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Year : 2016 Month : July Volume : 5 Issue : 60 Page : 4148-4153

CLINICAL CORRELATION AND LABORATORY DIAGNOSIS OF BACTERIAL VAGINOSIS.

Kirti Malpekar1, Kumar Vivek2, Jayanthi Shastri3

1Associate Professor, Department of Microbiology, Topiwala National Medical College, Nair Hospital, Mumbai.
2Senior Registrar, Department of Microbiology, Topiwala National Medical College, Nair Hospital, Mumbai.
3Professor and HOD, Department of Microbiology, Topiwala National Medical College, Nair Hospital, Mumbai.

CORRESPONDING AUTHOR

Dr. Kirti Malpekar,
Email : kirtimalpekar@yahoo.co.in

ABSTRACT

Corresponding Author:
Dr. Kirti Malpekar,
#404, Audumber So,
3rdAkkalkot Lane,
Khadilkar Road,
Girgaum, Mumbai-400004.
E-mail: kirtimalpekar@yahoo.co.in

ABSTRACT

Bacterial vaginosis characterised by a heavy overgrowth of gram-negative and gram-positive anaerobes with no signs of inflammation has been regarded as a microbiological and immunological enigma.

AIMS AND OBJECTIVES

1. To identify the causative organisms from clinically suspected cases of bacterial vaginosis. 2. To evaluate the accuracy of Amsel’s clinical criteria and Nugent’s Gram stain criteria for diagnosis of bacterial vaginosis. 3. To correlate with other associated sexually transmitted infections.

MATERIAL AND METHODS

This prospective study involving total 600 non-pregnant women with abnormal vaginal discharge, clinically suspected cases of bacterial vaginosis (BV) attending the Gynaecology OPD at a tertiary care hospital was done over a period of one year. The following samples were collected from each subject: Three vaginal swabs and one blood sample (5 mL). Three vaginal swabs were taken and immediately sent to the microbiology laboratory for processing, by microscopy and culture on appropriate media. For serological diagnosis of Hepatitis B and C, HIV, and VDRL testing, 5 mL blood was collected from cubital vein with aseptic precautions. The following parameters were noted - age, marital status, contraceptive use, presence of abnormal, recurrent, and/or foul-smelling vaginal discharge and clue cells. BV was diagnosed using Amsel’s clinical criteria and Nugent’s Gram stain criteria. Data was analysed using SPSS version 13, Fischer’s exact test, and chi-square test.

RESULTS

BV occurred in 142/600 (23.7%) women with abnormal vaginal discharge, 55/135 (40.7%) were in age group 36-45 yrs., 140/593 (23.6%) married women and in 92/259 (35.5%) women with recurrent vaginal discharge. Abnormal discharge was most commonly seen (193/600) women who had not used any contraceptives. Foul-smelling discharge was more in BV patients as compared to pain, itching, and burning micturition, which was more common in non-bacterial vaginosis (NBV) and had statistically significant p value (<0.05). Clue cells were seen in total 19/142 (13.3%) patients suffering from BV. Based on Nugent’s score, the 600 cases were classified as normal (223), intermediate (161), and BV (142). Based on aetiology, it was noted that mixed infection occurred in 42/600 cases: Vulvovaginal candidiasis (VVC) + intermediate in 24, BV+VVC in 16, and BV+Trichomoniasis in 2. Majority of anaerobes, i.e. 100/136 (73.5%) were found in BV patients. This association of anaerobes in BV was found to be significant with p-value (<0.05). Yeast was grown in 105/600 (17.5%); 16/105 (11.3%) were significantly associated with BV (p<0.05).

CONCLUSION

The Gram stain as interpreted by Nugent’s criteria provides an objective, reproducible laboratory based test, and should be used in addition to clinical criteria for diagnosis of BV.

KEYWORDS

Bacterial Vaginosis (BV).

