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Original article / research
Year : 2011 Month : August Volume : 5 Issue : 4 Page : 752 - 754

The Significance of the Secretory Immunoglogulin A to Gardnerella vaginalis in the Pathogenesis and Diagnosis of Bacterial Vaginosis
Udayalaxmi, Gopalkrishna Bhat, Subannayya Kotigadde

Corresponding Author. Associate professor, Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, India - 575001. Professor, Department of Microbiology, KVG Medical College, Kurunjibag, Sullia, India - 574327.
 
Correspondence Address :
Dr. Udayalaxmi, Senior Grade Lecturer
Department of Microbiology, Kasturba Medical College,
Manipal University, Mangalore-575001, India.
Tel: 091-0824-2423654 extn 5557; Fax: 091-0824-2428183
E-mail address: udayalaxmi68@gmail.com

 
Abstract
Background: Although bacterial vaginosis is prevalent, not much progress has occurred in identifying the factors which are responsible and are associated with bacterial vaginosis and its pathophysiology. Studies on the development of better methods for the diagnosis of bacterial vaginosis are still on.

Objective: To estimate the levels of secretory immunoglobulin A (sIgA) antibodies against G.vaginalis in vaginal discharge and its correlation with the clinical condition.

Materials and Methods: We developed an indirect ELISA by using whole cell antigen to detect sIgA antibodies against G.vaginalis in vaginal washings which were collected from 170 women who attended two hospitals in south India for antenatal care or any other complaint. The women were categorised into bacterial vaginosis, intermediate and normal by using Nugent’s criteria. Statistical analysis was done by using the Chi- Square test, analysis of variance and Scheffe’s multiple range tests, as appropriate.

Results: Of the 170 women under study, 42 were cases of bacterial vaginosis, 30 had intermediate flora and 98 were normal women. Of the 42 women with bacterial vaginosis, 16 (38%) showed a significantly high titre and 26 (62%) had low titre. Of the 30 women with intermediate flora, 10 (33.3%) showed a significantly high titre and 20 (66.7%) had low titres of the 98 women with normal flora, 84 (85.7%) showed low titres and 14 (14.3%) showed high titres of sIgA antibodies against G.vaginalis. The results of the ELISA test correlated statistically with the cases of bacterial vaginosis when analysed by Chi-Square test (p < 0.05). Analysis of variance showed that there was significant variation (p < 0.05) between the titers of sIgA antibodies detected in the women categorised by Nugent’s criteria.

Conclusion: All the women with bacterial vaginosis or with intermediate flora do not show significant titres of sIgA antibodies to G.vaginalis. So, the detection of sIgA to G.vaginalis in the vaginal discharge cannot be used as a means for the diagnosis of bacterial vaginosis.
Keywords : Bacterial vaginosis, Diagnosis, Gardnerella vaginalis, Pathogenesis, Secretory Immunoglobulin A
How to cite this article :
Udayalaxmi, Gopalkrishna Bhat, Subannayya Kotigadde. THE SIGNIFICANCE OF THE SECRETORY IMMUNOGLOGULIN A TO GARDNERELLA VAGINALIS IN THE PATHOGENESIS AND DIAGNOSIS OF BACTERIAL VAGINOSIS. Journal of Clinical and Diagnostic Research [serial online] 2011 August [cited: 2014 Jul 29 ]; 5:752-754. Available from
http://www.jcdr.net/back_issues.asp?issn=0973-709x&year=2011&month=August&volume=5&issue=4&page=752-754&id=1437  
 
Bacterial vaginosis represents a unique and complex change in the flora of the vagina, which is characterised by a reduction in the prevalence and the number of lactobacilli and an increase in the concentration of Gardnerella vaginalis and resident anaerobic bacteria (1). In India, the prevalence of bacterial vaginosis in the general population is 18-20% (2),(3),(4). Although bacterial vaginosis is prevalent, not much progress has occurred in identifying the factors which are responsible and are associated with bacterial vaginosis and its pathophysiology (5). Bacterial vaginosis is associated with a variety of obstetric and gynaecological complications such as preterm birth, low birth weight, post partum endometritis and pelvic inflammatory disease (PID) (1),(6).

