A pilot study of HTLV-I infection in high-risk individuals & their family members from India.

BACKGROUND & OBJECTIVES: Human T lymphotropic virus-I (HTLV-I) has been associated with adult T cell lymphoma/leukemia (ATLL). There are Indian studies on HTLV-I infection among people with sexually transmitted infection, but no large study has been conducted on individuals with haematological malignancies. In this group of individuals, serology is known to under-diagnose HTLV-I infection. This study was carried out to identify serologically and where possible with molecular techniques, HTLV-I infection in individuals with haematological malignancies. To understand the modes of transmission, family members of individuals with proven HTLV-I infection were also studied. Individuals with sexually transmitted infection (STI), blood donors and pregnant women were also studied. METHODS: Particle agglutination test was used to detect antibody to HTLV-I. HTLV genome was amplified by polymerase chain reaction (PCR) and detected with probes by digoxiginin (Dig) ELISA. RESULTS: There was no serological evidence of HTLV-I infection among the healthy blood donors and pregnant women studied. High prevalence of anti-HTLV-I antibody was identified in the patients with haematological malignancies (8 of 86 patients, 9.3%) and a lower prevalence in individuals with STI (8 of 670 individuals, 1.2%). In the STI group, all 8 individuals seroreactive to HTLV-I were coinfected with human immunodeficiency virus (HIV). In the group with haematological malignancies, three of 22 (13.6%) patients with leukemia, 3 of 11 (27.3%) with cutaneous T-cell lymphoma (CTCL) and 2 of 53 (3.8%) with lymphoma were reactive for anti-HTLV-I antibody. In this group, PCR identified all the seroreactive individuals tested. There were also seronegative infected individuals who were only identified by PCR. There was also a large number of seronegative family members who were only positive by PCR. INTERPRETATION & CONCLUSION: The study revealed a strong disease association of HTLV-I with haematological malignancies and evidence for both horizontal and vertical transmission of the infection in the Indian population. HTLV-I infection appears to be common among family members of individuals with HTLV-I associated haematological malignancies.

Abnormal insect bite reactions: a manifestation of immunosuppression of HIV infection?

Pruritic papular eruptions are a common manifestation in patients infected with HIV. The significance of these lesions is not known. We have many apparently normal individuals presenting with exaggerated insect bite reaction on the exposed parts of the body as the only manifestation of HIV disease. A pilot study was done to see whether the appearance of exaggerated insect bite reactions is an indicator of impending immune suppression in HIV infected individuals. CD4 count was done in 10 serial patients presenting to our clinic with exaggerated insect bite response of recent onset and HIV infection. Out of the 10 cases, 8 showed low CD4 counts (less than 400/cmm). Further studies may prove that exaggerated insect bite reaction can be a marker of impending immunosuppression.

Importance of RT-PCR for detection of hepatitis C virus among different patient groups.

A pilot study was undertaken to evaluate a sensitive single step reverse transcription polymerase chain reaction (RT-PCR) to detect hepatitis C virus (HCV) RNA in plasma samples as an adjunct to antibody detection. In this study, digoxiginin labelling of the amplicon was carried out during the PCR and the product was detected by an ELISA format (DIG-ELISA) as well as ethidium bromide stained gel detection. DIG-ELISA detection of PCR product compared favourably with gel detection. We tested 42 individuals with either parenteral risk factors or chronic liver disease and found the RT-PCR enhanced the detection rate by 19 per cent. Considering either plasma RNA or antibody positivity, HCV infection was detected in 64.3 per cent of patients.

Increasing prevalence of HIV antibody among blood donors monitored over 9 years in one blood bank.

To determine the time trend of HIV infection among non-professional blood donors at the Christian Medical College and Hospital, Vellore, the annual HIV prevalence among them for the years 1993-1997 was compared with that of the previous 5 yr. Since confirmed number of HIV positive blood donors is required for calculation of prevalence, the serum samples which were reactive for HIV 1/2 antibody by ELISA, were confirmed by Western blot. The annual prevalence of HIV antibody gradually increased from 1.6 per 1000 in 1988-1989, to 3.8 per 1000 in 1996-1997. The mean tri-annual prevalences for the years 1988-91, 1991-94 and 1994-97 were 1.3, 2.7 and 3.6 per 1000 respectively, suggesting a slow but steady increase in HIV prevalence over time. Our findings provide an insight into the dynamics of HIV infection in the general population of our country, whom the nonprofessional blood donors at our hospital represent.

A primate model of respiratory syncytial virus infection.