INTRODUCTION

Bacterial vaginosis is characterised by a heavy overgrowth of gram-negative and gram-positive anaerobes with no signs of inflammation and regarded as a microbiological and immunological enigma.1 It occurs in up to 25% of the general population with more than half of the women being

MATERIAL AND METHODS

This prospective study involving total 600 non-pregnant women with abnormal vaginal discharge attending the Gynaecology OPD at a tertiary care hospital was done over a period of one year.

Inclusion Criteria

Young non-pregnant sexually active females with excessive vaginal discharge in the reproductive age group of 15-45 years.

Exclusion Criteria

Age below 15 years, age older than 45 years, pregnant, menstruating, history of antibiotics, and/or topical vaginal creams within seven days prior to the date of examination.

The Institutional Ethics Committee approval was taken. Informed consent of the participants was obtained. Participants were asked about their symptoms, past illness, and previous treatment before undergoing gynaecological examination. In the gynaecological OPD, after assuring the patient, a clean unlubricated Cusco’s vaginal speculum was passed into the vagina to examine the condition of the vaginal wall, cervix, and characteristics of the discharge [with respect to amount, odour, and type of discharge, which was described as normal (Mucoid or floccular), purulent, curdy, or thin and homogenous. The following samples were collected from each subject: Three vaginal swabs and one blood sample (5 mL). The vaginal sample was collected by swabbing the posterior and lateral vaginal fornices with a cotton-tipped sterile swab. Three vaginal swabs were taken and immediately sent to the microbiology laboratory for processing. After aseptic precautions, 5 mL blood was collected from cubital vein, for serological diagnosis of Hepatitis B and C and the patient was directed to ICTC Centre for HIV and VDRL testing. In the laboratory, the processing was as follows: For the 3 vaginal swabs: 1. First swab was immediately processed by inoculating on Brucella blood agar with Hemin and Vitamin K1 supplement for anaerobes,13 Human Blood Bilayer (HBT) agar for detection of Gardnerella vaginalis, New York City agar for Neisseria gonorrhoeae, and Sabouraud dextrose agar for Candida. 2. A second swab was used to inoculate a tube of thioglycollate broth by gently introducing the swab into the lower half of the tube and rubbing it against the wall of the tube. The swab was subsequently mixed with two drops of sterile saline on a clean glass slide and a coverslip was placed over it. This wet mount was immediately examined using bright field microscopy under high power objective (x40) for clue cells and jerking motility of Trichomonas vaginalis. 3. Third swab was used for smear, pH test, whiff test, and KOH mount the swab was then mixed with 2 drops of 10% potassium hydroxide on a slide and immediately held close to nose to detect the fishy odour associated with volatile amines (Whiff amine test). A diagnosis of BV was made using Amsel’s clinical criteria5, if three of the following four criteria were present: A thin, homogenous discharge with milk-like consistency tending to adhere to the vaginal vault, Vaginal pH >4.5, positive whiff amine test, and presence of clue cells. For diagnosis by Nugent’s Gram stain criteria: Morphotypes were scored as the average number seen per oil immersion field. A total of 100 fields were examined for each slide. Total score=Lactobacillus+Gardnerella and Bacteroides spp. + curved rods.14 Women who had a score of 7 or higher were considered to have BV, score of 4-6 were termed as intermediate vaginal flora and 0-3 as normal flora. For detection of yeast, Gram stain was screened for the presence of gram-positive budding yeast cells, and pseudohyphae. The presence of intracellular gram-negative diplococci within the polymorphonuclear leucocytes was presumptively diagnosed as Neisseria gonorrhoeae. For culture identification of anaerobes, Brucella blood agar with Hemin and Vitamin K1 supplement was incubated for 48 hours and examined for growth. The plates were kept for 7 days before final examination and then discarded. The thioglycollate broth was kept for 7 days at 370 C. If no anaerobes were isolated from the primary plates after 7 days incubation, then the broth was subcultured onto Brucella blood agar with Hemin and Vitamin K1 supplement plates. For identification of anaerobes: Each distinct anaerobe colony was examined and its morphology noted. A portion of the colony was Gram stained and inoculated onto the following media: 1) Chocolate agar plate incubated in a candle jar for 48 hours to test the isolate for aerotolerance. 2) Brucella blood agar with Hemin and Vitamin K1 supplement for the antibiotic identification test. 1 mg kanamycin disc, 5 µg vancomycin disc, and 10 µg Colistin disc were placed well separated from each other. The plate was incubated anaerobically for 48 hours at 370C. A zone diameter of ≤10 mm indicated resistance while a zone diameter >10 mm indicated sensitivity. For Gardnerella vaginalis: Human Blood Bilayer with Tween (HBT) agar with selective supplement (From HiMedia) and New York City (NYC) agar with NYC supplement (From HiMedia) were inoculated. Sodium polyanethole sulfonate (SPS) disc indicator was used. Both HBT and NYC agar plates were incubated at 370 C for 48 hrs. and 72 hrs. respectively in a CO2 enriched humid atmosphere achieved by using candle jar. The blood sample was kept in room temperature for a minimum 1 hr and the serum was used for the following tests: ELISA for anti-HCV detection (3rd generation) (SD Bioline) and ELISA for HBsAg detection (Sun Pharma).

