Detection of mutations in mtrR gene in quinolone resistant strains of N. gonorrhoeae isolated from India.

BACKGROUND AND OBJECTIVES: Emergence of multi-drug resistant Neisseria gonorrhoeae resulting from new genetic mutation is a serious threat in controlling gonorrhea. This study was undertaken to identify and characterise mutations in the mtrR genes in N.gonorrhoeae isolates resistant to six different antibiotics in the quinolone group. MATERIALS AND METHODS: The Minimum inhibitory concentrations (MIC) of five quinolones for 64 N.gonorrhoeae isolates isolated during Jan 2007-Jun 2009 were determined by E-test method. Mutations in MtrR loci were examined by deoxyribonucleic acid (DNA) sequencing. RESULTS: The proportion of N.gonorrhoeae strains resistant to anti-microbials was 98.4% for norfloxacin and ofloxacin, 96.8% for enoxacin and ciprofloxacin, 95.3% for lomefloxacin. Thirty-one (48.4%) strains showed mutation (single/multiple) in mtrR gene. Ten different mutations were observed and Gly-45 → Asp, Tyr-105 → His being the most common observed mutation. CONCLUSION: This is the first report from India on quinolone resistance mutations in MtrRCDE efflux system in N.gonorrhoeae. In conclusion, the high level of resistance to quinolone and single or multiple mutations in mtrR gene could limit the drug choices for gonorrhoea.

Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease.

PURPOSE: Genital Ulcer Disease (GUD) is common sexually transmitted infection (STI). Multiple studies have shown that GUDs are strongly associated with the transmission and the acquisition of HIV infection. An accurate diagnosis of common etiology of GUD namely Herpes, syphilis and Chancroid is possible using Multiplex PCR (M-PCR). However, frequent presence of Polymerase Chain Reaction inhibitors in the ulcer swab specimen limits the performance of the assay. In order to overcome this problem, alternative specimen preparation method was used. MATERIALS AND METHODS: To determine the common etiology, GUD specimens obtained under an STI operations research study were tested with M-PCR after the samples were prepared using Roche Amplicor specimen preparation kit. PCR inhibiting samples were identified from that, which showed negative results. These samples were subjected to phenol-chloroform extraction and ethanol precipitation before the conduct of M-PCR on them. RESULTS: Of the 237 GUD specimens tested, in 145 etiologies could be detected, whereas 92 samples were found negative. Further spiking with one of the target DNA, 128 of the negative samples were found to contain the inhibitors. These 126 samples were then subjected to phenol chloroform extraction and ethanol precipitation followed by M-PCR. Using this method for sample preparation, etiology could be determined in 46 (23%) additional samples. This success rate of altered sample preparation method has been lower than that has reported. CONCLUSION: The results indicate that sample preparation using phenol chloroform extraction and ethanol precipitation, prior to M-PCR helps to eliminate the inhibitors and increase the yield of the assay. However, being a laborious procedure, it may be used for samples giving negative results after the screening by Roche Amplicor specimen preparation kit.

Prevalence and assessment of clinical management of sexually transmitted infections among female sex workers in two cities of India.

OBJECTIVE: Control of sexually transmitted infections (STIs) among female sex workers (FSWs) is an important strategy to reduce HIV transmission. A study was conducted to determine the prevalence and assess the current clinical management of STIs in India. METHODS: FSWs attending three clinics for regular checkups or symptoms were screened for study eligibility. A behavioral questionnaire was administered, clinical examination performed, and laboratory samples collected. RESULTS: 417 study participants reported a mean number of 4.9 (SD 3.5) commercial clients in the last week. 14.6% reported anal sex in the last three months. Consistent condom use with commercial and regular partners was 70.1% and 17.5%, respectively. The prevalence of gonorrhea was 14.1%, chlamydia 16.1%, and trichomoniasis 31.1% with a third of all infections being asymptomatic. Syphilis seropositivity was 10.1%. CONCLUSIONS: At study sites, presumptive treatment for gonorrhea, chlamydia, and syphilis screening should continue. Presumptive treatment for trichomoniasis should be considered. Consistent condom use and partner treatment need to be reemphasized.

