Shilpa Kerkar

Bacterial vaginosis: a cause of infertility?

Bacterial vaginosis (BV) is a common disorder of the genital tract in women characterized by an alteration of the normal acidic lactobacilli-predominant vaginal ecosystem to a vaginal environment dominated by Gardnerella vaginalis, mycoplasma species and anaerobes, with an increase in pH. The present study evaluated whether BV is associated with reproductive complications in women. BV was screened with a Gram stain of vaginal smear and interpretation was done using the Nugent score. Wet mount and polymerase chain reaction were used to screen other infections. Among 510 enrolled women, 72 (14.1%) had BV. Statistical analysis between the BV negative and positive population revealed a significant association (P = 0.0001) with infertility. In pregnant women, the infection rate was low (P = 0.01). Multiple infections such as Candida, Chlamydia and human papilloma virus were observed in 4.2%, 15.3% and 8.3% of BV-infected women, respectively. Results suggest that BV infection is associated with infertility and its absence leads to pregnancy, emphasizing its screening and treatment.

Use of vaginal pH in diagnosis of infections and its association with reproductive manifestations.

Increase in vaginal secretion pH is an indicator of bacterial vaginosis (BV), but is yet to be in use as a diagnostic tool by clinicians. Similarly, no reports are available on the effect of cervical chlamydia infection and different reproductive manifestations on vaginal secretion pH. This study evaluated the use of vaginal pH for screening of BV, the effect of Chlamydia trachomatis (C. trachomatis) infection, and different reproductive manifestations on vaginal pH of women attending the gynecology outpatient department of a general hospital. Vaginal pH was recorded while diagnosing infections in 358 women, among which 45 were with repeated spontaneous abortion, 79 with infertility, 185 had sign and symptoms of lower genital tract infection, and 49 had no history or symptom of any complications or infections. Normal vaginal pH, BV, and C. trachomatis infection were observed in 72.6, 21.5, and 10.1% of women, respectively. BV and C. trachomatis were observed in 78.6 and 4.1% of women, respectively, with high vaginal pH; 12.3% of women with normal vaginal pH had C. trachomatis infection. C. trachomatis infection or different reproductive manifestations do not lead to change in vaginal pH but high vaginal pH correlated with BV and should be used as a simple tool for its diagnosis. J. Clin. Lab. Anal. 22:375–379, 2008.

Human Papillomavirus infection in asymptomatic population

OBJECTIVES The present study aimed to evaluate type specific Human Papillomavirus (HPV) strains in women with different clinical manifestations but with normal cervical cytology, attending a gynecology out patient clinic and HPV infection in males attending a private pathology laboratory for routine check up in Mumbai. METHODS Cervical swab specimens from 470 women with normal cervical cytology as detected by Pap were used for detection and typing of HPV by PCR, southern blotting and sequencing. In 104 males, 30 ml of first void/random urine specimens were used for HPV screening. RESULTS Thirty-eight women (8.1%) tested positive for HPV. HPV 16, 18, 6, 11 and mixed infection was observed in 26.3%, 10.5%, 36.8%, 5.2% and 15.8% of these infected women, respectively, while 36.8% had other HPV types, indicating high rate of high-risk HPV types 16/18. Among the 104 males, 12 (11.5%) had HPV infection, 50% (n=6) of them were below 30 years. Nine of them were married and three were unmarried. CONCLUSIONS The present study revealed presence of high risk HPV infection in women with normal cervical cytology. This is the first report from the Western region of India on HPV infection in males using urine specimen.

CTLA-4 gene polymorphism at position +49 A>G in exon 1: a risk factor for cervical cancer in Indian women

