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.

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.

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

Background & objectives: 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/μl, C. parvum was the most commonly observed (54%) pathogen. Proportion of opportunistic pathogens in patients with CD4 count <200 cells/μl was significantly higher as compared with other two groups of patients with CD4 count >200-499 and ≥ 500 cells/μl (P=0.001, P=0.016) respectively. Interpretation & conclusions: 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.

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.

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.

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.

Evidence for high prevalence & rapid transmission of HIV among individuals attending STD clinics in Pune, India.

Prevalence and incidence of HIV-1 infection among persons attending two STD clinics in Pune between May 1993 and October 1995 are reported. On screening 5321 persons, the overall prevalence of HIV-1 infection was found to be 21.2 per cent, being higher in females (32.3%) than in males (19.3%). Analysis of behavioural and biological factors showed that old age, sex work, lifetime number of sexual partners, receptive anal sex, lack of circumcision, genital diseases and lack of formal education were related to a higher HIV-1 seroprevalence. The observed incidence rate of 10.2 per cent per year was very high, much higher in women than in men (14.2% and 9.5% per year respectively) and over three times higher among the sex workers. Females in sex work, males having recent contacts with female sex workers (FSWs) and living away from the family and persons with previous or present genital diseases had a higher risk of seroconversion. Condom usage was shown to have a protective effect in seroprevalence and seroincidence analysis. With limited available resources and lack of a suitable vaccine or a drug, long-term prevention policy of creating awareness in the community must be supplemented by strengthening STD control measures and promotion of condom use and safe sex. Factors related to availability and utilization of condoms must be carefully investigated.

Measles outbreak in a tribal population of Thane district, Maharashtra.

In March 1992, an outbreak of measles, in the tribal population of Vavar village, Mokhada Taluk, Thane district, Maharashtra, was investigated. Two hamlets of Vavar village namely Sagpanipada (epidemic in October, November 1991) and Behedpada (epidemic in January, February 1992) were affected. In both hamlets, measles cases were confined to children below 10 yrs and 96% of the cases occurred in children below 6 yrs. Attack rates were 52.7% and 51.4% and case fatality rates were 31.2% and 15.6% at Sagpanipada and Behedpada, respectively. All the convalescent patients' sera possessed IgM antibodies against measles. A clear drop in IgM and a rise in IgG antibodies against measles was observed in 35 paired samples from convalescent patients. Fifty four per cent of sera from controls, possessed IgM antibodies. Migrating population appeared to have imported measles which flared up in an epidemic among the susceptibles. Priority immunization of the children of remote isolated populations may prevent such epidemics.

Serological response to Japanese encephalitis vaccine in a group of school children in South Arcot district of Tamil Nadu.

A trial with Biken Japanese encephalitis (JE) vaccine made in Japan was carried out in South Arcot district of Tamil Nadu state, India. A total of 113 school children were included in the trial. The efficacy (as determined by serological response) and safety of the vaccine were evaluated. Side effects, though minor, were noted in 54.9 per cent of the children after each dose. The serum antibody titres were determined by mouse neutralization test, plaque reduction neutralization test and haemagglutination inhibition test. An antibody response to two-dose primary vaccination schedule was observed in 72.7 per cent, whereas 87.8 per cent of the vaccines responded positively after the booster dose administered one year after. Only about 20 per cent of the children had persisting antibodies one year after the primary vaccination. The results indicated a probable need of the third dose in the primary vaccination schedule.

Non A non B hepatitis epidemic in Rewa district of Madhya Pradesh.

An epidemic of viral hepatitis occurred at Rewa district of Madhya Pradesh during December 1989 through April 1990. A total of 302 cases were admitted to Gandhi Memorial Hospital. Few cases were reported from the adjacent rural areas. Twenty six of the 40 wards of the city were affected and 7 wards were most affected with attack rates ranging from 3-7 per cent. Seventy one per cent of the hospitalised cases were 15 to 35 years of age. Males constituted 72.2% of the cases. In 37 patients (12.2%) the illness had a fatal outcome. The cases fatality rate was 7.9% in males and 20.6% in females. There were leakages in water supply pipe lines at many places which ran parallel to or were laid across open gutters. The source of infection appeared to be water contaminated by sewage. Results of serological tests indicated a non-A, non-B hepatitis viral aetiology of the epidemic.

Detection of virus specific IgG subclasses in Japanese encephalitis patients.

During the Japanese encephalitis (JE) epidemic in 1988 at Gorakhpur, Uttar Pradesh, 34 cerebrospinal fluid (CSF) samples with 16 matching sera from 34 anti JEV IgM positive (confirmed JE) and 24 CSF samples with 4 matching sera from 24 anti JEV IgM negative (clinical encephalitis) patients were collected and tested for presence of JEV specific IgG by ELISA. Eighteen CSF samples and 8 matching sera from confirmed JE and 5 CSF samples and one matching serum from clinical encephalitis patients positive for JEV specific IgG were further assayed for subclass specificity using specific murine monoclonal antibodies. Almost all the samples exhibited IgG1 as the virus specific subclass. In addition to IgG1, one serum and one CSF sample each from two different confirmed JE patients showed the presence of virus specific IgG4 and IgG3 respectively. Half of the confirmed JE and clinical encephalitis patients exhibited intrathecal synthesis as evident from either elevated IgG index or CSF IgG/CSF albumin ratio. Most of the patients who recovered had predominantly virus specific IgG1 in CSF. It seems likely that IgG1 might have a protective role in clearance of virus from the central nervous system.

Outbreak of dengue fever in rural areas of Parbhani district of Maharashtra (India).

Outbreak of dengue fever in Chikalthana, Pimpalgaon and Waloor villages in Parbhani district of Maharashtra (India) were investigated. Clinically, the illness was typical of dengue fever except for the absence of maculopapular rash. A total of 42 acute, 14 late acute, 73 convalescent and 19 sera from contacts were collected. Of the 15 virus isolates, 12 were identified as dengue virus type 2 and 1 as dengue virus type 1. Serological tests confirmed the etiological role of dengue virus in the outbreak. House-to-house survey was carried out in Chikalthana and Pimpalgaon villages. Overall, 15.09 per cent of the surveyed population was affected during the outbreak and attack rate was higher at Pimpalgaon. A tendency of water storage was observed in the households and concomitant entomological studies proved Aedes aegypti breeding. Higher prevalence of dengue fever was noted among larger families and in families that had two or more patients, the commonest duration between the first and the last patient was often less than 5 days.

Post-epidemic serological survey for JE virus antibodies in south Arcot district (Tamil Nadu).

Following an extensive epidemic of Japanese encephalitis (JE) in 1981, a serological survey was carried out in the South Arcot district of Tamil Nadu in April 1982. Serum specimens were obtained from 10 villages with serologically proven infections and 6 villages with no reported case of encephalitis during the 1981 epidemic. The serum specimens tested for haemagglutination inhibition, complement fixing and neutralizing antibodies, showed a high prevalence of JE (49.17%), West Nile (40.78%) and dengue (18.14%) viruses with predominance of JE. The prevalence did not differ between the villages with or without Japanese encephalities. There was also no difference between the family members and neighbours.