day of exposure to stress factors the blood fibrinogen underwent a large jump [graph.2].

Graph 2: Dynamics of Fish Coagulogram during the Experiment Click here to View Figure

TT on the 1^st day of the study had significant differences with the indicators of the 3^rd and 4^th days_; while on the 2^nd day it had significant differences only from the 3^rd one. Analyzing TT in dynamics it can be noted that, like APTT, it has noticeably decreased by the 4^th day, what indicates an acceleration of the processes in the third phase of coagulation [tabl. 2].

Table 2: – Dynamics of Carp Coagulogam

The number of SFMCs on the 2^nd and 4^th days of the experiment was significantly different from the 3^rd day. Contrary to the assumptions about the occurrence in stages of changes SFMCs of blood plasma decreased by the 4^th day.

Correlation analysis revealed the presence of a moderate to close correlation relationship between indicators of stress reactions [tabl. 3]. Analyzing the data of the table, we can conclude that all three stress markers are quite strongly interconnected, this makes it possible to use any of them to identify stress conditions. The highest quality regression models were obtained by analyzing the dependence of cortisol and glucose on hemoglobin level (R2 was 0.44 and 0.45, respectively). Correlation analysis revealed the presence of medium and high links between some indicators of the secondary link of hemostasis and the content of stress markers in the blood serum of carps [tabl. 4]. The strongest relationship was found between plasma hemostasis and levels of hemoglobin and cortisol. The correlation between the hemoglobin content and the hemostatic parameters in the blood of the tested fish was revealed from noticeable to very close. Cortisol most noticeably affects the duration of PV (r = 0.4) and inversely correlates with fibrinogen level (r = -0.3).

Table 3: The correlation Relationship between Stress Markers in Carp

Table 4: Correlation Parameters between Stress Markers and Coagulogram Indices in Carp

Studying the complex effect of stress factors, it was found that the dynamics of the levels of cortisol and glucose used in this experiment as stress markers corresponds to the classical occurrence in stages of stress, where all three stages are visible. The main interest for us is the adaptive reaction of a coagulogram of carps to critical environmental conditions. According to numerous indicators of plasma coagulation of blood, it can be seen that its speed increased significantly during the days of the experiment and closer to the stage of exhaustion, and the prothrombin time, which plays a significant role in the formation of a blood clot in carps, deviated slightly compared to other indicators.

Based on the correlation and regression analysis, we, first of all, established a close relationship between the hemostasis indices and the hemoglobin level in the blood of stressed carps. Furthermore, the less hemoglobin level is, the more actively the internal and general links of the fish coagulation system function. Besides the indicator of the work of the external coagulation link – PT will lengthen with a decrease in hemoglobin. All these changes are likely to help stop possible bleeding, as the causes of low hemoglobin, but, at the same time, prevent excessive activation of the coagulation system, as the cause of thrombosis. Cortisol most noticeably affects the duration of PT and inversely correlates with fibrinogen level.

It is equally important that the correlation analysis revealed a moderate to close correlation between the indicators of stress reactions: cortisol and plasma glucose. All three stress markers are highly interconnected, this makes it possible to use any of them to identify stressful conditions.

An important factor was the presence of medium and high links between some indicators of the secondary link of hemostasis and the content of stress markers in the blood serum of carps, and therefore, these indicators of the coagulogram can also be used as bioindicators of critical conditions in fish.

Conclusion

Data on hemostatic indications of commercial fish in the literature, as before, remain fragmentary. The results of this work confirm the thesis of the sources cited on the large role of cortisol in the stress response, and on the acceleration of coagulation in fish in stress.

Since fish, both in natural and in artificial growing conditions, is subjected to multiple stress factors of different nature and origin, the work carried out may become the basis for further study of the impact of various conditions on the blood physiology of these animals. It is also promising to develop alternative assessments of fish health status, including hemostasis indicators.

Acknowledgments

Thanks RFBR for the source of financial support this research.

Conflict of interest

We certify that we have no Conflict of interest.

Funding Source

The reported study was funded by RFBR, project number 19-34-90109.

