India is undergoing an epidemiologic, demo-graphic and health transition. The expectancy of life has increased, with consequent rise in degenerative diseases of aging and life-styles. Nevertheless, communicable diseases are still dominant and constitute major public health issues.
The research strategy adopted by the Council is a balance between the upstream (fundamental and basic) and downstream (product development/evaluation and operational) research.
Through the network of its disease-specific Institutes/Regional Medical Research Centres and extramural research programme, the Council is supporting and encouraging biomedical research in communicable diseases. New viral and bacterial infections have been identified. Monitoring of anti-microbial resistance to commonly used drugs is being extended to include more organisms. Disease surveillance at the molecular level has been expanded and strengthened. Studies to assess disease burden not only in terms of morbidity and mortality but also economic are high on the Council’s agenda. Feasibility of effective strategies under field conditions for control of infectious diseases is being demonstrated. Research support to eradicate target diseases has been intensified. Development and evaluation of diagnostic tools, drugs and vaccines is being undertaken. Programme relevant research to strengthen the national health programmes and human resource development are an integral part of the efforts of the Council towards control of communicable diseases.
Tuberculosis accounts for a loss of approximately 11 million disability adjusted life years (DALYs). The burden of disease may increase further with the emergence of the HIV epidemic. The Revised National TB Control Programme (RNTCP) which covers more than 120 million population has successfully treated approximately 80% of patients in 48 districts of 16 states and Union Territories. Treatment success rates have more than doubled and death rates have decreased by 75 per cent. The ICMR’s Tuberculosis Research Centre (TRC) at Chennai is providing research support to the RNTCP through the conduct of basic, applied and operational research to develop better tools and training strategies for tuberculosis control.
The TRC initiated a study to examine the feasibility of shortening the duration of treatment for smear positive pulmonary tuberculosis from the present 6-8 to 3-5 months with regimens using ofloxacin in a randomized controlled clinical trial. The regimens were (i) three months of ofloxacin, isoniazid (H), rifampicin (R) and pyrazinamide (Z) daily (Reg.1); (ii) three months of ofloxacin, isoniazid, rifampicin and pyrazinamide daily followed by one month of isoniazid and rifampicin twice a week (Reg 2); (iii) three months of ofloxacin, isoniazid, rifampicin and pyrazinamide daily followed by two months of isoniazid and rifampicin twice a week (Reg 3); and (iv) two months of ofloxacin, isoniazid, rifampicin and pyrazinamide daily followed by two months of isoniazid and rifampicin twice a week (Reg 4). All drugs were given under supervision. Patients who defaulted for treatment were visited at home and motivated to attend. Interim analysis in 352 of the 529 patients admitted to the study showed that at the end of treatment, overall 96 to 99% of the patients had a favourable response. Forty two patients had pre-treatment resistance to isoniazid. Six patients had resistance to rifampicin and isoniazid (MDR tuberculosis), one of whom was also resistant to ofloxacin. Of these six patients, two had an unfavourable response, one relapsed and the other three had favourable response. After amalgamating the results of regimens 1, 2 and 3, all of which had an intensive phase of three months, and comparing it with the fourth regimen, which had an intensive phase of only two months, it was found that the rate of culture conversions was very similar. Culture conversion at two months ranged from 94 to 97%. Analysis in 346 patients in whom results were available showed that the relapse rates were 9, 4, 4 and 13% respectively in the four regimens. Even though the relapse rates in regimens 1 and 4 are higher compared to regimens 2 or 3, the difference was not statistically significant. The follow up of patients would continue upto 5 years.