Clinical response of newly diagnosed HIV seropositive & seronegative pulmonary tuberculosis patients with the RNTCP Short Course regimen in Pune, India.

BACKGROUND & OBJECTIVES: In the Revised National Tuberculosis Control Programme (RNTCP) in India prior to 2005, TB patients were offered standard DOTS regimens without knowledge of HIV status. Consequently such patients did not receive anti-retroviral therapy (ART) and the influence of concomitant HIV infection on the outcome of anti-tuberculosis treatment remained undetermined. This study was conducted to determine the results of treatment of HIV seropositive pulmonary tuberculosis patients with the RNTCP (DOTS) regimens under the programme in comparison with HIV negative patients prior to the availability of free ART in India. METHODS: Between September 2000 and July 2006, 283 newly diagnosed pulmonary TB patients were enrolled in the study at the TB Outpatient Department at the Talera Hospital in the Pimpri Chinchwad Municipal Corporation area at Pune (Maharashtra): they included 121 HIV seropositive and 162 HIV seronegative patients. They were treated for tuberculosis as per the RNTCP in India. This study was predominantly conducted in the period before the free ART become available in Pune. RESULTS: At the end of 6 months of anti-TB treatment, 62 per cent of the HIV seropositive and 92 per cent of the HIV negative smear negative patients completed treatment and were asymptomatic; among smear positive patients, 70 per cent of the HIV-seropositive and 81 per cent of HIV seronegative pulmonary TB patients were cured. Considering the results in the smear positive and smear negative cases together, treatment success rates were substantially lower in HIV positive patients than in HIV negative patients, (66% vs 85%). Further, 29 per cent of HIV seropositive and 1 per cent of the HIV seronegative patients expired during treatment. During the entire period of 30 months, including 6 months of treatment and 24 months of follow up, 61 (51%) of 121 HIV positive patients died; correspondingly there were 6 (4%) deaths among HIV negative patients. INTERPRETATION & CONCLUSIONS: The HIV seropositive TB patients responded poorly to the RNTCP regimens as evidenced by lower success rates with chemotherapy and high mortality rates during treatment and follow up. There is a need to streamline the identification and management of HIV associated TB patients in the programme with provision of ART to achieve high cure rates for TB, reducing mortality rates and ensuring a better quality of life.

Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific and South East Asian regions, 2007-2008.

Long-term surveillance of antimicrobial resistance in Neisseria gonorrhoeae has been conducted in the World Health Organization (WHO) Western Pacific Region (WPR) to optimise antibiotic treatment of gonococcal disease since 1992. In 2007 and 2008, this Gonococcal Antimicrobial Surveillance Programme (GASP) was enhanced by the inclusion of data from the South East Asian Region (SEAR) and recruitment of additional centres within the WPR. Approximately 17,450 N. gonorrhoeae were examined for their susceptibility to one or more antibiotics used for the treatment of gonorrhoea by external quality controlled methods in 24 reporting centres in 20 countries and/or jurisdictions. A high proportion of penicillin and/or quinolone resistance was again detected amongst isolates tested in North Asia and the WHO SEAR, but much lower rates of penicillin resistance and little quinolone resistance was present in most of the Pacific Island countries. The proportion of gonococci reported as 'resistant', 'less susceptible' or 'non-susceptible' gonococci to the third-generation cephalosporin antibiotic ceftriaxone lay in a wide range, but no major changes were evident in cephalosporin minimal inhibitory concentration (MIC) patterns in 2007-2008. Altered cephalosporin susceptibility was associated with treatment failures following therapy with oral third-generation cephalosporins. There is a need for revision and clarification of some of the in vitro criteria that are currently used to categorise the clinical importance of gonococci with different ceftriaxone and oral cephalosporin MIC levels. The number of instances of spectinomycin resistance remained low. A high proportion of strains tested continued to exhibit a form of plasmid mediated high level resistance to tetracyclines. The continuing emergence and spread of antibiotic resistant gonococci in and from the WHO WPR and SEAR supports the need for gonococcal antimicrobial resistance surveillance programs such as GASP to be maintained and potentially expanded.

Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhoea by the level of immunesuppression.

BACKGROUND & OBJECTIVE: Enteric parasites are major cause of diarrhoea in HIV infected individuals. The present study was undertaken to detect enteric parasites in HIV infected patients with diarrhoea at different levels of immunity. METHODS: The study was carried out at National AIDS Research Institute, Pune, India, between March 2002 and March 2007 among consecutively enrolled 137 HIV infected patients presenting with diarrhoea. Stool samples were collected and examined for enteric parasites by microscopy and special staining methods. CD4 cell counts were estimated using the FACS count system. RESULTS: Intestinal parasitic pathogens were detected in 35 per cent patients, and the major pathogens included Cryptosporidium parvum (12%) the most common followed by Isospora belli (8%), Entamoeba histolytica/Enatmoeba dispar (7%), Microsporidia (1%) and Cyclospora (0.7%). In HIV infected patients with CD4 count < 200 cells/microl, C. parvum was the most commonly observed (54%) pathogen. Proportion of opportunistic pathogens in patients with CD4 count <200 cells/microl was significantly higher as compared with other two groups of patients with CD4 count >200-499 and >or= 500 cells/microl (P=0.001, P=0.016) respectively. INTERPRETATION & CONCLUSION: Parasitic infections were detected in 35 per cent HIV infected patients and low CD4 count was significantly associated with opportunistic infection. Detection of aetiologic pathogens might help clinicians decide appropriate management strategies.

Expanded safety study of Praneem polyherbal vaginal tablet among HIV-uninfected women in Pune, India: a phase II clinical trial report.

BACKGROUND: Praneem, a candidate vaginal microbicide with reported anti-HIV and contraceptive properties was evaluated for its long-term safety through a double-blind, placebo-controlled, randomised phase II trial. METHODOLOGY: 142 women were screened and 100 HIV-uninfected, eligible women were enrolled, after obtaining written informed consents. Of these, 50 were randomly assigned to the study product arm and 50 to the placebo arm. Participants were requested to use the study product at least half an hour before each sexual act for 6 months. RESULTS: Participants in both treatment groups contributed 601 person-months of follow-up and 95% of the participants completed the last follow-up. Although transient genital discomfort was the main reported adverse experience, none of the study participants discontinued product use. Most of the adverse events were mild and there were no serious adverse events related to study product use and no HIV seroconversions. DISCUSSION: Praneem polyherbal tablet was safe for vaginal use up to 6 months with each act of sex among low-risk women. In light of the failure of Nonoxynol-9, cellulose sulphate and Carraguard in preventing HIV infections among women, additional preclinical evaluations should be undertaken urgently before moving into effectiveness studies.

High rates of syphilis among STI patients are contributing to the spread of HIV-1 in India.

BACKGROUND: Recent syphilis outbreaks have raised concern regarding the potential enhancement of HIV transmission. The incidence of syphilis and its association with HIV-1 infection rates among a cohort of sexually transmitted infection (STI) clinic attendees was investigated. METHODS: 2732 HIV-1 seronegative patients attending three STI and one gynaecology clinic, were enrolled from 1993-2000 in an ongoing prospective cohort study of acute HIV-1 infection in Pune, India. At screening and quarterly follow up visits, participants underwent HIV-1 risk reduction counselling, risk behaviour assessment and HIV/STI screening that included testing for serological evidence of syphilis by RPR with TPHA confirmation. Patients with genital ulcers were screened with dark field microscopy. RESULTS: Among 2324 participants who were HIV-1 and RPR seronegative at baseline, 172 participants were found to have clinical or laboratory evidence of syphilis during follow up (5.4 per 100 person years, 95% CI 4.8 to 6.5 per 100 person years). Independent predictors of syphilis acquisition based on a Cox proportional hazards model included age less than 20 years, lack of formal education, earlier calendar year of follow up, and recent HIV-1 infection. Based on a median follow up time of 11 months, the incidence of HIV-1 was 5.8 per 100 person years (95% CI 5.0 to 6.6 per 100 person years). Using a Cox proportional hazards model to adjust for known HIV risk factors, the adjusted hazard ratio of HIV-1 infection associated with incident syphilis was 4.44 (95% CI 2.96 to 6.65; p<0.001). CONCLUSIONS: A high incidence rate of syphilis was observed among STI clinic attendees. The elevated risk of HIV-1 infection that was observed among participants with incident syphilis supports the hypothesis that syphilis enhances the sexual transmission of HIV-1 and highlights the importance of early diagnosis and treatment of syphilis.