To determine whether bonnet monkeys are susceptible to infection and disease due to respiratory syncytial virus (RSV), 4 juvenile bonnet monkeys (Macaca radiata) were inoculated with RSV intratracheally and sacrificed at 3, 5, 7 and 9 days post infection. RSV was cultured from pre-autopsy broncheoalveolar lavage fluid from all 4 animals with a peak titre of virus on day 9. Serum RSV neutralizing antibody was present by day 7. Animals developed tachypnoea and chest retractions by 5th day post infection and 2 animals had lobular pneumonia on chest radiography. The pathological changes were of a bronchovascular inflammation, interstitial pneumonia and alveolitis, akin to that seen in humans. These findings show that bonnet monkeys can be infected with RSV, and can develop immune response and clinical and pathological changes similar to those seen in human infants with RSV disease. Thus intractracheal RSV inoculation of juvenile bonnet monkeys appears to be a good model to study pathogenesis of RSV disease.

Diagnosis of typhoid fever by the detection of anti-LPS & anti-flagellin antibodies by ELISA.

In a developing country like ours where typhoid fever is endemic and there are very few microbiology laboratories to provide diagnosis by culture, a search for non culture techniques for rapid and reliable diagnosis continues. The Widal test has a low sensitivity. We have attempted to adapt the well established enzyme linked immunosorbent assay (ELISA) technique to provide a serological test of better sensitivity and specificity. The ELISA described here to detect anti-LPS antibodies was found to have a sensitivity of 89.4 per cent and a specificity of 94.9 per cent. The sensitivity for the antiflagellin ELISA was 68.1 per cent and specificity was 97.4 per cent. The likelihood ratio was 17.5 for anti-LPS ELISA and 26.2 for the anti-flagellin ELISA. Considering the positivity of either one ELISA as diagnostic, the sensitivity was 93.6 per cent and specificity was 94.9 per cent.

Epidemiological features of acquired immunodeficiency syndrome in southern India.

AIDS was diagnosed in 187 men and 24 women (M:F = 8:1) from April 1987 till December 1994 at the Christian Medical College Hospital, Vellore. The doubling time of the occurrence of AIDS cases was 14 months; during 1987-90 there were an average of 5.7 cases per year; in 1991-93 there were 28 per year; in 1994 there were 104 cases. The mean age of patients was 33 yr for men and 31 for women. Among men, the primary mode of infection was heterosexual contact with female commercial sex workers. Among women, the most common source of infection was their husbands. There were 4 bisexuals and one homosexual subject who might have acquired infection by having sex with other men. There were 135 subjects from urban and 76 from rural communities. Most subjects belonged to the lower socio-economic classes. These data show that HIV infection had been very widespread in this region, both urban and rural.

Clinical profile of AIDS in India: a review of 61 cases.

Among 61 patients with AIDS seen in our hospital from February 1986 to June 1992, 38 (62%) had significant weight loss, 34 (56%) had fever of more than a month and 19 (31%) had chronic diarrhoea; 43 (70%) patients had at least one of the above symptoms. Tuberculosis diagnosed in 32 (52%) patients was the commonest secondary infection: 10 had only pulmonary, 15 had pulmonary and extrapulmonary and 7 only extrapulmonary tuberculosis. Secondary infections commonly found in the general population, predominantly with pathogenic bacteria were found in 27 (44%) patients, 45 (74%) patients had opportunistic infections such as oropharyngeal candidiasis 25 (41%) cryptococcal meningitis 4 (7%), recurrent zoster 1 (1.6%). Two (3.3%) patients had interstitial pneumonitis of unknown aetiology. One (1.6%) patient each had oral hairy leukoplakia or immunoblastic lymphoma. Kaposi's sarcoma was not seen in any patient. At the time of writing this, 33 (54%) patients have died; the mean duration from diagnosis of AIDS to death was 4.5 months.

Increasing prevalence of human immunodeficiency virus infection among patients attending a clinic for sexually transmitted diseases.

Of 5883 patients screened during 1986 to 1993 in the sexually transmitted diseases (STD) clinic at a hospital in Vellore, south India, 105 (1.79%) were positive for human immunodeficiency virus (HIV) antibody. The prevalence of HIV infection increased from 0.26 per cent in 1986 to 2.64 per cent in 1993. In 1992 the prevalence was even higher, namely 3.94 per cent. The overall prevalence in these 8 yr was 1.98 per cent among men (n = 4392) and 1.21 per cent among women (n = 1491), the difference being statistically significant (P < 0.05). The prevalence in men rose from 0.36 per cent in 1986 to 4.24 per cent in 1992, and in women from 0 to 3.04 per cent during the same period. The major source of infection in men was female commercial sex workers, but among women it was mostly their husbands. The increase in prevalence of HIV infection is alarming, and indicates that the massive educational campaigns and other preventive measures have not resulted in decreased transmission.