Studies on Drug Resistance in Tuberculosis
The TRC has completed a study on surveillance of drug resistance in TB in Tamil Nadu to determine the proportion of initial and acquired drug resistance. Out of 400 patients for whom drug susceptibility tests were done, 384 (96%) had no history of previous anti-TB treatment while 16 had had previous treatment. Resistance to isoniazid alone or in combination with other drugs, was observed in 15.4% of the former and 50% of the latter. Any resistance to rifampicin was observed in 4.4% of previously untreated patients as against 25% of patients with previous treatment. These included 3.3% patients with H and R resistance in the untreated group and 25% in the treated group. This study made available, for the first time, authentic basic data on initial and acquired drug resistance from Tamil Nadu.
A study is being conducted at the All India Institute of Medical Sciences (AIIMS), New Delhi to define the magnitude of drug resistant tuberculosis amongst patients with active disease. A total of 215 strains isolated during the year under report were speciated using different biochemical characteristics viz. growth rate, pigmentation, niacin production, nitrate reduction, catalase production and growth on MacConkey medium. Susceptibilities to rifampicin (R), isoniazid (H), streptomycin (S) and ethambutol (E) at their respective critical concentrations were assayed by 1% proportionate method performed on L-J slants. The interim results indicated that among 22 Mycobacterium tuberculosis isolates, 9 were sensitive to all drugs, 3 were resistant to isoniazid alone, 4 were MDR (resistant to R+H), 2 were resistant to H+R+S, 1 resistant to R+E and 1 to streptomycin alone. Two isolates were resistant to all drugs. The study is continuing.
Enzymes involved in the de novo purine biosynthesis are important for infectivity, growth and virulence of certain pathogenic bacteria. The guaA gene encoding GMP synthetase in M.tuberculosis is being studied to help in understanding its role in pathogenicity. The promoter clones isolated during the early phase of this study were sequenced and the transcription start points were determined using primer extension analysis. One of the promoters exhibited very high homology to the putative guaA gene encoding GMP synthetase from M.tuberculosis. A 1.6 kb coding region along with 400 bp of the upstream region was PCR amplified using specific primers and cloned into pCR2.1 vector. Sequence analysis and restriction mapping confirmed the identity of the cloned fragment to be the M.tuberculosis guaA gene (Fig.1). The upstream region of this gene was cloned separately into a promoter-probe vector and studies are underway to determine the transcription start point of this promoter and to study its regulation. The coding region of the gene was cloned into an expression vector and transformed into Escherichia coli for overproduction of the gene product. The results show that expression of this gene is lethal to E.coli. Hence, attempts are being made to express this gene under the control of heat shock promoter (hsp60) in M.smegmatis using a shuttle plasmid vector (Fig.2). The promoter clones isolated in the initial part of this study were further characterised using primer extension analysis. Experiments are in progress to study the ability of these promoters to drive a candidate mycobacterial gene. The immunodominant 38 kDa protein antigen was chosen for this purpose. A 1.1kb coding region of the 38 kDa protein antigen was PCR amplified using specific primers and the product was cloned into pCR2.1 vector. This gene will be used to study the transcriptional strengths of the isolated promoters. The study is continuing.
A study is being supported at the V.P. Chest Institute, Delhi for the purification of plasmid(s) and characterisation of one of the plasmids from Mycobacterium avium-intracellulare complex (MAC) isolates obtained from Indian patients having mycobacterial infection. Ninety isolates were analyzed biochemically and were subjected to plasmid screening. Twenty two isolates showed the presence of the plasmid, of which two gave multiple bands which were lost on repeated subculturing. One isolate consistently gave a plasmid band that was purified using the Qualigens column kit. The purified DNA was subjected to transmission electron microscopy that endorsed the circular nature of the plasmid. From restriction digestion using EcoR1, HindIII, Pst1, EcoRV, BamH1 and double digests (BamH1 and Pst1) the exact sizes of individual fragments generated were calculated and the size of the intact plasmid was found to be 24 kb. To localize the origin of replication, a 37-mer oligoprobe designed from the ori of pLR7, the M.avium plasmid that has been partially sequenced so far, was hybridized using the ECL non-radiolabelled direct labelling and detection system. Signals were obtained with two fragments of 4 kb each of BamH1 and Pst1 double digest plasmid DNA blotted on nylon membrane. Based on these observations, a tentative restriction map could be constructed for the 24 kb wild plasmid that has been isolated from Indian MAC. The methodology standardized for plasmid isolation may be used to screen large number of indigenous MAC isolates for further studies. Probes developed from these plasmids could be of much use in the epidemiology of MAC and it paves the way for the development of a cloning/expression vector and thus opens up a whole new chapter in mycobacterial diagnostics and vaccines.
