ABSTRACT

Corresponding Author:
Dr. Amit Singh,
Associate Professor,
Department of Microbiology,
UPRIMS & R, Saifai,
Etawah-206130, Uttar Pradesh.
E-mail: dramitsingh.uprims@gmail.com

ABSTRACT

BACKGROUND

Bacterial vaginosis (BV) is the most common cause of abnormal vaginal discharge among women of child bearing age and is associated with adverse obstetric and gynaecologic outcomes.

OBJECTIVES

The aim of the study was to determine the prevalence of BV by use of Nugent’s criteria and to identify modifiable and non-modifiable, the risk factors associated with BV in women of reproductive age.

METHODOLOGY

A descriptive cross sectional study was conducted from January 2013 to December 2013, among women of child bearing age with complaints of vaginal discharge, attending Gynaecology and Obstetric OPD at UPRIMS & R, Saifai, Etawah. Bacterial morphotypes indicative of BV were identified by Nugent’s criteria. A pre-coded questionnaire was used to collect demographic and behavioural characteristics (including contraceptive usage, douching practice) in the study participants.

DATA ANALYSIS

Bivariate and multivariate analyses by logistic regression method performed. Crude Odds ratio and Adjusted Odds Ratio for the association between BV and demographic or behavioural characteristics was calculated using Poisson regression. Sensitivity, specificity, PPV and NPV were calculated keeping Nugent score of ³7 for BV as ‘Gold standard.’

RESULTS

A prevalence of 31.5.0%, (95% CI 25.6-38.2) was obtained from the study population. Sensitivity, specificity, PPV and NPV of Amsler’s criteria for clinical diagnosis of BV was 67.7%, 89.39%, 74.58% and 85.02% respectively. Low socioeconomic status including occupation, illiteracy, Intrauterine Contraceptive Device (IUCD) usage, douching practice and condom usage were significantly associated with BV.

CONCLUSIONS

The prevalence of BV was 31.5% in this population. Concordance between Amsel’s criteria and Nugent’s was low (67.67%). Among individual Amsel’s parameters, vaginal pH >4.5 had highest sensitivity (84.61%) and demonstration of ‘Clue Cell’ was most specific (92.2%). Risk factors for BV ought to be evaluated in larger population for development of interventional strategies.

KEYWORDS

Bacterial Vaginosis, Amsel’s Criteria, Nugent’s Criteria, Reproductive Age Group, Risk Factors.

INTRODUCTION

Bacterial Vaginosis (BV) is the among the common vaginal condition among women of child bearing age.¹ It is an imbalance in the ecology of the normal vaginal flora that is characterized by the depletion of lactobacilli and the proliferation of anaerobic bacteria such as Gardnerella vaginalis, Mobiluncus species, Prevotella species, Mycoplasma

hominis and the recently identified Atopobium vaginae. Megasphaera spp., Lachnospira spp., and Sneathia spp.^2,3 It most often manifests clinically as an increase in thin whitish homogeneous, malodorous vaginal discharge.⁴ The pathogenesis of BV remains controversial. It has been hypothesized that G. vaginalis create a biofilm community and successfully competes with lactobacilli for dominance in the vaginal environment.⁵ A symbiotic relationship between G. vaginalis and anaerobes exists where G. vaginalis, metabolically produces amino acids through its proteolytic action. These amino acids are utilized by strict anaerobes such as Prevotella bivia as fuel source and as a result produce ammonia, which in turn is used by G. vaginalis.⁶ Moreover, this symbiotic relationship with the production of ammonia would cause a shift to a more alkaline pH, which is inhospitable to lactobacilli.⁷ Studies have shown that the addition of anaerobes to a G. vaginalis biofilm enhances the growth of G. vaginalis.⁸ These symbiotic relationships could be responsible for the microbiological findings that define BV as well as the typical vaginal signs (Sloughing of the vaginal epithelium visualized as ‘clue cells’) and amine odor resulting from metabolic by-products of the increased numbers of BV-associated anaerobes.⁹

BV has been shown to increase the risk of gynaecological morbidity and adverse obstetrical outcomes such as preterm delivery, Pelvic Inflammatory Disease (PID) following surgical procedures, hysterectomy and upper genital tract infections.^10-13