References

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15. Schreck, Carl B., and Lluis Tort. “The concept of stress in fish.” Fish physiology. Vol. 35. Academic Press, 2016. 1-34.
16. Petitjean, Quentin, et al. “Stress responses in fish: From molecular to evolutionary processes.” Science of the Total Environment684 (2019): 371-380.
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Introduction

Kohl, also known as surma, surmi, kajal etc. has been considered as one of the ancient cosmetics which have been used by various civilizations since prehistoric times.¹ The earliest use of Kohl as eyeliner was reported in the ancient Egypt and had great importance in Egyptians that even they buried Kohl containers alongside the dead bodies.^2,3 Kohl has also got importance in Islam and its use is described as “Sunnah” in Abu DawudTib and TirmidhiTib. The use of Kohl Al-Ethmed before sleeping was recommended by Prophet Muhammad (Peace be upon Him) for enhanced vision and hair growth.^4,5 Initially, the source of Kohl was a black stone “Ithmed”, composed of stibnite ore (Sb₂S₃), also known as Antimony which was believed to have beneficial aspects⁶. This stibnite ore was used alone as Kohl or used in combination with other ingredients such as camphor, menthol, almond seeds, wood essence, charcoal or other vegetable ashes. Later, stibnite ore was gradually replaced by the Galena ore (lead sulphide) which owns the same grey black colour and shiny appearance like stibnite⁷. However, the exact composition of Kohl has long been a matter of dispute within the scientific community.⁸

The beneficial potentials of Kohl have been proved by the studies that fully supported the medicinal benefits of the multiple ingredients found in the Kohl.⁹ Earlier, Kohl mixed with rose water was also reported to be instilled in the eyes by community people to treat smallpox pustules¹⁰. Kohl has also been used for keeping eyes cool and clean, also as a prevention from several eye diseases such as cataract, conjunctivitis, chalazion, trachoma and blepharitis. Moreover, there are some places in world with a common practice to apply Kohl on the umbilicus of newborns to help the healing process.¹¹ The black and shiny particles of galena protected the eyes from the harmful effects of ultra violet radiations from sunlight and dust particles.¹² Most of the ingredients such as different chemicals, herbs and gemstones are also added in Kohl because of their beneficial influence on the eyesight along with astringent, antibacterial and antiviral effects. Some plant extracts such as Coptisteeta, Fennel (Foeniculumvulgare) and Saffron (Crocus sativus) extracts were also used in the composition of Kohl and considered highly beneficial for improving weak eyesight and to fight against many eye diseases such as catarrhal and rheumatic conjunctivitis.²

Besides, Kohl has also gained a negative reputation after the use of galena stone as a major constituent rather than antimony ore, as it resulted in lead intoxication due to high content of lead in Kohl samples.¹³ Many studies have discouraged the use of Kohl due to its possible health hazards. The possible lead toxicity in eye cosmetic (Kohl) consumers was studied in California, where the users were mainly the children from Pakistan and India as their lead level was observed as 12.9 µg/dL while it was only 4.3 µg/dL among children not using Kohl cosmetics.¹⁴ However, certain studies also reported no association of increased blood lead level with application of Kohl.¹⁵ The contamination in Kohl samples with pathogenic bacterial and fungal strains has also been reported earlier as a reason of spreading ocular infections among many individuals.¹⁰

The objective of this study was conceived after the incident reported in Sydney (Australia) where three children got severely ill due to lead toxicity caused by regular use of Kohl.The current study was designed to conduct the microbiological and chemical analysis of the Kohl samples available in different local markets of Karachi City, Pakistan.

Materials and Methods

Collection of the Kohl Samples

A total of 20 samples (branded and non-branded) of the traditional Kohls were collected, randomly, from different local markets of Karachi city, and transported to the microbiology research laboratory, Federal Urdu University of Arts, Science and Technology (FUUAST) for further analysis.

Detection of Antimicrobial Potential of the Kohl Samples

Antimicrobial potential of different Kohl samples was detected against different bacterial and fungal reported pathogens of ocular infections, as mentioned below.