Single nucleotide polymorphisms (SNPs) in the CTLA-4 gene exert differential effects on T-cell response to viral infection. We aimed to evaluate the association of two SNPs of the CTLA-4 gene with cervical cancer in Indian women. The two polymorphic loci, one in the promoter region -318 C>T, rs5742909 (100 cervical cancer cases and 101 controls) and the other in exon 1 +49 A>G, rs231775 (104 cervical cancer cases and 162 controls) were genotyped using polymerase chain reaction-restriction fragment length polymorphism methods. Haplotype block structure was determined using Haploview 4.2. The statistical analyses were performed using a commercially available statistical software package, whereas PyPop was used to calculate the haplotypic frequencies. In this case-control study, the A/A genotype frequency (30.76% vs. 17.6%, P = 0.01) as well as the allelic frequency for A (52.8% vs. 43.5%, P = 0.04) was significantly higher in cases compared to controls. No significant association was seen in the -318 C>T polymorphism. In forward stepwise binary logistic regression analysis considering age and parity as potential confounders, significant association was demonstrated between +49 A/A and cervical cancer. Most likely, this is the first study from India to highlight the significant association between the CTLA-4 gene +49 A/A SNP and cervical cancer, thus adding to the global knowledge of the association of this SNP with cervical cancer.

Cervical cancer in Indian women reveals contrasting association among common sub-family of HLA class I alleles.

We studied the relationship between human leukocyte antigen (HLA) class I alleles and cervical cancer among Indian women. Seventy-five cervical cancer cases were compared with 175 noncancer controls. Cervical biopsy tissue specimen from cancer cases and cervical swab specimen from controls were collected for HPV detection and typing. Blood was taken for HLA typing by PCR-SSOP method. The impact of HLA class I alleles on cervical cancer risk was evaluated using StatCalc program (Epi Info version 6.0.4. CDC Atlanta, GA, USA), and confirmed with Bonferroni correction. Results revealed HLA-B*37, HLA-B*58 were associated significantly with increased risk while HLA-B*40 with decreased risk for cervical cancer. At high-resolution analysis after Bonferroni correction, HLA-B*37:01 allele was associated with increased risk, whereas HLA-B*40:06 was with decreased risk for cervical cancer. HLA-B*37:01 and HLA-B*40:06 belong to the same superfamily of HLA-B44. In silico analysis revealed different binding affinities of HLA-B*37:01 and HLA-B*40:06 for the epitopes predicted for E6 and L1 proteins of HPV16. The higher binding affinity of epitopes to B*40:06, as revealed by docking studies, supports the hypothesis that this allele is able to present the antigenic peptides more efficiently than B*37:01 and thereby can protect the carriers from the risk of cervical cancer. Thus, there is a clear indication that HLA plays an important role in the development of cervical cancer in HPV-infected women. Identification of these factors in high-risk HPV-infected women may help in reducing the cervical cancer burden in India.

Human leukocyte antigen B distribution in HIV discordant cohort from India.

Limited reports are available on association of HLA-B with HIV infection from India, a home to the third largest population of HIV infected people in the world. This emphasizes the need to have more information specifically the genetic constitution of HIV serodiscordant couples (DCs), where one spouse is seropositive (HSP) while the other remains seronegative (HSN) even after repeated exposure. Hence, aim of this study was to document association of HLA-B with HIV infection in DCs living in Mumbai, India. A cohort was designed to enroll DCs attending the ICTC/Shakti Clinic of KEM Hospital, Mumbai. A group of unexposed volunteers were also enrolled as healthy controls (HC). HLA-B alleles were typed using sequence-specific oligonucleotide probes. Allele frequency comparison was done using 2×2 contingency tables. Results were considered significant, when p<0.05 with two-tailed Fishers exact test. At HLA-B locus, the frequencies of HLA-B*40;-B*35;-B*07;-B*15;-B*51;-B*44;-B*52;-B*37 and -B*57 were found in decreasing order in the population. Frequency of HLA-B*35 allele was significantly higher (HSP vs HSN; p<0.02 and HSP vs HC; p<0.04) in HSP. HLA-B*40 (HSN vs HSP; p<0.01 and HC vs HSP; p<0.01) and HLA-B*18 (HSN vs HSP; p<0.02) were significantly associated with HSN. Both HSN and HC had similar HLA-B*35 and -B*40 allele frequency. HLA-B*57 allele was observed in 15 individuals (3.69%). However, HLA-B*57:01 which is known to be associated with adverse reactions against Abacavir was observed in 7 of them. HLA-B*39 was observed exclusively in HSP. Our observation in DCs confirmed the association of HLA-B*35 with susceptibility while HLA-B*40 (specifically *B40:06), -B*18 with protection. These identified alleles can be used as possible marker associated with HIV transmission. In India, HLA screening is not carried out before initiation of HIV treatment. However, the presence of HLA-B*57:01 in the population emphasizes the importance of such screening to predict/avoid Abacavir hypersensitivity.