RESULTS

In this prospective study involving a total of 600 non-pregnant women with abnormal vaginal discharge, 142/600 (23.7%) had BV. The distribution of cases based on Nugent’s score was as follows: normal group: 223 followed by intermediate: 161 and BV: 142 (Table 1). Based on aetiology, it was seen that mixed infection occurred in 42/600 cases: Vulvovaginal candidiasis (VVC)+Intermediate in 24, BV+VVC in 16 and BV+ Trichomoniasis in 2 (Table 2). BV was commonly seen in 36-45 yrs. age group: 55/135(40.7%), which was statistically significant p value (<0.05). Married women comprised 593/600(98.8%); BV occurred in 140/593(23.6%) of them. Recurrent vaginal discharge occurred in 259/600 of which 92(35.5%) were associated with BV. This association was significant with Pearson chi-square (χ2) 35.405, continuity correction 34.305, df-1 and p-value of 4.71E-09. Abnormal discharge was most commonly seen (193/600) in women who had not used any contraceptives. Majority of the women with BV were sterilised with TL (55/142); very few used OC pills (1/142), condom (10/142), and other methods (10/142) such as diaphragm, sponge, spermicidal jelly/foam, etc. Foul smelling discharge was more in BV patients as compared to pain, itching, and burning micturition, which was more common in NBV, and was statistically significant p value  (<0.05 ). Clue cells were seen in a total of 19/142 (13.3%) BV patients of which majority i.e. 13/19 had Nugent’s score of 8 followed by 4/19 with Nugent’s score of 7 and one each with Nugent’s score of 9 and 10. Amsel’s criteria were able to detect BV in 167/600 (27.8%) [Table 3]. Of these, Nugent’s Gram stain criteria was negative in 50. On the contrary, 25 patients who did not satisfy the Amsel’s clinical criteria were positive by Nugent’s Gram stain. The inter-rater agreement statistic (Kappa) was determined between the Amsel’s criteria and Nugent’s score (Kappa=0.674). Majority of anaerobes i.e. 100/136 (73.5%) were found in BV patients. This association of anaerobes in BV was found to be significant with p-value (<0.05). Curved gram-negative rods suggestive of Mobiluncus spp. were most commonly seen microscopically in BV cases (30.3%) followed by Peptostreptococcus spp. (27.5%) and Bacteroides spp. (21.8%). Gardnerella vaginalis was not isolated. Yeast was grown in 105/600 (17.5%), 16/105 (11.3%) were significantly associated with BV (p<0.05). In our study, HIV infection was seen in 0.7%, HBV infection occurred in 0.3%, and HCV occurred in 0.2%.

(In the present study, BV was diagnosed in 23.7% based on Nugent’s criteria).

*N=Non-pregnant; P=Pregnant; SAW=Sexually active women.

 

Diagnosis (3 Groups)

No.

Percentage

BV

142

23.7%

Intermediate

161

26.8%

Normal

297

49.5%

Total

600

100.0%

Table 1: Distribution Based on Nugent’s Score Criteria*

 

*Nugent’s score: BV 7-10; Intermediate-4-6; Normal 0-3.