Lane 1 & 15-000 bp mol. Wt. Marker; Lane 14-pJEM13 vector cut with Apa1 & Kpn1; Lanes 2-13-Promoter clones cut with Apa1 & Kpn1.
Fig2: SDS-PAGE analysis of pMV/GMP (pMV 261 + gua A gene) recombinant from M. smegmatis(mc2 155) . The arrow indicates the presence of a doublet in induced cultures (lanes 2 & 3) which is absent in the vector control (lane 5).
Lane I-protien mol. wt. marker; Lane 2-induced pMVGMP1 in mc2 155 for 15 min; Lane 3-induced pMVGMP1 in mc2 155 for 30 min; Lane 4-induced pMVGMP1 in mc2 155 for 60 min; Lane5-pMV 261 in mc2 155 (vector control).
Studies on Molecular Biology of Mycobacteria
Studies were carried out at Central JALMA Institute for Leprosy (CJIL), Agra to study the relationship of different levels of rifampicin resistance associated with certain mutations earlier observed in the Indian strains of M.tuberculosis. Extended studies have provided newer information about type of mutations in case of quinolone resistance and transporter genes. ABC transporters have been amplified by polymerase chain reaction (PCR) from different strains of M.tuberculosis. Data show structural as well as functional differences in these transporters in the resistant vs sensitive strains thus highlighting the need for investigating other efflux pumps and their functional status. Based on the original data about the type of mutations present in Indian strains of M.tuberculosis, new probe systems are being designed.
Studies on developing PCR based ribosomal DNA fingerprinting techniques for pathogenic mycobacteria progressed. Based on the testing of new PCR-RFLP (restriction fragment length polymorphism) system(s) designed at CJIL, two rDNA-RFLP assays targetting 16 S rRNA gene and spacer region have been found to be potentially useful for rapid identification of clinical isolates of M.tuberculosis and other mycobacteria even directly from the lesions specially in tuberculosis.
Application studies on the earlier developed ribosomal DNA fingerprinting techniques as well as random amplification of polymorphic DNA (RAPD), IS 6110 DNA fingerprinting methods on M.tuberculosis have made significant progress. These have been used to type large numbers of mycobacterial strains collected in the Mycobacterial Repository Centre at CJIL from different parts of the country and complementary protocol for DNA fingerprinting of Indian strains of M.tuberculosis is being formulated. No clustering of any drug resistant type and any particular RFLP type has been observed so far indicating random distribution and selection by drug pressures.
The potential role of natural killer (NK) cells in the killing of mycobacteria is being studied at the TRC, Chennai. There was a significant decrease in NK activity in patients as compared to controls, whereas there was no difference between the contact and patient groups. Further, in M.tuberculosis infection, it has been proved in vitro that reactive oxygen intermediate (ROI) levels were increased. This increased ROI by monocytes or neutrophils may cause suppression of NK activity possibly explaining the decreased activity seen in tuberculosis patients. The mechanism of the defect in tuberculosis patients (cytotoxic effect) was not observed at the recognition/binding stage but probably lies in the subsequent events involved in the lethal hit.