HPV16 E6 variants: Frequency, association with HPV types and in silico analysis of the identified novel variants

High‐risk human papillomavirus (HPV) types, specifically HPV 16 E6 variants are involved in viral persistence and the development of cervical lesions. India contributes to 1/3rd of the global cervical cancer deaths; however, information on E6 variants in the Indian population is limited. Information on these variants is essential for successful implementation of cervical cancer immunization programs. The E6 variants and their possible biological implications to the outcome of infection were studied in women attending the Tata Memorial Hospital, Mumbai, India. Cervical cancer patients with HPV 16 as a single infection (n = 33), co‐infection with another HPV type (n = 20) or with multiple types (n = 10) were examined for HPV16 E6 variants using PCR and sequence analysis. The variants were identified using the prototype sequence (HPV 16R) belonging to the European lineage. The results revealed that the European T350G was the most common variant (50%) followed by the European prototype (40.3%) and the North‐American (N = 3; 4.8%). The European prototype was significantly more frequent in patients infected with HPV16 alone (P < 0.05, C.I. 1.2–13.6), while the European T350G variants were seen in women with co‐infections. The North‐American lineage was found in women infected with HPV16 and 33. Three novel variants were identified of which two were non‐synonymous. Phylogenetic analysis revealed that the variant F69L + L83V is not related to any of these lineages, while the variant M137L + L83V is closely related to the North American variant. This study found a difference in the prevalence of E6 variants compared to earlier Indian studies and their association with type of infection. J. Med. Virol. 86:968–974, 2014.

Polymorphisms in major cytokine genes: a study among human immunodeficiency Virus-1 serodiscordant couples in Mumbai, India.

PURPOSE Polymorphism in cytokine genes may affect its production, which play an important role in modulation of human immunodeficiency virus (HIV) infection. Evaluation of these polymorphisms might help to understand why some individuals remain uninfected in spite of several exposures to HIV infection, such as the negative spouses of discordant couples. The aim of this study was to evaluate the association of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine genes and their receptors with HIV infection in serodiscordant couples, attending the Integrated Counselling and Testing Centre of a Municipality Hospital. MATERIALS AND METHODS At the end of at least 2 years of follow up, 42 couples were confirmed as being serodiscordant. Genotyping was carried out in blood samples of these couples using the polymerase chain reaction-sequence-specific amplification method. RESULTS Significantly high frequency of interleukin-1 receptor antagonist IL-1RA mspa 11100 CC (P=0.04), tumor necrosis factor-alpha TNF-α -238 AG (P=0.01) and IL-4 -33 TT (P=0.01) was observed in HIV seropositives (HSP) while frequency of TNF-α -238 GG (P=0.02) was significantly high among the exposed uninfected (EU). However, application of Bonferroni correction identified only two SNPs i.e., TNF-α -238 AG and IL-4 -33 TT to be significantly associated with the acquisition of HIV. In remaining cytokine genes, no significant association was observed. CONCLUSION Our study highlighted possible association of certain specific polymorphisms with HIV transmission, whereas presence or absence of certain other polymorphism in EU individuals might be offering protection from HIV infection. These variations at the genetic level might help to explore new insights into treatment and HIV prevention strategies.

Multi-centric validation of an in-house-developed beacon-based PCR diagnostic assay kit for Chlamydia and Neisseria and portable fluorescence detector

Objective. The development of an accurate, sensitive, specific, rapid, reproducible, stable‐at‐room‐temperature and cost‐effective diagnostic kit, and a low‐cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries. Methods. We developed the ‘Chlamy and Ness CT/NG kit’ based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi‐centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)‐based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 °C and the inter‐day reproducibility of results were also analysed. Results. The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the ‘inter‐rater’ &kgr;=0.976 for Chlamydia and &kgr;=0.943 for Neisseria. Conclusion. Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource‐limited settings of developing countries.