 

For diagnosis by Nugent’s Gram stain criteria14: Morphotypes were scored as the average number seen per oil immersion field. A total of 100 fields were examined for each slide. Total score = Lactobacillus + Gardnerella and Bacteroides spp.+curved rods. Women who had a score of 7 or higher were considered to have BV, score of 4 - 6 were termed as intermediate vaginal flora, and 0-3 as normal flora.

Diagnosis

No.

Percentage

Intermediate*

137

22.8%

BV (Bacterial Vaginosis )*

124

20.7%

VVC (Vulvovaginal candidiasis)

65

10.8%

VVC+Intermediate

24

4.0%

BV+VVC

16

2.7%

Trichomoniasis

7

1.2%

BV+Trichomoniasis

2

0.3%

Gonorrhea

2

0.3%

Normal*

223

37.2%

Total

600

100.0%

Table 2: Distribution of Cases of RTIs

(Reproductive Tract Infections)

 

 


 

 

 

Clinical Sign

BV

NBV

Total

Chi-Square Tests

Value

Df

p-Value

Association is-

Thin Grey Homogenous Discharge

No.

101

119

220

Pearson Chi-Square

95.127

1

1.79E-22

Significant

%

71.1%

26.0%

36.7%

Continuity Correction

93.193

1

4.74E-22

Significant

pH >4.5

No.

126

121

247

Pearson Chi-Square

173.777

1

1.11E-39

Significant

%

88.7%

26.4%

41.2%

Continuity Correction

171.214

1

4.02E-39

Significant

Whiff Test

No.

124

71

195

Pearson Chi-Square

254.875

1

2.25E-57

Significant

%

87.3%

15.5%

32.5%

Continuity Correction

251.612

1

1.16E-56

Significant

Clue Cells

No.

19

0

19

Pearson Chi-Square

63.286

1

1.79E-15

Significant

%

13.4%

0.0%

3.2%

Continuity Correction

58.997

1

1.58E-14

Significant

Table 3: Based on Amsel’s Clinical Criteria: (Cases With and Without BV)

 