Nugent’s gram’s stain scoring system is considered as the gold standard for the diagnosis of bacterial vaginosis (4), (7). Nugent’scriteria divides women into bacterial vaginosis (Nugent’s score > 7), intermediate (Nugent’s score of 4-6) and normal women (Nugent’s score of ≤ 3) (7). The chromogenic filter paper method for sialidase detection and quantitative PCR are some of the newer methods for the diagnosis of bacterial vaginosis, which are being developed (8),(9).

There are studies on the immune response in the vaginal discharge in cases of bacterial vaginosis. But in these studies, ELISA was done by using purified haemolysin as an antigen (10),(11). In these studies, a specific immunoglobulin A response was detected in 60% of the women with overt bacterial vaginosis, and in 18.5% of the women with intermediate vaginal flora and 91% of the normal women showed the absence of specific sIgA antibodies. In the present study, we used whole cell antigen ELISA to detect the sIgA antibodies against G.vaginalis in the vaginal discharge, with theintention of getting better results as haemolysin is highly unstable than the whole cell antigen (12).
 
Material and Methods
One hundred and seventy women who attended two hospitals in south India for antenatal care or IUD insertion or removal, with complaints of discharge, abdominal pain or any other complaint, formed the study population. Married women between the age group of 21–35 years and women with or without vaginal discharge complaints were included. Women who were menstruating at the time of the specimen collection andwomen who were on medication for any bacterial, fungal, parasitic or viral infection for up to one month prior to the specimen collection were excluded. The study had the approval of the Institutional Ethics Committee.

Vaginal swabs and vaginal washings were collected. The vaginal walls were irrigated with 10 mL of sterile saline after inserting a sterile speculum which was lubricated with water. The resulting fluid was collected by using a sterile syringe and was stored in storage vials at –70°C until use (11). The diagnosis of bacterial vaginosis was based on Nugent’s criteria (7).

G.vaginalis ATCC 14018 was inoculated on human blood agar plates and incubated at 37°C for 48 hrs. The colonies were harvested from human blood agar plates by using 0.15 M sterile saline. The bacterial cells were washed twice with 0.15 M sterile saline. The saline suspension of the bacteria was adjusted to the turbidity OD540 of 2.5. Flat bottom, 96 well microtitre plates were coated with this whole cell antigen suspension by incubating at 37°C for 2 hrs and then overnight at 4°C.

ELISA was done as described earlier (10). Two hundred microlitres of vaginal washings were added to each well and incubated at 37˚C for 2 h. The ELISA plate was washed 3 times with the wash buffer – phosphate buffered saline pH 7.3 with 0.05% (v/v) Tween 20 (Hi Media Laboratories, Pvt. Ltd., Mumbai, India) and 100 μl of the conjugate – goat anti human IgA antibody which was conjugated with the alkaline phosphatase enzyme (Sigma, St. Louis), was added. The plate was incubated at 37˚C for 1 hr and was washed 3 times with the wash buffer. Then, 100 μl of the substrate –0.1% (w/v) ρ-nitrophenyl phosphate (Sigma, St. Louis) in 0.1M glycine buffer containing 1mmol/L of magnesium chloride (MgCl2) (Hi Media Laboratories, Pvt. Ltd., Mumbai, India) and 1mmol/L of zinc chloride (Hi Media Laboratories, Pvt. Ltd., Mumbai, India), pH 10.6 was added and this was incubated in the dark for 30 min. Then, 16 μl of the stop solution –3 mol/L NaOH solution was added and the absorbance was read at OD405 by using an ELISA reader (Biotech, USA, Model ELX 800). All the tests were performed in duplicate. Wells without the vaginal washings served as the blanks. The cut off value was calculated as the mean absorbance of the normal women plus one standard deviation (10).