Bacterial vaginosis has been shown to correlate well with Amsel clinical criteria and is an effective way of screening cases. The four diagnostic elements are: A vaginal fluid of pH>4.5, presence of “clue cells” (Epithelial cells with unclear borders, dotted with bacteria) on microscopic examination of vaginal swab samples in normal saline, milky homogeneous, adherent vaginal discharge and a positive ‘whiff’ test, which is accentuation of an amine or ‘fishy’ odor of discharge after addition of 10% potassium hydroxide. The presence of three out of four criteria is recommended by Amsel for diagnosis.⁹ However, the Amsel’s criteria, has been criticized because two of the four criteria, in particular the appearance of the discharge and the appraisal of the odour are rather subjective and hence may lead to misdiagnosis. By contrast, a pH greater than 4.5 is considered the most sensitive criterion, whereas the presence of clue cells has been considered the single most specific predictor of BV.¹⁴ Nugent et al described a Gram stain scoring system of vaginal smears to diagnose BV.¹⁵ The Nugent score is calculated by assessing for the presence of large gram-positive rods (Lactobacillus morphotypes; decrease in Lactobacillus scored as 0 to 4), small gram-variable rods (G. vaginalis morphotypes; scored as 0 to 4), and curved gram-variable rods (Mobiluncus spp. morphotypes; scored as 0 to 2) and can range from 0 to 10. A score of 7 to 10 is consistent with BV. Compared to the Amsel criteria, the Nugent score allows for assessment of alteration in vaginal flora as a continuum rather than a dichotomy. Because Amsel criteria are dependent on the acumen of the clinician, the Nugent score has been favoured for diagnosing BV due to superior reproducibility and sensitivity.¹⁴

Previous studies have identified a number of risk factors and behaviour associated with BV including the number of lifetime male sexual partners, recent partner change, lower age of first intercourse, working as sex worker, douching, use of an intrauterine device, non-usage of condoms and smoking.^16-19 Other factors that have been associated with BV are low socio-economic status, poor personal hygiene, marital status, HIV infection, STIs most commonly trichomoniasis.²⁰

In India, reported prevalence of BV varies widely among different population. It ranges from 17.8% to 45% among sexually active women with vaginal discharge, 11.53% to 38.5% among pregnant women.^21-24 Many studies have investigated the association of risk factors and bacterial vaginosis elsewhere; however, no such study has been done on population in and around Etawah district of Western Uttar Pradesh. Therefore, the present study was carried out to determine the prevalence of bacterial vaginosis and the associated risk factors.

MATERIALS AND METHODS

A descriptive prospective cross sectional study was conducted at Department of Microbiology and Department of Gynaecology and Obstetrics, UPRIMS and R, Saifai, Etawah. All adult women of child bearing age (18-45) with complaints of vaginal discharge, attending the Gynae and Obs OPD, UPRIMS and R, Saifai from January 2013 to December 2013 were included. Pregnant or puerperal women, those taking medications for STIs or having abnormal uterine/vaginal bleeding were excluded from the study. Sample size of 206 was calculated using the formula²⁵ {n=4pq/ (20% of p)}² assuming the prevalence of vaginal discharge 32.7%²⁶ and margin of error 20%.

Ethical clearance was obtained by the Institute. Informed written consent was taken from patient before their enrolment in the study. For illiterate women consent information was read and explained, then thumb print was taken on consent form. A semi-structured schedule was prepared for interviewing women and information about demographic, socio-economic status, obstetric and gynaecological history, douching, contraceptive use was recorded. After history taking, general physical examination, per abdomen, per speculum and per vaginum examinations were done. During per speculum examination vaginal mucosa was inspected for presence of erythema, lesions and discharge. Vaginal material was collected from the posterior fornix with help of sterile cotton-swab. Three swabs per patient were collected.

From the first swab, vaginal pH was determined by touching a pH strip and comparing the change against a reference reader. Then few drops of 10% potassium hydroxide were added to it to determine amine/fishy odor (whiff test). The second swab was smeared on to a glass slide for Gram staining. They were then methanol fixed, coded and sent to the Department of Microbiology, UPRIMS and R for Gram’s staining. Gram stained smears were evaluated for bacterial vaginosis as well as for candida spp. (Budding yeast-like cells).