Rate of hospitalization and inpatient care costs for HIV-1-infected patients in Pune, India.

BACKGROUND: The transition of human immunodeficiency virus (HIV) infection to acquired immune deficiency syndrome (AIDS) has begun in India, and an increase in AIDS-related hospitalizations and deaths is an anticipated challenge. We estimated the rates of hospitalization and inpatient care costs for HIV-1-infected patients. METHODS: Data were analysed on 381 HIV-1-infected persons enrolled in a HIV-1 discordant couples' cohort between September 2002 and March 2004. Inpatient care costs were extracted from select hospitals where the study patients were hospitalized and the average cost per hospitalization was calculated. RESULTS: A majority of the patients were in an advanced state of HIV-1 disease with the median CD4 counts being 207 cells/cmm (range: 4-1131 cells/cmm). In all, 63 participants who did not receive antiretroviral therapy required hospitalization, 53 due to HIV-1-related illnesses and the remaining 10 due to worsening of pre-existing conditions. The overall HIV-1-related hospitalization rate was 34.2 per 100 person-years (95% CI: 26.94-42.93). The median duration of HIV-1-related hospitalization was 10 days (range 2-48 days) and the median cost was Rs 17,464 (range: Rs 400-63,891). CONCLUSION: It is necessary to strengthen the inpatient care infrastructure and supporting diagnostic set-up, and work out economically optimized treatment algorithms for HIV-1-infected patients. Although this analysis does not cover all costs and may not be generalizable, these baseline data might be a useful reference while planning related studies accompanying the government-sponsored programme to roll out antiretroviral therapy to AIDS patients.

Low HIV-1 incidence among married serodiscordant couples in Pune, India.

Unlike commercial sex workers and patients attending sexually transmitted infection (STI) clinics, married couples are not typically targeted for HIV risk reduction programs in India. Thus, married partners of HIV-infected persons are at particularly high risk for HIV infection. Between September 2002 and November 2004, 457 HIV-1 sero-discordant, married couples were enrolled in a one-year prospective study of HIV transmission in Pune, India. The HIV incidence among uninfected partners was 1.22 per 100 person-years (95% CI 0.45-2.66), which is much lower than what has been previously reported among discordant couples in Africa. This may be due to higher rates of condom use, lower rates of STIs and higher CD4 T lymphocyte counts, among the Indian HIV sero-discordant couples.

Prevalence of HIV infection in pregnant women in remote rural areas of Maharastra State, India.

In a study of 304 pregnant women, the prevalence of HIV infection in remote rural areas of western India was 0.7% (confidence interval 0.08-2.3%). It is nearly 2(1/2) times higher than the presumed prevalence for this part of the country.

High-risk behaviour in young men attending sexually transmitted disease clinics in Pune, India.

The present study reports sexual risk factors associated with HIV infection among men attending two sexually transmitted disease (STD) clinics in Pune, India and compares these behaviours between young and older men. Between April 1998 and May 2000, 1872 STD patients were screened for HIV infection. Data on demographics, medical history and sexual behaviour were collected at baseline. The overall HIV prevalence was 22.2%. HIV risk was associated with being divorced or widowed, less educated, living away from the family, having multiple sexual partners and initiation of sex at an early age. The risk behaviours in younger men were different to older men. Younger men were more likely to report early age of initiation of sex, having friends, acquaintances or commercial sex workers as their regular partners, having premarital sex and bisexual orientation. Young men were more educated and reported condom use more frequently compared with the older men. Similar high HIV prevalence among younger and older men highlights the need for focused targeted interventions aimed at adolescents and young men and also appropriate interventions for older men to reduce the risk of HIV and STD acquisition.