Statistical analysis was done by using the Chi Square test, Analysis of variance and Scheffe’s multiple range tests as appropriate. A two tailed p value of less than 0.05 was considered to be significant. SPSS version 11.5 was used to do the statistical analysis.
 
Results
Of the 170 women under study, 42 were cases of bacterial vaginosis (Nugent’s score of ≥ 7), 30 had intermediate flora (Nugent’s score of 4 – 6) and 98 were considered as normal (Nugent’s score of 0 - 3) when diagnosed by Nugent’s method. Of the 42 women with bacterial vaginosis, 16 (38%) had significantly high titres and 26 (62%) had a low titre of sIgA antibodies against G.vaginalis. Ofthe 30 women with intermediate flora, 10 (33.3%) had significantly high titres and 20 (66.7) had low titres of sIgA antibodies. Of the 98 women with normal flora, 84 (85.7%) showed low titres and 14 (14.3%) showed high titres of antibodies (Table/Fig 1).

The results of the ELISA test statistically correlated with the cases of bacterial vaginosis when analysed by Chi-Square test (p < 0.05). Analysis of variance showed that there was a significant variation (p < 0.05) between the titres of antibodies detected in women categorised by Nugent’s criteria (Table/Fig 2). Scheffe’s multiple range tests showed that there was a statistically significant difference in the titer of sIgA antibodies to G.vaginalis, between normal women and women with bacterial vaginosis (p < 0.05) and also between normal women and women with intermediate flora (p < 0.001). But, there was no significant difference between the titer of the sIgA antibodies against G.vaginalis of women with bacterial vaginosis and those with intermediate flora (p = 0.735).
 
Discussion
We developed an indirect enzyme immunoassay (ELISA) for the estimation of sIgA antibody levels in the vaginal discharge against G.vaginalis by using whole cell antigen. The vaginal discharge samples were tested in duplicate and also the ELISA tests were repeated by using both G.vaginalis ATCC 14018 and also vaginal isolates as antigens. The intra assay and inter assay variations have been found to be minimum.

We found that only 38% of the women with bacterial vaginosis showed elevated titers of the sIgA antibodies against G.vaginalis. The reason for low positivity may be because of IgA depletion as observed in cases of bacterial vaginosis in past studies. In studies which were conducted earlier, many women showed extensive IgA and IgM degradation in the vaginal washings, whereas the IgG titer was intact. The IgA degradation correlated with a high titer of the sialidase enzyme in the vaginal discharge, which was produced by the organisms which were associated with bacterial vaginosis (11),(13).

High titres of the sialidase enzyme and low titres of sIgA correlated with an adverse pregnancy outcome, especially preterm delivery and low birth weight. High titres of sIgA and low levels of the sialidase enzyme were followed by a good pregnancy outcome. So, the sIgA antibodies against G.vaginalis appear to be protective (11),(13). There is only one Indian study on the detection of sIgA in the vaginal discharge of women with bacterial vaginosis (14). This study showed that women with a low BMI (body mass index) did not show elevated titers of sIgA in their vaginal discharge (14). In our study, most of the women belonged to the low socioeconomic status. It is possible that the women with low titres of sIgA may be having a low body mass index.

It is not certain whether women with intermediate flora can revert back to the normal flora or complicate into bacterial vaginosis. In the present study, 33.3% of the women with intermediate flora showed a significantly high titre of sIgA antibodies. It is possible that women with intermediate flora, who do not have sIgA antibodies, may have high titres of the sialidase enzyme activity in their discharge and hence, may develop bacterial vaginosis. Further studies are needed to prove this aspect of the pathogenesis of bacterial vaginosis.