DISCUSSION

In our study involving 600 non-pregnant women with abnormal vaginal discharge, BV was diagnosed in 23.7% (Based on Nugent’s criteria). Among the Indian studies, Adamson et al15 Modak et al16 Uma et al17 and Kosambiya et al18 reported similar results (Fig. 1). Higher rate have been reported by Becker et al19 Thulkar et al20 and Aggrawal et al21 Slightly lower rate was reported by Indu et al22 Madhivanan et al4 Patel et al23 whereas very low infection was noted by Mania et al24 Shethwala et al25 and Ray et al26 Highest number (68%) was noted in a study by Aggrawal et al21 where Amsel’s clinical criteria was used as standard. Also, a meta-analysis by Gillet et al27 showed a higher rate (32%). In our study, Vulvovaginal Candidiasis (VVC) was noted in 14.8%; similar findings were reported 14% by Kosambiya et al18 in Surat, India and 15.7% River et al28 in USA. Higher rate of VVC, 17.4% was noted by Esim et al29 and Xiao et al30 (21.87%) in China. Indu et al22 reported 9%, which was low. In our study, 2.27% cases had mixed infection of BV/VVC, which was close to 4.4% reported by River et al28 3% by Indu et al22 in North India and 4.2% by Mania et al24 in Mumbai; slightly less (1.36%) was found by Xiao et al.30 In our study, Trichomoniasis was seen in 9/600 (1.5%) and mixed infection of BV/Trichomoniasis in 2/600 (0.3%). Similar reports from India are: 1.4% by Sunita et al31 1.2% by Patel et al23 1.18% by Rao et al32 1.2% by Ray et al.26 Reports by Shethvala et al25 (2%), Bhalla et al33 2.8%, Thulkar et al20 6.74%, Adamson et al 8.5%,15 Bogearts et al 2%,34 and Kosambiya et al 22%18 showed higher number of cases. Studies from China reported lower infection 1.7% Youngin et al35 and 0.67% Liu et al.36 In the present study, majority of women with abnormal discharge 240/600 (40%) belonged to the 26-35 yrs. age group among them BV occurred in 66/240 (27.5%). Older age group (36-45 yrs.) comprised 135/600 (22.5%) and BV occurred in 55/135 (40.7%) of them. Sumati et al37 noted that 60.11% patients were between 26 to 40 years of age and BV occurred in 52% in this age group. The mean SD (Standard deviation) and median age (Yrs.) of all cases are 29.59, 6.97, and 29 respectively whereas in BV it was 32.46, 6.10, and 33 respectively. This difference was significant with p-value 1.59 E-09 (<0.05). Modak et al16 found the mean SD and median of overall cases to be 30.7, 10.46, and 30 almost similar to us; however, in BV cases these were 28.33, 7.90, and 29.5 respectively, which is less compared to us. In our study, married women were 593/600 (98.8%) and 7 were widowed, which is similar to Madhivanan et al4 and Modak et al16 Abnormal discharge was higher among women who had not used any contraceptives (193/600). Maximum women associated with BV had undergone sterilization with TL (55/142). Patel et al23 reported similar findings. We noted that abdominal pain, itching, and burning micturition, all three inflammatory signs were less in BV compared to NBV, which was significant. In present study, 36.62% of BV patients had abdominal pain whereas it was higher (58.52%) in NBV patients. Bhalla et al33 reported abdominal pain in 24.3% BV patient and Patel et al23 in only 18.6%. Itching was noted in 247/600 (41.2%) of total patients and 17/142 (11.97%) of BV cases. Kosambiya et al18 reported itching in 10/42 (23.8%) patients with discharge and Patel et al23 in 19.3% of BV cases. In present study, burning micturition was seen in 241/600 and 30/241 (12.4%) had BV, which was lower than 20.4% by Patel et al23 In our study, 88.03% BV cases had foul-smelling discharge compared to 18.12% in NBV, which was similar to 68.6% and 100% by Figueiredo et al38 and Hapsari et al39 in BV. The present study showed that grey thin homogenous discharge was more common in BV patients (71.12%) similar to 84% seen by Aggrawal et al21 Grey thin homogenous discharge had sensitivity, specificity, positive, and negative predictive values of 71.13%, 74.02%, 45.91%, and 89.21% respectively similar to Modak et al16 (66.67%, 71.05%, 42%, and 87% respectively). Whiff test in present study had sensitivity, specificity, positive and negative predictive value of 87.32%, 84.50%, 63.59%, and 95.56% (Table 3). Modak et al16 findings have shown sensitivity, specificity, positive, and negative predictive value of 41.67%, 100%, 100%, and 84% respectively; sensitivity being lower. Aggrawal et al21 reported sensitivity of 68% while Hainer et al40 observed a sensitivity and specificity of 77% and 93% respectively. Clue cells found in our study was 19/600 (3.2%) similar to 1.07% by Sunita et al.31 The presence of clue cells was the most specific of all the criteria (Specificity=100%). It also had the highest predictive value of a positive test (100%). But, presence of clue cells was not found to be very sensitive (Sensitivity=13.38%) and gave a large number of false negative cases (86.6%) with negative predictive value of 78.82%. In our study, pH >4.5 had sensitivity, specificity, positive, and negative predictive values of 88.73%, 73.58%, 51.01%, and 95.47% respectively. Modak et al16 noted similar findings: 83.33%, 86.84%, 67%, and 94% respectively. 