The TRC in collaboration with the Tamil Nadu State TB Programme has established a model centre for directly observed treatment short-course (DOTS) implementation, tuberculosis control, training and research. Five panchayat blocks in Tiruvallur district covering a population of 5,00,000 were selected for the model DOTS project. There are 225 villages in these blocks. Since the area has been under observation for a long period of time (as it is the same area where the earlier Chingleput BCG study was conducted), the epidemiological impact of the DOTS programme can effectively be measured here. By the end of the third quarter in 1999, 220 patients had been registered for treatment, of whom 124 were allocated to CAT I, 43 to CAT II and 53 to CAT III. This exercise is continuing.
Further in order to assess the epidemiological impact of DOTS, a base-line and annual risk of infection (ARI) survey was started in December 1998 which is still continuing. The TRC has undertaken a massive training programme as part of the project activity.
Studies conducted earlier at the TRC indicated that 88% of private medical practitioners (PPs) in Chingleput district and Chennai are willing to participate in RNTCP while only 52% are willing for DOTS. During the year, studies were carried out to identify the mode of involvement of PPs in the RNTCP and to assess its feasibility.
In addition the TRC has established an agency called Advocacy for Control of Tuberculosis (ACT) in collaboration with ‘THE HINDU’. The agency has trained and encouraged private practitioners to follow RNTCP guidelines in their private practice.
As a result of the introduction of multidrug therapy (MDT) in the national disease control programme, 98 of 122 countries have reached the goal of elimination of leprosy as a public health problem. However, the prevalence of leprosy in India is still around 5/10,000 population. The new case detection rate has also not shown any appreciable decline. Twenty four other endemic countries share a similar situation. For all such countries, the deadline for elimination of leprosy has been extended by the WHO Leprosy Elimination Project to the year 2005. The Council’s Central JALMA Institute for Leprosy (CJIL), Agra is focusing its research activities to find solutions to problems related to the persistence of leprosy in India through better understanding of the disease process.
The follow up of patients treated with a one year MDT regimen for multibacillary leprosy designed at CJIL (comprising rifampicin, ofloxacin, minocycline, clofazimine and dapsone) showed that this regimen was well tolerated and effective in killing the bacilli. Most patients have completed a follow up of more than 3-3.5 years on placebo. Episodes of erythema nodosum lepromatosum (ENL) requiring the use of steroids in highly bacillated group have been observed. The patients have been divided into different groups depending on the initial bacillary index (BI). Some of the patients with initial high BI continued to be bacteriologically positive for a longer period, while others had episodes of ENL. Follow up is continuing.
Studies conducted earlier at CJIL had shown that the duration of MDT therapy, as suggested by the WHO may not be adequate to cure a patient. The efficacy of alternate regimens is being compared with the WHO regimen in terms of persisters, late reactions and relapses. Follow up of patients of paucibacillary (PB) leprosy treated with a six monthly regimen comprising dapsone, clofazimine and rifampicin has confirmed that the patients treated with this regimen have less residual activity, lower reaction rates and no relapses during the first 3.5-4 years of follow up. This regimen appears to be a good alternate regimen for the treatment of PB leprosy.
In a study conducted earlier at National Institute of Epidemiology (NIE), Chennai, single dose of rifampicin, ofloxacin and minocycline (ROM) has been shown to be as effective as six months of MDT for patients with mono lesion leprosy. Subsequently, the study was extended to cover patients with 2 and 3 lesions. The regimen has been found to be equally effective in such patients. A multi-centre double-blind randomized controlled trial is being conducted to evaluate the therapeutic efficacy of single dose ROM for patients with PB leprosy with 2-5 lesions.
Major ICMR Research Projects in Communicable Diseases
In a field study conducted by NIE, Chennai, the WHO design for leprosy elimination monitoring (LEM) activities was tested. The main purpose of this study was to collect information on a limited number of indicators that can describe the performance of the MDT programme for leprosy. The study carried out in 10 randomly selected PHCs of Villupuram district, Tamil Nadu in 1997-98, showed that 97% of 518 patients were treated with M DT. Of the 96 health facilities, 84% provided MDT. All the blister-packs were of good quality. Of the 518 registered patients, 43% had grade 2 disabilities, 22% had multibacillary (MB) leprosy, 22% had single lesions, and 30% were children. There was complete flexibility in delivering MDT. Cure rates were 50 and 64% respectively in patients of MB (115) and PB (403) leprosy. The results suggest that the WHO design is useful to improve the quality of routine reporting and can be incorporated in the National Leprosy Eradication Programme records.