All women with bacterial vaginosis or with intermediate flora do not show significant titres of the sIgA antibodies to G.vaginalis. So, the detection of sIgA to G.vaginalis in the vaginal discharge cannot be used as a means for the diagnosis of bacterial vaginosis.
 
Key Message
All women with bacterial vaginosis or with intermediate flora do not show significant titres of sIgA antibodies to G.vaginalis. The detection of sIgA to G.vaginalis in the vaginal discharge cannot be used as a means for the diagnosis of bacterial vaginosis
 
Acknowledgement
The authors are grateful to Manipal University for providing the facilities to perform the study. We are grateful to the clinicians who were working in the obstetrics and gynecology departments of the hospitals which were under study for helping with sample collection. We thank the statistician, Mr. Shashidhar Kotian Associate Professor, Department of Community Medicine, Kasturba Medical College, Mangalore, Manipal University, for helping us with the statistics.
 
References
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Sobel JD. Bacterial vaginosis. Annu Rev Med 2000;51:349-56.
2.
Madhivanan P, Krupp K, Chandrasekaran V, et al. Prevalence and correlates of bacterial vaginosis among young women of reproductive age in Mysore, India. Indian J Med Microbiol 2008; 26(2):132-7.
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Rao PS, Devi S, Shriyan A, Rajaram M, Jagdishchandra K. Diagnosis of bacterial vaginosis in a rural set up: comparision of clinical algorithm, smear scoring and culture by semiquantitative technique. Indian J Med Microbiol 2004;22(1):47-50.
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Demba E, Morison L, Maartem Schim VDL, et al. Bacterial vaginosis, vaginal flora patterns and vaginal hygiene practices in patients presenting with vaginal discharge syndrome in the Gambia, West Africa. BMC Infect Dis 2005;5:12-24.
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Hay P. Life in the littoral zone: lactobacilli losing the plot. Sex Transm Infect 2005;81:100-2.
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Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 2007;21(3):375-90.
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Mohanty S, Sood S, Kapil A, Mittal S. Interobserver variation in the interpretation of Nugent scoring method for diagnosis of bacterial vaginosis. Indian J Med Res 2010;131:88-91.
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Wiggins R, Crowley T, Horner PJ, Soothill PW, Miller MR, Corfield AP. Use of 5-Bromo–4- Chloro-3-Indolyl–α-D-N–Acetylneuraminic Acid in a novel spot test to identify sialidase activity in vaginal swabs from women with bacterial vaginosis. J Clin Microbiol 2000;38:3096-7.
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Beverly ES, Hua YC, Qiong JW, Zariffard MR, Mardge HC, Gregory TS. Utility of Amsel Criteria, Nugent Score, and Quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for Diagnosis of Bacterial Vaginosis in Human Immunodeficiency Virus- Infected Women. J Clin Microbiol 2005;43:4607–12.
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Cauci S, Scrimin F, Driussi S, et al. Specific immune response against Gardnerella vaginalis hemolysin in patients with bacterial vaginosis. Am J Obstet Gynecol 1996;175:1601–5.
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Cauci S, Thorsen P, Schendel DE, Bremmelgaard A, Quadrifoglio F, Guaschino S. Determination of immunoglobulin A against Gardnerella vaginalis hemolysin, sialidase, and prolidase activities in vaginal fluid: implications for adverse pregnancy outcomes. J Clin Microbiol 2003;41:435-38.
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Cauci S, Monte R, Ropele M, Missero C, Not T, Quadrifoglio F, Menestrina G. Pore-forming and haemolytic properties of the Gardnerella vaginalis cytolysin. Mol Microbiol. 1993;9(6):1143-55.
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Cauci S. Vaginal immunity in bacterial vaginosis. Curr Infect Dis Rep 2004;6(6):450-6.
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Yasodhara P, Raghunath M, Sreeramulu D, Venu L, Hemalatha R, Krishna TP. Local immunity in Indian women with bacterial vaginosis. J Reprod Immunol 2006;70:133-41.
 
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DOI and Others
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