Rising trend in the prevalence of HIV infection among blood donors.

The prevalence of HIV infection among blood donors was analysed, for the five year period from April 1988 to March 1993. All donors were patients' relatives or volunteers; no paid/commercial donors were accepted. Each year between 14,084 and 15,544 donor blood samples were screened by ELISA and those found reactive were tested by Western blot. Western blot positive samples were considered to be infected with HIV. The prevalence rates were 1.5 per 1000 (1988-89), 1.1 per 1000 (1989-90 and 1990-91) 1.9 per 1000 (1991-92) and 3.1 per 1000 (1992-93). When the mean prevalence rate over the first three years [1.3 per 1000 (1988-89 to 1990-91)] was compared to the prevalence in 1991-92 (1.9/1000), increase was statistically significant (P < 0.05). The prevalence rate in 1992-93 (3/1000) was significantly higher than that of the previous year (P < 0.01). These data suggest that the prevalence of HIV infection in blood donors is increasing and this could be a reflection of the rising prevalence of HIV infection in the general population.

Comparison of the indirect immunofluorescence assay with western blot for the detection of HIV antibody.

With the increase in the prevalence of human immunodeficiency virus (HIV) infection in India, there is an urgent need to explore cost-containing methods of HIV antibody detection. We compared the indirect immunofluorescence assay (IFA) with the widely used but expensive confirmatory test, the Western blot (WBT). A panel of sera comprising 42 WB positive, 46 antibody negative (27 ELISA negative and 19 ELISA reactive but WB negative) and 58 sera with indeterminate WB profiles were tested by a commercial IFA kit. The sensitivity of IFA was found to be 97.6 per cent and specificity 97.8 per cent. Of the indeterminate sera, 18 (31%) were positive in IFA while 26 (45%) were negative and 14 (24%) were equivocal. In general, the IFA positivity was associated with the presence of the gag and env antibodies of HIV while negativity associated with the presence of lone antibodies. The cost of testing by IFA is less than 20 per cent of that of WB testing. Thus IFA appears to be a cost-containing alternative to WB in centres in India where immunofluorescent microscopy is routinely done.

Transfusion associated HIV infection in patients with haematologic disorders in southern India.

Seventy patients with congenital coagulation disorders (group A) and 202 other patients (group B) attending the Haematology clinic at the Christian Medical College and Hospital, Vellore (India) were screened for HIV infection between March 1989 and April 1991. Fifty five patients in group A and 131 patients in group B had received blood or blood products in the past. Nineteen transfused patients (9 in group A and 10 in group B) had received blood or blood products exclusively from the hospital blood bank and none of them was HIV infected. Among the remaining 167 transfused patients, 14 (30.4%) of the 46 patients in group A and 6 (4.9%) of the 121 patients in group B were found to be positive for HIV. In group A, 13 of the 14 infected patients had received commercially available cryoprecipitate which was thus found to be the most frequent source of infection. In group B the source of infection was most probably unscreened HIV infected blood which was transfused.

Prevalence of HIV infection in pregnant women in Vellore region.

All pregnant women attending the antenatal clinic of a large hospital in Vellore, India (Christian Medical College Hospital) were screened for HIV infection between October 1987 and June 1992. A total of 36,953 blood samples were thus screened and 20 infected women were identified. Among these 18 women had acquired HIV infection from their husbands, who were also detected to be HIV infected. While these 18 women were monogamous, all the husbands had multiple sex partners. Two of the 20 women in this series were commercial sex workers. Among the 20, 17 (85%) women belonged to low socio-economic status, while three were from well-to-do families, with the husbands being businessmen or teachers. As the overall prevalence (0.054%) of HIV infection among pregnant women was relatively high and equal to or higher than many States in the USA and in areas outside London in the UK, the authors recommend that strict universal precautions be instituted in all obstetric practice in India.

High prevalence of HTLV antibodies in wild-caught bonnet monkeys in southern India.

The prevalence of human T-lymphotropic virus (HTLV) type-1 antibodies was determined in the bonnet monkeys, living naturally, within about 30 km radius of Vellore (south India). Sera from 157 animals, collected between January 1982 and May 1993 were screened for the presence of HTLV-I infection by a particle agglutination test (PAT). When sera repeatedly reactive in PAT were subjected to indirect immunofluorescence and western blot tests, 63 (40%) were confirmed to be positive for HTLV-1 antibody. These findings are significant in the light of recent reports that HTLV infection is endemic to southern India.