Antibacterial Potential of the Kohl Samples

The antibacterial effect of the Kohl samples was investigated against different clinical strains of three Gram-negative (K. pneumoniae, P. aeruginosaand P. mirabilis) and two Gram-positive bacteria (S. aureus and S. epidermidis), obtained from Lab of Applied Microbiology and Clinical Mycology, Department of Microbiology, FUUAST. The suspensions of kohl samples were prepared by adding one gram of kohl in 2 ml of sterile distilled water and vortexed well.The antibacterial activity of Kohl samples was assessed by Agar-well diffusion method ¹⁶.

Antifungal Potential of the Kohl Samples

Antifungal effect of the Kohl samples was investigated against pathogenic isolates reported for ocular infections, including two yeasts (C. albicansand C. tropicalis) and three molds (A. flavus, F. oxysporum, and Mucorsp.) obtained from Lab of Applied Microbiology and Clinical Mycology, Department of Microbiology, FUUAST. The suspensions of kohl samples were prepared by adding one gram of kohl in 2 ml of sterile distilled water and vortexed well.  The antifungal activity of these Kohl samples was determined by Agar-well diffusion method¹⁷.

Isolation and Identification of Microorganism from Kohl Samples

For the isolation of microorganisms from the Kohl samples, a serial dilution of each Kohl sample was prepared up to 10^-2 dilution and processed. Identification of the isolated bacterial and fungal colonies was done macroscopically by observing the color, texture and pigmentation of media by microorganisms, while microscopically by staining (Gram staining in case of bacteria and Lactophenol blue staining for fungi isolates), according to established protocol¹⁸.

Heavy Metal Analysis of the Kohl Samples

The chemical analysis of Kohl samples was carried out by Flame Atomic Absorption Spectrophotometer (FAAS) (Perkin Elmer-A Analyst 700). The Kohl samples were prepared for this analysis by using wet digestion method, and analyzed, in triplicate, for the estimation of trace metals including arsenic (As), cadmium (Cd), lead (Pb), copper (Cu), iron (Fe) and sodium (Na), using established protocol¹⁹. The results were expressed as mean ± SD and compared with BVL standards. According to BVL standards, toxic metals in cosmetic products such as arsenic, cadmium, lead, Mercury exceeding the values 0.5, 0.1, 0.1 and 5 ppm (mg/kg), respectively, should be avoided.²⁰

Results                                                                    

In current study, a total of 20 different Kohl (Surma) samples were purchased from different local markets in the Karachi city. Among these Kohl samples, 85% were branded while 15% were unbranded. These Kohl samples were available within the price range of 20 to 90 Pakistani rupees, manufactured locally or imported from India and Saudi Arabia.

The antimicrobial potentials of collected Kohl samples were tested against some clinically important pathogens, reported for causing predominant ocular infections. Amongst all, 75% of the total Kohl samples revealed good antibacterial activity. The highest activity was reported against P. mirabilis and S. epidermidis(Fig. 1& 3). Moreover, 30% of the samples demonstrated antagonistic potential against fungal pathogens. The highest activity was observed against Candida and Mucorsp. (Fig 2& 3).

Figure 1: Antibacterial potential of Kohl samples against different ocular bacterial pathogens Click here to View Figure

Figure 2: Antifungal  potential of Kohl samples against different ocular fungal pathogens Click here to View Figure

Figure 3: Antimicrobial activity of Kohl samples by agar well diffusion method against (A) S.epidermidis  (B) C.albicans Click here to View Figure

The microbial contamination in collected Kohl samples was also examined by spread plate method. Based on the colony forming unit (CFU), the samples were interpreted as contaminated or free from contamination. Bacterial contamination alone was found in 45% of the Kohl samples while contamination with fungi was observed in 15% of the samples. However, contamination with both bacteria and fungi was detected in 15% of the Kohl samples (Fig.4).

Figure 4: Microbial contamination in Kohl samples. Click here to View Figure

Based on their morphological shape, visualized by Gram’s staining, the bacterial isolates were identified as rod shaped (Bacillus) bacteria, while fungal strains were identified as Aspergillusflavus and A. niger, on the basis of their microscopic as well as macroscopic characteristics.