The third swab was placed in screw-cap plastic tubes containing 0.5 mL of 0.9% saline to carry out wet mount microscopy for detection of Trichomonas vaginalis. Swab was vigorously rotated in the saline and pressed against the side of the tube to express as much fluid as possible. A drop of the expressed fluid was placed on glass slide with a cover slip and examined at magnification of 400x within 15 minutes of collection of the sample. The positive result was defined as the presence of one or more Trichomonads with characteristic morphology and jerky motility.²⁷

For the purpose of the study, the Nugent’s score¹⁵ was taken to be the gold standard. The Nugent’s score was assessed as following:

• Small Gram-negative/Gram-variable rods (G. vaginalis morphotypes) more than 30 bacteria per oil immersion field (OIF) was scored as 4; a count of 6-30 bacteria per OIF scored 3; and 1-5 bacteria per OIF scored 2. Less than 1 per OIF scored 1 and their absence scored 0.
• For large Gram-positive rods (Lactobacillus morphotypes), the scoring was reversed with their absence scored as 4, fewer than 1 per OIF scored 3; a count of 1-5 per OIF scored 2; a count of 6-30 per OIF scored 1; and more than 30 per OIF scored 0.
• For curved Gram-variable rods (Mobiluncus spp. morphotypes), the presence of five or more bacteria was scored 2, less than 5 scored 1 and absence of bacteria was scored as 0.

The sum of the 3 scores was taken and a score of 7 or more was considered as a case of bacterial vaginosis.

DATA ANALYSIS

Conventional descriptive statistics were used to assess the characteristics of study participants. For numerical data, mean±SD was calculated at 95% CI level. Univariate associations of baseline characteristics with BV were made using Pearson Chi-square test or Fisher-exact method for categorical or ordinal variables. Continuous variables were compared using Student’s t-test or the Mann-Whitney test for non-parametric data. Covariates were considered if they were associated with BV in the literature. Nugent’s scores of 0-3 and 4-6 were clubbed and categorized as 0 and category 7-10 as 1 to denote absence and presence of BV respectively. The following characteristics of the subjects were examined: age, education, parity, contraceptive use. Socio-economic status was assessed according to modified BG Prasad classification.²⁸ and entered into binary logistic regression method to determine the association of various covariates and BV. The association was reported with Crude Odds Ratio (COR). Multivariate analyses were performed on variable with p-values <0.1. Adjusted Odds Ratio (AOR) and respective p-value were calculated with p-values <0.05 considered significant. Data were analysed using IBM SPSS statistics vs 21 software (USA).

RESULTS

Among of 206 adult women of child bearing age enrolled in the study, BV was diagnosed in 65 (31.5%) patients by Nugent’s criteria, while 59 (28.64%) patients were positive by Amsel’s criteria [Table 1]. Maximum concordance 84.6% with BV was seen for vaginal pH >4.5 and least 52.3% for the characteristic homogeneous vaginal discharge [Table 2]. The sensitivity, specificity, PPV and NPV of overall and individual parameters of Amsel’s criteria are shown in Table 3. Vaginal pH >4.5 had the highest sensitivity, while highest specificity was observed for ‘clue cells’. Budding yeast like cells (candida spp) were seen in 8 subjects (3.8%) and only 2 were present in BV. No trichomonal co-infection was seen in our study.

The mean age of the individual was 29.5±5.5 years. The age group 25-30 years maximally presented with 38.8%, while least representation 3.9% was in age group 40-45 years. Majority (49%) of respondents had elementary level education with 21.8% had no formal education; 17% had high school education, whereas 12.1% were graduate.

An updated BG Prasad socioeconomic classification with Consumer Price Index, Oct. 2015 (Industrial worker) of 259, was used as an indicator of socioeconomic status. More than half (56.8%) of the respondents earned less than Rs 2000/ month. Over half of the women enrolled were housewife (51.5%), followed by unskilled labourers 21.8%, while 14.6% were salaried and 12.1% were self-employed [Table 4].