88% of sensitivity was also observed by Aggrawal et al21 Ultimately, pH seemed to be the best indicator of bacterial vaginosis, if all sensitivity, specificity, positive, and negative predictive values are taken into consideration. It was found to be most sensitive and had the best predictive value of a negative test. Furthermore, it is the one, which could be objectively measured at the bedside. In this study, the Amsel’s criteria had a sensitivity of 82.39%, specificity of 89.08%, positive and negative predictive values of 70.06% and 94.23% respectively when compared to the Nugent’s criteria. Modak et al16 also found sensitivity, specificity, positive, and negative predictive values 66.67%, 94.47%, 80%, and 90% respectively. Similar parameters were reported by Schwebke et al41 with 70.40%, 94.40%, 89%, and 83.10% respectively. Gallo et al42 found the sensitivity and specificity of Amsel’s criteria as 60% and 90% respectively; sensitivity being less compared to present study. Schwebke et al41 compared Amsel’s clinical criteria with Nugent’s criteria and showed that the Nugent criteria had a higher sensitivity of 89% and Amsel’s criteria had a higher specificity of 94%. It is suggested by many reports to consider Nugent’s score as standard criteria as the comparatively low sensitivity of Amsel’s criteria results in the decrease of true positive cases causing ineffective treatment.16,24,20,15,43,44 Pus cells were seen in 32/142(22.5%) of BV patients compared to 104/161(64.6%) of intermediate and 108/297(50.2%) of normal group suggestive of non-inflammatory characteristic of BV. Sachdeva et al45 also observed that “vaginal discharge of patients with BV is notable for its lack of (Polymorphs) PMNs, typically 1 or less than 1 PMN per vaginal epithelial cell.” Distribution of anaerobes was as follows: curved gram-negative rods suggestive of Mobiluncus spp. were most commonly seen microscopically in BV cases (30.3%) followed by Peptostreptococcus spp. (27.5%) and Bacteroides spp. (21.8%). Evidence of association of anaerobic bacteria with BV is mounting. Aggrawal et al21 reported Peptostreptococcus spp. (53.30%) as most common followed by Bacteroides spp. (16.7%). Rao et al32 found Peptostreptococcus spp. and Prevotella spp. to be common among the anaerobes isolated. Sumati et al37 found Bacteroides spp. to be more common followed by Peptococcus spp. Curved gram-negative rods were also noted by Rao et al in 8.45% BV cases. In present study, Gardnerella vaginalis was not isolated. This may be due to inadvertent error in transport or inhibition by high concentration of NACl in the Columbia base agar used in the culture medium (Catnil et al)9 Various studies have reported isolation of Gardnerella vaginalis ranging from 10.2% Esin et al29 7.32% Rao et al32 28% Khan et al46 to as high as 96.8% Figueiredo et al38 Among the STIs in our study, gonorrhea occurred in 0.3%, which was less compared to 0.5% of Bogaerts et al34 1.9% of Patel et al23 1% of Bhalla et al33 1.7% of Youngin et al35 15.8% of Wang et al47 and 16.11% of Liu et al.36 In our study, HIV infection was seen in 0.7%, which was less than 0.87% of Sunita et al31 11.6% of Shethwala et al25 and 0.95% of Bhalla et al.33 In the present study, HBV infection occurred in 0.3%, which was low compared to 3.33% by Shethwala et al25 (India), 0.9% by Wang et al47 (China), 0.67% by Liu et al114 (China) and highest 35% reported by Bogaerts et al.34 In our study, HCV occurred in 0.2%, which was low compared to 0.5% by Wang et al47 (China), 0.67% by Liu et al36 (China), and 0.9% reported by Bogaerts et al34 The limitation of the study was that we could not include Herpes and Chlamydia due to lack of funds. Further studies are required especially for detection of HBV and HCV infection in BV.

 

CONCLUSION

India has a high burden of reproductive morbidity and bacterial vaginosis has been documented as a risk factor for both adverse birth outcomes and HIV. Proper diagnosis of BV is challenging. It is often misdiagnosed using clinical criteria alone because the components are subjective. Many studies have suggested that the Gram stain be considered the gold standard for diagnosis of BV. Recently, although, newer diagnostic molecular methods have been devised, Nugent’s and Amsel methods remain the most practical, viable, and economic option especially in developing countries.

ACKNOWLEDGEMENT

We acknowledge the cooperation of the faculty and staff of Obstetrics and Gynaecology and Microbiology Department, TN Medical College and BYL Nair Hospital, Mumbai.

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