The contamination of different heavy metals such as arsenic, cadmium, lead, copper, iron and sodium in the Kohl samples was analyzed by Flame Atomic Absorption Spectrophotometer (FAAS). The Kohl samples where metals were detected in Below Detection Limit (BDL) were declared as free from heavy metal contamination. Amongst lethal heavy metals (As, Cd and Pb), 90% of the Kohl samples were found contaminated with arsenic metal, however cadmium and lead contamination was detected in 65 and 40% of Kohl samples, respectively. Other than these metals, Cu, Fe and Na metals were also detected from these samples. Both the iron and sodium were detected in 95 to 100 % of the Kohl samples. The results showed complete absence of copper metal in all the Kohl samples (Table1 & Fig 5).

Table 1: Determination of heavy metals content (ppm*) in kohl samples

*Ppm: Parts per million, **BDL: Below Detection Limit

Figure 5: Detection of different metals in kohl samples. Click here to View Figure

Medicinal Importance of KohlDiscussion

Kohl, generally known as Surma/Kajal in Indo-Pakistan, has been traditionally used since antiquity in most of the Middle and Far-east countries, having a strong cosmetic, medicinal and religious importance. In respect of medicinal importance, Kohl has been widely used for its therapeutic effects against different eye diseases and for the enhancement of eye vision.²¹ Apart from its beneficial importance, detrimental effects of Kohl have also been reported on human health progressively from last few decades due to the presence of microbial contaminants and presence of heavy metals such as lead, arsenic, copper, cadmium etc.in its composition¹⁰.The present study was focused to investigate both beneficial and injurious aspects of Kohl in various branded and non-branded samples, while the brand names are not mentioned to avoid any possible legal complications. The antimicrobial effect of Kohl samples was investigated against microbial pathogens which are usually involved in ocular infections being well recognized as leading cause of conjunctivitis, blepharitis, dacryocystitis, keratitis and endophthalmitis in the previously reported literature.²² The study revealed excellent bioactivity of Kohl samples against most of the bacterial strains particularly P. mirabilis and S. epidermidiswhich confirm previous researches, where Kohl showed good antibacterial effect against Staphylococcus and Proteus species.⁶ However, previous finings regarding antifungal bioactivity of Kohl is lacking although in the present study many Kohl samples showed antifungal capacity against a number of ocular fungal pathogens. According to current findings, Candida spp.and Mucorspp. were highly inhibited by the Kohl samples. These fungal species are well known ocular pathogens and were recently reported to cause a severe Oculo-rhino-cerebral mucor mycosis in immune-compromised patients.²³ Moreover, Candida and Aspergillus species are also found to be associated with fungal keratitis which has been considered as a common ocular mycosis responsible for blindness in most of the cases.²⁴

Microbial Contamination in Kohl Samples

Apart from beneficial importance of Kohl, injurious aspects due to microbial and chemical contamination have also been reported earlier. In current findings, we isolated bacteria and fungi from many Kohl samples. Bacillus and Aspergillus species were found to be the most common contaminants in Kohl samples, which also coincide with a previous study where Kohl, mascara and eyeliner samples were found contaminated with these species.¹⁰ These species are present in environment and therefore their contamination might be due to improper packaging or storage of these kohl samples.

Chemical Contamination in Kohl Samples

The heavy metals are non-essential and non-biodegradable elements and can induce toxicity even at very low doses. Presence of heavy metals in cosmetic products has been associated with many health-related issues due to heavy metals toxicity.²⁵ However, these toxic metals are now banned or at least restricted by the concerned regulatory authorities of many countries, though permissible limits of metals might be different in many products with respect to various countries.²⁶ The metal impurities in cosmetic products are usually compared with the standard limits of United States Food & Drug Administration (USFDA) but recently the Federal Office of Consumer Protection and Food Safety of Germany (BVL) has reduced the standard levels and demonstrated new limits for metals content in cosmetic products.²⁰

Arsenic is one of the most lethal heavy metal having the highest rank in the list of risky substances. Its continuous exposure to humans may lead to severe illnesses such as it reduces the synthesis of erythrocytes and leukocytes, developed skin lesions, neurological problems, pulmonary, peripheral, vascular, cardiovascular diseases, diabetes mellitus and certain types of cancers.²⁷ In our study, arsenic metal was detected in 90% of the total Kohl samples and 80% of these samples were detected with high metal content (5.912 ppm) according to BVL published standard limits. These findings correlate with the previous studies in which the arsenic content was detected higher within different cosmetic products.²⁸