The majority 85.9% of women in our study were married; unmarried and widowed/divorced being 9.7% and 4.4% respectively. A small proportion of women were nulliparous, whereas 25.3% had one or two child and 56% had more than two children [Table 4].

There were various family planning methods used and reported by the study participants. These included condoms,

IUCD and oral pills. A small proportion 2.9% of participants did not use any method of contraception and/or used rhythm method. Among contraceptives used, oral pills 29.1% were most preferred followed by condom and IUCD, 27.7% and 15.5% respectively. Terminal sterilization by tubal ligation was seen in 12.7% of women. About one-third (33.0%) of the women gave history of regular douching practice. The frequency of planning methods, reproductive characteristics, behaviour and BV prevalence is shown in Table 5.

Overall prevalence of BV was 31.5% (95% CI 25.6-38.2) and the highest proportion 31/65 (47.9%) was observed in age group of 25-30 years compared with the lowest proportion 2/65 (3.07) % in age group 40-45 years. Women with bacterial vaginosis were significantly younger than non-bacterial vaginosis group (mean 27.8±4.7 years vs 30.3±5.8 years, p<0.0001).

The demographics of BV group and non-BV group is compared and shown in Table 4. On performing the bivariate analysis (Chi-square) of various characteristics among BV and non-BV subgroups, age (p=0.48), marital status (p=0.81), parity (p=0.13), non-use of any contraception method (p=0.42), usage of oral pills (p=0.6), tubal ligation (p=0.35), did not give any statistically significant difference (p£0.05). For characteristics showing significant difference, bivariate and multivariate analysis for their strength of association (Odds Ratio, OR) with BV was calculated. In multivariate analysis after adjusting for factors that were significant in unadjusted analyses, increased odds of being diagnosed with BV were seen among non-literate women, low socioeconomic (BG Class 5), labour class, IUCD usage and history of regular douching practice with adjusted OR of 2.3 (p=0.001), 2.1 (p=0.02), 1.9 (p=0.03), 1.7 (p=0.023) respectively. Condom usage had AOR of 0.26 (p-value 0.029) indicating its protective role in BV [Table 6].

^*According to updated BG Prasad Socioeconomic criteria, 2015

p-values in bold are statistically significant (p<0.05).

P-values in bold are statistically significant (p<0.05).

P-values in bold are statistically significant (p<0.05).

Fig. 1: Wet Mount showing ‘Clue Cell’

Fig. 2: Candida spp. (BYLC & Pseudohyphae, Gram Stain)

DISCUSSION

Bacterial vaginosis is the most common cause of vaginal symptoms among women.²⁹ The present study attempts to assess the prevalence of BV in rural setup and the risk factors associated with it. The prevalence of BV among women reproductive age group in our study was 31.5%. Using Nugent’s criteria as diagnostic tool, prevalence of BV seems to vary significantly from study to study.^30-32 Our findings were in concurrence with Bhalla et al,³³ who reported 32.8% prevalence of BV among women in Delhi and with Verma et al²⁶ who observed prevalence of 29.2% among women with vaginal discharge. These variations in the rate could be different to geographical distribution or systematic differences among various population samples. The average age of the bacterial vaginosis group in our study (mean age=27.8±4.7 years) was lower than that of the non-suffering group (Mean age=30.3±5.8 years), and it was statistically significant (p=0.001). Studies have suggested that women of childbearing age are more prone to developing BV.³⁴ This might be seen as reason for earlier age of presentation of BV in our study.

All parameters of Amsel’s clinical criteria were satisfied by 59 subjects; however, concordance for BV by Nugent’s criteria was seen in 44 (67.69%) cases only. Thus, overall sensitivity, specificity, PPV and NPV of Amsel’s criteria were 67.69% and 89.36%, 74.58% and 85.02%, respectively. Each individual parameter of Amsel was analysed for diagnosis of BV against Nugent’s criteria, vaginal pH>4.5 improved the sensitivity of diagnosis but at expense of its specificity [Table 3]. Finding of Clue cells were highly specific (92.2%) for BV. Similar findings have been reported from Sha et al.³⁵ Nugent score is preferred for diagnosing BV as better reproducibility and sensitivity. However, smear evaluation is also subjective and requires considerable expertise.