Gene probes have been developed for detection of early forms of leprosy in histopathological sections by in situ hybridization. Additional data confirm that this method is promising and is able to detect suspicious and indeterminate cases efficiently.
The rationale of classifying leprosy patients on the basis of the number of skin lesions alone was completed during the year. Clinico-bacteriological analysis of a large number of patients has shown that with the increase in the number of skin lesions, the skin smear positivity increases. Further analysis confirms that inadvertently a proportion of patients with MB leprosy could be treated for PB leprosy. These observations have therapeutic implications as these patients are likely to be undertreated and may relapse later.
Immunology of Leprosy
The CJIL expanded the study on IgG levels to heat shock protein antigens. Additional data further confirmed that there is no difference in the levels of IgG in patients with borderline tuberculoid (BT)/tuberculoid (TT) and lepromatous (LL) leprosy and in those with and without reactions.
An attempt is being made at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh to study the mechanism of T cell anergy or impaired immune response in leprosy patients. Lymphocyte phenotyping of T cells from untreated patients was done by flow cytometry. Eight leprosy patients and 10 healthy controls were studied. Expression of adhesin molecules LFA-1a, Mac-1, LFA-1b and ICAM-1 was also checked on both the lymphocytes and the monocytes of these patients and the expression of the adhesin molecules was compared with the control group. The interim results indicated no significant difference in the per cent values of lymphocyte phenotyping. It was also observed that the adhesin molecules are critical in ensuring the optimum activation of T cells. They have an important role in T cell adhesion and initiation of signal transduction events and may also have a role in immune suppression in leprosy patients. The study is continuing.
In another study at PGIMER, Chandigarh, the mechanism and regulation of apoptosis in the peripheral blood mononuclear cells (PBMCs) of leprosy patients is being studied with the aim of controlling the progression of disease. PBMCs from different categories of leprosy patients were cultured with 2 mg/ml (standardized) of antigen in 5% CO2 for 24 and 48 h. Spontaneous apoptosis at 0 h was also checked. Of the three antigens, viz. PGL-1, membrane antigen and cytosolic antigen, membrane antigen has been observed to cause noticeable apoptosis. Phenotypic analysis of apoptotic cells was carried out using anti-CD3, anti-CD4, anti-CD8, anti-CD45 and anti-CD69 each with CD95 antibodies conjugated to FITC and phycoerythrin. It has been observed that the lymphocytes with CD3, CD4 markers labelled with CD95 antibodies show apoptosis. The study is continuing.
Immunoprophylaxis and Immunotherapy
A two-arm double blind, randomized, controlled clinical trial of the ICRC vaccine was recently completed at the Cancer Research Institute, Mumbai. The study was conducted in south-eastern Maharashtra, (Latur, Osmanabad, and Sholapur districts) in which the relative efficacy of the ICRC vaccine (0.5 x 109 bacilli per dose) was compared against one fifth the standard dose of BCG. The immunoprophylaxis trial was launched in August 1986. Nearly 34,000 healthy household contacts of leprosy patients formed the study population. The vaccinees were between 1-65 yr of age, of both sexes, and received either of the two vaccines. Four resurveys were carried out in 1993, 1994, 1997 and 1998 for detection of new cases.
Distribution of vaccinees according to age (child and adult), sex, and BCG scar status was similar in the 2 arms. There were 62 eligible incidence cases (29 belonged to ICRC group and 33 to BCG group) giving an attack rate of 2.1 per 10,000 person years, as against the expected rate of 3.5 per 10,000 person years. The overall protection offered by ICRC vaccine was 18% (95%CI - 35% to 50%, statistically not significant), more than the one fifth dose of BCG vaccine.