Cadmium is biologically a non-essential heavy metal which is well recognized for its adverse influence on the enzymatic systems of cells.²⁹ Our results revealed cadmium metal in 65% of the total Kohl samples, in which 30% samples possessed high level of cadmium (1.430ppm) than standard limits. Another study also reported a little lower concentration of cadmium (0.942 ppm) as compared to our findings.³⁰ Similarly, lead (Pb) is also a non-essential metal, having no nutritional value to living organisms and ranks fifth in the list of metals³¹. Lead is a recognized carcinogen and its minimal exposure to humans can be very toxic causing severe diseases such as headache, hypertension, arthritis, birth defects, paralysis, brain and kidney damages, and even the death.^32,33 Kohl has been reported as one of the major source of lead poisoning mainly in children and it can be absorbed across the conjunctiva by eye rubbing, tearing (lacrimation) while finger licking could further enhance the lead absorption in living systems. Many researchers after laboratory confirmation reported the existence of lead in many Asian Kohls¹³. An analytical study of different types of Kohls in Pakistan also revealed lead concentration within the range of 0.03% to 81.37%.³⁴ In literature of more than 20 years, many case studies of plumbism (lead poisoning) have been reported, most frequently in infants and children that upon investigation have been attributed to Kohl usage were found with high concentration (199.9 ppm) of lead which is much higher than mandated levels. Another study, however, reported the further higher concentrations (1005 ppm) in Kohl samples.³⁰ Copper is also known to play a vital role in enhancing and strengthening of eye vision and can inhibit activity of lactate dehydrogenase, which is a sensitive marker of cataract formation³⁵. Although this beneficial metalwas not detected in any of our Kohl samples. However, in previous studies, it was detected in high levels from various Kohls. Similarly, another important and beneficial metal Iron was also detected in 95% of the Kohl samples but in very little amount (23.14 ppm) as compared to the previously reported studies.³⁰

Conclusion

Briefly in this study, we observed that most of the Kohl samples possessing antimicrobial potential against ocular pathogens had high contents of heavy metals. While the Kohl samples with lower content of heavy metals exhibited weak or no inhibitory effect against microbial pathogens. Therefore, the antimicrobial activity of Kohl samples might be due to the presence of high contents of heavy metals which are known to produce bactericidal or fungicidal activities since antiquity.³⁶ Furthermore, two Kohl samples, SE16 and SE17 demonstrated excellent antimicrobial effect in the absence of toxic metals in their composition. Therefore, the antimicrobial potential of these Kohl suggested their use in ocular infections. However most of the samples were found contaminated with heavy metals particularly samples: SE1, SE2, SE3, SE4, SE5, SE6, SE14 and SE15 were found heavily contaminated with heavy metals such as Lead, Arsenic and Cadmium and all of these Kohl samples were from very famous brands of Pakistan. It was concluded that detection of high contents of heavy metals such as lead, arsenic and cadmium in most of the Kohl samples suggest proper monitoring of locally manufactured or imported cosmetic products in order to ensure the safety of public health.

Acknowledgement

The authors gratefully acknowledge Lab of Applied Microbiology and Clinical Mycology, Department of Microbiology, FUUAST for the provision of microbial cultures for the study.

Funding Source

The study was partially supported by Higher Education Commission, through NRPU: 4497 awarded to one of the author.

Conflict of Interest

There is no conflict of interest.

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Introduction

Surfactants are an important class of chemical compounds synthesized in large part by petroleum derivatives (Gudiña et al., 2015a; Padilha et al., 2015) They are formed by hydrophobic and hydrophilic portions, which are distributed at the interface between liquid phases causing the decrease of surface and interfacial tensions ( Akbari et al., 2018; Ehinmitola et al., 2018; Gudiña et al., 2016; Grüninger et al., 2019; Mondal et al., 2015; Mondal et al., 2016, Mondal et al., 2017a). The surfactant production is expected to increase to 24 million tons and be worth approximately $ 120 million by 2020 (Jiang et al., 2020).