We analysed various socio-demographic characters, contraceptive preference, douching practices as risk factors for BV. Multivariate analysis revealed age, marital status, parity, oral contraceptive use and tubal ligation were not associated with BV. Many studies have reported similar findings with age group, parity and marital status, oral pills and tubal ligation.^36,37

In our study, non-literate woman with low socioeconomic status, working as unskilled labour had almost 2 times the risk of being associated with BV in comparison to literate and economically well to do women. Similar observations were made by various researchers.³⁸ Ganjoei et al, analysing at the risk factors for bacterial vaginosis found that low education level and low socioeconomic status was a significant risk factor for bacterial vaginosis with OR 3.8 and 2.7 respectively.³⁹ Literate and economically well-being women are likely to be more health conscious, can afford and maintain good personal hygiene and seek early medical advice in comparison to illiterate women.

Among contraceptives, IUCD usage was significantly associated with BV with crude OR and AOR of 2.3 and 1.7 respectively. This was in congruence with earlier studies. Om HS et al reported significant increase in risk of BV (p=0.017; OR= 1.70) among IUCD users.³⁷ Joesoef MR et al⁴⁰ investigated risk of BV among IUCD user and non-users at Indonesia and concluded BV to be commonly associated with IUCD user with an AOR of 2. However, studies having contradictory finding have been reported from Turkey.⁴¹ and USA.⁴² Our data supports the hypothesis that IUCD might change endogenous cervico-vaginal environment, which may lead to vulvovaginal infection and BV.

In our study, condom usage had an AOR of 0.26 (p<0.05), which indicates its protective role against acquisition of BV. Earlier studies had reported association of condom use with decrease in risk for BV.^17,43 Ma L et al investigated the effect of condoms and IUCD on vaginal Lactobacilli colonization and concluded that consistent condom use increases the colonization of Lactobacillus crispatus in the vagina and may protect against BV.⁴⁴

An increased risk for BV prevalence (AOR 2.06, p<0.05) was observed among those subjects who practiced vaginal douching. Earlier studies had reported association between the vaginal cleaning practices using stream of liquid with higher risk of acquisition of BV.^45,46 Frequent douching may alter the vaginal ecology and may enhance the risk for BV. However, only few published studies of the effects of douching on the vaginal environment have been done. Earlier study has reported that douches containing povidone–iodine have a profound inhibitory effect on vaginal Lactobacillus than did douches containing saline or acetic acid. Thus, significant reductions in BV might be achieved by decreasing the frequency of vaginal washing.

CONCLUSION

Bacterial vaginosis prevalence was relatively high in our study population (31.5%). Factors like low socioeconomic status, illiteracy, IUCD usage and douching practices were associated with increased BV, whereas condom usage had protective role for identification of modifiable risk factors could help to develop interventions that could improve vaginal heath and reduce risk of BV and its associated co-morbidities in women.

Study Limitation

Analyses were done on cross-sectional study, hence inference of causal association between risk factor and BV could not be drawn. We did not explore role of multiple sexual partners, sex with women or oro/anal sex, as these sexual behaviours have considerable social stigma attached to them, especially so in our study population which was in rural setting and patients tends to obscure or even refuse to divulge information. Qualitative or quantitative culture of the causative agents for BV were not done as microbiology of BV is heterogeneous and many of them are part of normal vaginal flora. Further, it is technically difficult to culture and speciate all the associated fastidious bacteria of BV.