The detection of HIV-2 infection in southern India.

To determine the prevalence of HIV-2 infection in southern India, we tested two sets of sera, selected from among the samples which had been collected between January 1988 and October 1990 from high risk subjects and tested for HIV-1 antibody. They were screened for HIV-2 antibody by ELISA and repeatedly reactive sera confirmed by HIV-2 Western blot and line immunoassay. In the first set of 604 sera, only one (0.16%) was positive for HIV-2. In the second set of 24 sera, selected on the basis of having indeterminate HIV-1 Western blot profiles, again one (4%) was positive for HIV-2. The two HIV-2 infected subjects were residents of Madras or Visakhapatnam. Residents of Vellore region constituted 88 and 75 per cent of the two sets of subjects; none was positive for HIV-2. Our results show the prevalence of HIV-2 in the port-cities of southern India. Since it will spread to other regions continuous monitoring for this infection is essential in order to determine when to establish HIV-2 screening in addition to the existing HIV-1 screening of donor blood for transfusion.

Reduction of the cost of testing for antibody to human immunodeficiency virus, without losing sensitivity, by pooling sera.

The sensitivity of testing pooled sera instead of individual sera for antibody against human immunodeficiency virus (HIV) was evaluated using a non-competitive enzyme-linked immunosorbent assay (ELISA). For this purpose, 42 HIV antibody positive sera were titrated and introduced into 42 sets of pools of 2, 4, 8, 16, 32 or 64 sera in such a manner that each pool had one positive sample and the rest, HIV antibody negative sera. When the pools were tested in ELISA, all pools with high titred antibody positive sera were reactive irrespective of pool size, while some of the pools containing medium or low titred sera were non-reactive when pool size exceeded 16. Subsequently the pool size was limited to 16. When 208 previously unscreened samples were tested in 52 pools of 4, 26 pools of 8 or 13 pools of 16 sera, or individually, 6 antibody positive sera were correctly identified. Thus, it was found that the pooling method did not reduce the sensitivity of ELISA test, whereas the cost was reduced to less than half of that of individual testing.

T-lymphocyte mediated injury of beta cells in dual-aetiology diabetes mellitus in Swiss albino mouse.

Earlier we had described a dual aetiology diabetes mellitus (DADM) in mice injected with a sub-diabetogenic dose of streptozotocin (SD-SZN) and afterwards infected with coxsackie B3 virus (CBV). Further experiments were conducted to understand the mechanism of diabetogenesis. In in vitro stimulation and proliferation tests, the splenic lymphocytes (SLC) of mice given either SD-SZN or CBV infection showed lower responses to two T cell mitogens than those of control mice, indicating an immunosuppressive effect. Unexpectedly, SLC of mice given both SD-SZN and CBV showed enhanced response, indicating immunoactivation; they were not stimulated to proliferation in response to CBV antigen, indicating that the immunoactivation was not directed against CBV, but against streptozotocin or cellular elements. When mice were depleted of T cells by injecting with anti-thymocyte serum, the diabetogenic effect of SD-SZN and CBV infection was abrogated, without diminishing the replication of virus in the pancreas. Thus beta cell injury in DADM appears to be T cell-mediated.

HIV seropositivity among patients with sexually transmitted diseases in Vellore.

In a prospective, systematic investigation, 2215 new patients with sexually transmitted diseases (STD) were screened for human immuno deficiency virus (HIV) infection during April 1986 to December 1988. Among them 9 were positive for HIV antibody giving a rate of 4/1000. In addition, 5 patients had been referred to our clinic with a diagnosis of HIV infection and STD. Among these 14, there was one married couple. Of the remaining 5 married individuals, 4 were men; the wives of 3 of them were infected but asymptomatic, and were not included in this report. The rate of conjugal infection was 71 per cent. Most (57%) patients belonged to poor socio-economic background; 78 per cent of patients were young, in the age group 15-25 yr. The presenting STD were syphilis, venereal warts, gonorrhoea, chancroid, genital herpes, non-gonococcal urethritis and candidiasis. It appeared that the course of associated STD had not been altered in these HIV infected individuals.

Lack of evidence for seroconversion to human immunodeficiency virus in recipients of anti-D immunoglobulin containing HIV antibody.

Twenty nine Rh negative women who had received injections of anti-D (Rho) immunoglobulin preparations (from an Indian firm) contaminated with antibody to human immunodeficiency virus (HIV), were tested for seroconversion to HIV, 14-38 wk after injections. All 29 were negative for HIV antibody, indicating that infectious HIV or immunogenic concentrations of inactivated HIV were not present in the investigated immunoglobulin product.