Currently, the studies about biosurfactants have been expanded due to the high environmental impact caused by some chemical surfactants. In addition to having similar properties to chemical surfactants, these amphiphilic bioproducts have advantages such as biodegradability, low toxicity and stability under extreme conditions of pH, temperature and salinity (França et al., 2015). Surfactants have potential to be applied in numerous products or fields, such as, detergents, paints, paper products, pharmaceuticals, cosmetics, petroleum, food, and water treatment (Costa et al., 2010; Mondal et al., 2017b).

Biosurfactants can be produced by different strains of microorganisms (bacteria, filamentous fungi and yeasts) using renewable raw materials with low cost as a substrate (Abdel-Mawgoud et al., 2010; Araújo et al., 2013). These molecules are classified into five major groups: lipopeptides, glycolipids, fatty acids, phospholipids and polymeric biosurfactants (Geetha et al., 2018).

Among the glycolipids there are the rhamnolipids, composed of rhamnose molecules and one or two units of β-hydroxydecanoic acid, which are present mainly in four isoforms (Mulligan, 2005). The production of these molecules occurs predominantly by Pseudomonas aeruginosa and the z is classified as mono and di-rhamnolipids according to the amount of rhamnose present in the structure. In addition, the proportion of these two forms can be influenced by nutritional and environmental conditions of microbial growth (Oluwaseun et al., 2017; Varjani and Upasani, 2017). Mono-rhamnolipids congeners show increase emulsification and antimicrobial properties in comparison to di-rhamnolipids (Sood et al., 2020). Other species of Pseudomonas have also been reported as producing rhamnolipids, such as P. chlororaphis, P. plantarii, P. putida and P. fluorescens (Randhawa and Rahman, 2014). The main characteristics of these biosurfactants are related to their ability to reduce the surface tension of water to between 28 and 30 mN/m, reduce interfacial tension between water and hydrocarbons, and have a critical micelle concentration (CMC) between 10 and 200 mg/L (França et al., 2015; Gudiña et al., 2015a).

Although can be applied in different areas, the production of biosurfactant on a large scale is not yet totally feasible, since the cost with the production and recovery of this product is relatively high (Souza et al., 2018). However, an alternative to reduce production costs would be the use of low-cost raw materials such as frying oils, sugarcane and beet molasses and cassava wastewater (Banat et al., 2014). But, even with some limitations it is estimated that in 2023 approximately 524 tons of biosurfactant will be traded, which will be responsible for a turnover of US$ 2.7 billion (Felipe and Dias, 2017).

In this context, the objective of this study was to evaluate the production of rhamnolipids by Pseudomonas aeruginosa AP029-GLVIIA by varying the carbon/nitrogen ratio (C/N), based on a simple and affordable source of carbon and energy (glucose), and the percentage of inoculum. Thus, the produced rhamnolipids were characterized in terms of CMC, emulsification index, bioremediation and antifungal activity against the species Candida albicans and Candida tropicalis.

Material and Methods

Chemical

The main chemicals used during this study were corn oil (Cargil Co. – SP, Brazil), D-glucose (Synth Co. – SP, Brazil), hexadecane (Sigma Co., USA), iron sulfate II heptahydrate (Synth Co. – SP, Brazil), kerosene (Líder Co. – RN, Brazil), magnesium sulfate heptahydrate (Synth Co. – SP, Brazil), monobasic potassium phosphate (Synth Co. – SP, Brazil), motor oil (Petronas Co.-MG, Brazil), peptone (BD Co. – SP, Brazil), sodium chloride (Cinética Co. – PR, Brazil), sodium nitrate (Cinética Co. – PR, Brazil), sodium phosphate dibasic heptahydrate (Synth Co. – SP, Brazil), soybean oil (Bunge Co. – SP, Brazil), and yeast extract (BD Co. – SP, Brazil) they were all of analytical grade.

Microorganism and Maintenance

Pseudomonas aeruginosa AP029-GLVIIA was isolated from an oil well in the city of Mossoró (Rio Grande do Norte, Brazil) and deposited in the culture collection of the Department of Antibiotics in the Federal University of Pernambuco (UFPE – Brazil). The microorganism was maintained in petri dish with PCA (Plate Count Agar) at 5 °C (Araújo et al., 2017).