REFERENCES

1. Sobel JD. Vaginitis. N Engl J Med 1997;337(26):1896-903.
2. Fredricks DN, Fiedler TN, Marrazzo JM. Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005;353(18):1899-911.
3. Spear GT, Sikaroodi M, Zariffard MR, et al. Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis. J Infect Dis 2008;198(8):1131-40.
4. Eschenbach DA, Hillier S, Critchlow C, et al. Diagnosis and clinical manifestations of bacterial vaginosis. Am J Obstet Gynaecol 1988;158(4):819-28.
5. Schwebke JR, Muzny CA, Josey WE. Role of gardnerella vaginalis in the pathogenesis of bacterial vaginosis: a conceptual model. J Infect Dis 2014;210(3):338-43.
6. Pybus V, Onderdonk AB. Evidence for a commensal, symbiotic relationship between gardnerella vaginalis and prevotella bivia involving ammonia: potential significance for bacterial vaginosis. J Infect Dis 1997;175(2):406-13.
7. Nagy E, Petterson M, Mardh PA. Antibiosis between bacteria isolated from the vagina of women with and without signs of bacterial vaginosis. APMIS 1991;99(8):739-44.
8. Machado A, Jefferson KK, Cerca N. Interactions between lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation. Int J Mol Sci 2013;14(6):12004-12.
9. Amsel R, Totten PA, Spiegel CA, et al. Non-specific vaginitis. Diagnostic and microbial and epidemiological associations. Am J Med 1983;74(1):14-22.
10. Nelson DB, Hanlon A, Hassan S, et al. Preterm labor and bacterial vaginosis-associated bacteria among urban women. J Perinat Med 2009;37(2):130-4.
11. Klebanoff MA, Hillier SL, Nugent RP, et al. Is bacterial vaginosis a stronger risk factor for pre-term birth when it is diagnosed earlier in gestation? Am J Obstet Gynaecol 2005;192(2):470-7.
12. Ness RB, Kip KE, Hillier SL, et al. A cluster analysis of bacterial vaginosis-associated microflora and pelvic inflammatory disease. Am J Epidemiol 2005;162(6):585-90.
13. Peipert JF, Montagno AB, Cooper AS, et al. Bacterial vaginosis as a risk factor for upper genital tract infection. Am J Obstet Gynaecol 1997;177(5):1184-7.
14. Krohn MA, Hillier SL, Eschenbach DA. Comparison of methods for diagnosing bacterial vaginosis among pregnant women. J Clin Microbiol 1989;27(6):1266-71.
15. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991;29(2):297-301.
16. Hellberg D, Nilsson S, Mardh PA. Bacterial vaginosis and smoking. Int J STD AIDS 2000;11(9):603-8.
17. Calzolari E, Masciangelo R, Milite V, et al. Bacterial vaginosis and contraceptive methods. Int J Gynaecol Obstet 2000;70(3):341-6.
18. Ness RB, Hillier SL, Richter HE, et al. Douching in relation to bacterial vaginosis, lactobacilli, and facultative bacteria in the vagina. Obstet Gynaecol 2002;100(4):765-72.
19. Baeten JM, Nyange PM, Richardson BA, et al. Hormonal contraception and risk for sexually transmitted disease acquisition: results from a prospective study. Am J Obstet Gynaecol 2001;185(2):380-5.
20. Hart G. Factors associated with trichomoniasis, candidiasis and bacterial vaginosis. Int J STD AIDS 1993;4(1):21-5.
21. Patel V, Weiss A, Mabey D, et al. The burden and determinants of reproductive tract infections in India: a population based study of women in Goa, India. Sex Transm Infect 2006;82(3):243-9.
22. MasandDL, PatelJ, GuptaS. Utility of microbiological profile of symptomatic vaginal discharge in rural women of reproductive age group. J Clin Diagn Res 2015;9(3):4-7.
23. Purwar M, Ughade S, Bhagat B, et al. Bacterial vaginosis in early pregnancy and adverse pregnancy outcome. J Obstet Gynaecol Res 2001;27(4):175-81.
24. Mathew R, Kalyani J, Bibi R, et al. Prevalence of bacterial vaginosis in antenatal women. Indian J Pathol Microbiol 2001;44(2):113-6.
25. Mahajan BK. Methods in biostatistics. New Delhi: Jaypee Brothers Medical Publishers Pvt Ltd 1997;6^th edn.
26. Verma A, Gupta A, Goel S, et al. Clinicopathological correlation of infective vaginal discharges in non pregnant sexually active women of reproductive age group in a tertiary care centre of  western UP. Int J Reprod Contracept Obstet Gynaecol 2013;2(3):349-54.
27. Garber GE. The laboratory diagnosis of trichomonas vaginalis. Can J Infect Dis Med Microbiol 2005;16(1):35-8.
28. Agarwal AK. Social classification: the need to update in the present scenario. Indian J Community Med 2008;33(1):50-1.
29. Kouman EH, Stenberg M, Bruce C, et al. The prevalence of bacterial vaginosis in the United States 2001-2004: associations with symptoms, sexual behaviour and reproductive health. Sex Transm Dis 2007;34(11):864-9.
30. Bradshaw CS, Morton AN, Garland SM, et al. Evaluation of point-of-care test, BV blue, and clinical and laboratory criteria for diagnosis of bacterial vaginosis. J Clin Microbiol 2005;43(3):1304-8.
31. Chaijareenont K, Sirimai K, Boriboonhirunsarn D, et al. Accuracy of nugent’s score and each of amsel criteria in the diagnosis of bacterial vaginosis. J Med Assoc Thai 2004;87(11):1270-4.
32. Sha BE, Zariffard MR, Wang QJ, et al. Female genital-tract HIV load correlates inversely with lactobacillus species but positively with bacterial vaginosis and mycoplasma hominis. J Infect Dis 2005;191(1):25-32.