Phase I safety & preliminary acceptability of nonoxynol-9 vaginal pessary as a vaginal microbicide in low risk women in Pune, India.

BACKGROUND & OBJECTIVES: Though nonoxynol-9 (N-9) is available in India as a spermicidal pessary, data on its safety as a potential microbicide among Indian women are not available. Nonoxynol-9 containing compounds have shown anti-HIV activity in in vitro studies and protection against cervical infections. Nonoxynol-9 is being extensively evaluated as a vaginal microbicide world-wide. We assessed the safety and preliminary acceptability of nonoxynol-9 pessary as a vaginal microbicide in women at low risk for human immunodeficiency virus (HIV) infection and sexually transmitted diseases (STDs). METHODS: Twenty three HIV seronegative women enrolled in the study were given Today pessarys containing 5 per cent of nonoxynol-9 for vaginal use at bedtime for 14 days. Colposcopy was done at enrollment and on day 14 and speculum examination on day 7 to assess the local toxicity. RESULTS: Most of the women (16/23, 69.6%) did not experience any symptoms of genital irritation. The remaining 7 (30.4%, 95% CI 11.6-49.2) women reported 11 episodes of mild irritative symptoms of short duration. On clinical examination, three adverse events were reported of which one could have been product related. Eight (34.8%) women showed willingness to use the product for protection against HIV transmission if it was approved. INTERPRETATION & CONCLUSION: Nonoxynol-9 vaginal pessary was found to be safe and acceptable in once daily dose in low risk women after consecutive use for 14 days. Willingness for future use, if found safe and effective for HIV prevention was shown by 8 (34.8%) women.

Sensitivity and specificity of rapid HIV testing of pregnant women in India.

OBJECTIVE: Efforts to prevent HIV transmission from mother to infants in settings like India may benefit from the availability of reliable methods for rapid and simple HIV screening. Data from India on the reliability of rapid HIV test kits are limited and there are no data on the use of rapid HIV tests for screening of pregnant women. METHODS: Pregnant women attending an antenatal clinic and delivery room in Pune agreed to participate in an evaluation of five rapid HIV tests, including (a) a saliva brush test (Oraquick HIV-1/2, Orasure Technologies Inc.), (b) a rapid plasma test (Oraquick HIV-1/2) and (c) three rapid finger prick tests (Oraquick HIV-1/2; HIV-1/2 Determine, Abbott; NEVA HIV-1/2 Cadila). Results of the rapid tests were compared with three commercial plasma enzyme immunoassay (EIA) tests (Innotest HIV AB EIA, Lab systems/ELISCAN HIV AB EIA, UBI HIV Ab EIA). RESULTS: Between September 2000 and October 1, 2001, 1258 pregnant women were screened for HIV using these rapid tests. Forty-four (3.49%) of the specimens were HIV-antibody-positive by at least two plasma EIA tests. All of the rapid HIV tests demonstrated excellent specificity (96-100%). The sensitivity of the rapid tests ranged from 75-94%. The combined sensitivity and specificity of a two-step algorithm for rapid HIV testing was excellent for a number of combinations of the five rapid finger stick tests. CONCLUSION: In this relatively low HIV prevalence population of pregnant women in India, the sensitivity of the rapid HIV tests varied, when compared to a dual EIA algorithm. In general, the specificity of all the rapid tests was excellent, with very few false positive HIV tests. Based upon these data, two different rapid HIV tests for screening pregnant women in India would be highly sensitive, with excellent specificity to reliably prevent inappropriate use of antiretroviral therapy for prevention of vertical HIV transmission.