33. Bhalla P, Chawla R, Garg S, et al. Prevalence of bacterial vaginosis among women in Delhi, India. Indian J Med Res 2007;125(2):167-72.
34. Modak T, Arora P, Agnes C, et al. Diagnosis of bacterial vaginosis in cases of abnormal vaginal discharge: comparison of clinical and microbiological criteria. J Infect Dev Ctries 2011;5(5):353-60.
35. Sha BE, Chen HY, Wang QJ, et al. Utility of amsel criteria, nugent score, and quantitative PCR for gardenella vaginalis, mycoplasma hominis, and lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women. J Clin Microbiol 2005;43(9):4607-12.
36. Shayo PA, Kihunrwa A, Massinde AN, et al. Prevalence of bacterial vaginosis and associated factors among pregnant woman attending at Bugando medical centre, Mwanza, Tanzania. Tanzan J Health Res 2012;14(3):175-82.
37. Om HS, Singh A, Dhole TN, et al. Factors associated to bacterial vaginosis in non-pregnant women of north Indian population. J Biotechnol Biomater 2015;5:3. doi:http://dx.doi.org/10.4172/2155-92X.1000195.
38. Nigeen W, Bhat AS, Gulzar K, et al. Correlation of bacterial vaginosis with preterm labour: a case control study. Int J Reprod Contracept Obstet Gynaecol 2015;4(6):1868-74.
39. Ganjoei TA. Risk factors for bacterial vaginosis in women attending a hospital in Kerman, Islamic Republic of Iran. La Revue de Sante de La Mediterrance Orientale 2005;11(3):410-5.
40. Joesoef MR, Karundeng A, Runtupalit, et al. High rate of bacterial vaginosis among woman with intrauterine devices in Monado, Indonesia. Contraception 2001;64(3):169-72.
41. Ocak S, Cetin M, Hakverdi S, et al. Effects of intrauterine device and oral contraceptive on vaginal flora and epithelium. Saudi Med J 2007;28(5):727-31.
42. Madden T, Grentzer JM, Secura GM, et al. Risk of bacterial vaginosis in users of the intrauterine device: a longitudinal study. Sex Transm Dis 2012;39(3):217-22.
43. Hutchinson KB, Kip KE, Ness RB. Condom use and its association with bacterial vaginosis and bacterial vaginosis associated vaginal microflora. Epidemiology 2007;18(6):702-8.
44. Ma L, Lv Z, Su J, et al. Consistent condom use increases the colonization of lactobacillus crispatus in the vagina. PLoS One 2013;8(7):e70716. http://doi.org/10.1371/journal.pone.0070716.
45. Hutchinson KB, Kip KE, Ness RB. Vaginal douching and development of bacterial vaginosis among women with normal and abnormal vaginal microflora. Sex Transm Dis 2007;34(9):671-5.
46. Hassan WM, Lavreys L, Chohan V, et al. Associations between intravaginal practices and bacterial vaginosis in Kenyan female sex workers without symptoms of vaginal infections. Sex Transm Dis 2007;34(6):384-8.