Prevalence and incidence of hepatitis B virus infection in STD clinic attendees in Pune, India.

OBJECTIVES: To estimate the prevalence and incidence of hepatitis B virus (HBV) infection among patients attending three STD clinics in Pune, India, and to identify associated risk factors. METHODS: Of the 2098 patients screened at STD clinics in Pune during 1996, 497, who returned for at least one follow up visit, were screened for various markers of HBV infection (HBsAg, anti-HBs, anti-HBc), HIV antibody, and VDRL. RESULTS: Of the 497 participants 3.6%, 26.5%, and 43.2% were positive for HBsAg, anti-HBs, and anti-HBc respectively. Tattooing (AOR 1.64, 95% CI 1.03 to 2.64) was found to be independently associated with presence of core antibody. Additionally, history of being in commercial sex work and history of a genital ulcer were independently associated with a positive anti-HBc antibody test (AOR 12.45, 95% CI 5.58 to 27.82 and AOR 1.70, 95% CI 1.09 to 2.66, respectively). 72 out of 497 (14.5%) participants were HIV positive at baseline. HIV-1 antibody positive patients were more likely to have a positive anti-HBc test (69.4% v 39.0%, p<0.001). 30 out of 282 participants, negative for anti-HBc antibody at enrolment, seroconverted subsequently, resulting in an incidence of 10.86 per 100 person years (95% CI 7.2%, 14.5%) (mean and accumulated follow up of 11.7 months and 276.17 person years, respectively). CONCLUSIONS: A high prevalence and incidence of HBV infection, seen in STD clinic attendees underscore the need to provide HBV vaccine to commercial sex workers and their clients in India.

Changing trends in clinical presentations in referred human immunodeficiency virus infected persons in Pune, India.

AIMS: To study profile and trends of clinical presentations among human immunodeficiency virus (HIV) infected individuals seen in a HIV Reference Clinic in Pune. METHODOLOGY: In a cross-sectional study, 3574 subjects were seen at a HIV Clinic in Pune from January 1997 to December 1999. Data on clinical presentation of 2801 (78.4%) HIV seropositive subjects were evaluated. RESULTS: Clinical conditions like oral thrush, tuberculosis, skin rash and sexually transmitted diseases showed decreasing trends during the three years study period (p=0.03, 0.02, < 0.01 and < 0.01, respectively). Conversely a significant increase in the number of asymptomatic HIV positive persons at the time of detection was observed over the same period (p < 0.01). CONCLUSION: Temporal change in the clinical presentations in the HIV positive persons referred to our clinic probably reflects increased awareness and a high index of suspicion among clinicians. Early diagnosis of HIV infection in asymptomatic phase might help the clinicians to make timely decisions on prescribing chemoprophylaxis for prevention of opportunistic infections and to take appropriate measures for prevention of secondary HIV transmission to the uninfected sex partners/spouses.

A serological survey of arboviral diseases among the human population of the Andaman and Nicobar Islands, India.

In an attempt to determine the prevalence of certain arthropod-borne viruses of public health importance amongst the human population of the Andaman and Nicobar Islands of India, 2,401 sera were collected from six major localities. The sera were analysed by the hemagglutination inhibition (HI) and neutralization (N) tests, using Chikungunya (CHIK), Japanese encephalitis (JE), West Nile (WN), dengue (DEN-2), Langat (TP-21) and Kyasanur Forest disease (KFD) viral antigens. The highest prevalence of HI antibodies was detected against KFD virus (22.4%), followed by Langat (20.2%), JE (5.9%), DEN-2 (3.1%), CHIK (2.9%) and WN (0.8%) viruses. Cross-reactions to the viral antigens were also noted. The results of N tests indicated a high prevalence of DEN-2 (25.4%) virus, followed by Langat (17.5%), CHIK (15.3%), KFD (12%), JE (2.19%) and WN (1.8%). These results are discussed in relation to important epidemiological parameters like age, sex and geographical location. To our knowledge, this is the first report of an extensive serosurvey of arthropod-borne viruses on these islands.