The immunotherapeutic efficacy of the ICRC vaccine was analysed in LL patients who were clinically classified as non responders to MDT as they had not shown any fall in BI despite 3-4 years of MDT. Immune functions and clinical parameters were monitored in these patients before and 6-12 months after vaccination. Laboratory investigations focused on analysis of lymphocyte proliferative responses, cytokine production (IL-2, IFN-gd) and limiting dilution analysis to determine the frequency of ICRC/M.leprae reactive T cells in peripheral blood of LL and TT patients and in ICRC vaccinated LL patients. Lymphocytes from LL patients showed poor responses to M.leprae antigens but good response to ICRC antigens. Using the above immunological parameters of T cell function, it was noted that the patients exhibited a marked improvement in T cell responses after vaccination with a concomitant fall in BI and histological upgrading. An increased frequency of M.leprae/ICRC reactive T cells was observed in peripheral blood of vaccinated LL patients.
It was also observed that in the vaccinated group there was clonal expansion of lymphocytes expressing T cell receptors Vb6, Vb7 and Vb11 indicating that T cell anergy could be reversed after vaccination.
Studies on Drug Metabolism and Drug Permeability
Studies on clofazimine metabolism were continued at CJIL. In the experimental studies in mice model high levels of clofazimine were observed in the tissues having reti-culoendothelial components. In other tissues the levels were relatively lower. Significant amount of drug was detected in the mouse foot-pad where M.leprae is known to multiply. Traces of drug could be detected in pooled nerves. Concomitant administration of isoniazid was found to reduce the distribution profile of clofazimine. It was also noted that with a loading dose of 50 mg of clofazimine daily, the bioavailabilty of the drug in the plasma attained a plateau and did not rise even after further administration of clofazimine.
The National Institute of Cholera and Enteric Diseases (NICED), Calcutta and RMRC, Bhuban-eswar continued to pursue their research goals on different facets of diarrhoeal diseases. The NICED, Calcutta has earned an important affiliation with the Japanese International Collaborating Programme. Its active surveillance programme continues to monitor the newly emerging diarrhoeal pathogens and addresses unknown frontiers in clinical diagnosis and disease management.
During the course of invasive intestinal amoebiasis, E.histolytica actively penetrates the mucosa and submucosa of the host’s intestine. Since collagen is a major component of the extracellular matrix and basal lamina of the human intestine, it is thought that collagenase which was detected in pathogenic E.histolytica is one of the important factors of tissue lysis during invasive amoebiasis. Attempts were made by NICED, Calcutta to clone and sequence some of the genes that are differentially expressed during activation with human collagen type I and Ca2+. Expression of this gene and further characterization of recombined protein will help in immunodiagnosis of amoebiasis. Preliminary results showed approximately 15 differentially expressed bands in pathogenic amoeba incubated with human collagen type 1 and Ca2+. Incubation showed formation and release of electron dense granules (EDG). Analysis of EDG showed maximum collagenase activity. Purified EDG was used to raise antibodies in rabbit which were used in immunoscreening of the cDNA library constructed from pathogenic E.histolytica. Further sub cloning and characterization of cloned fragments is under progress.
The re-emergence of human group B rotavirus (HuGBR) in India was accompanied by a recognition of the need for the development of sensitive and rapid diagnostic techniques for the early detection of this potentially highly virulent pathogen. A number of primers were designed from different genes of a previously sequenced adult diarrhoea rotavirus (ADRV) strain for detection of HuGBR strains by reverse transcription polymerase chain reaction (RT-PCR). Altogether 32 primers were selected for detection of gene segments coding for viral structural proteins V4, VP6, VP7 and non structural proteins NSP1, NSP2, NSP3, NSP4 and NSP5. The standardization of the RT-PCR conditions has resulted in amplification of various genes in identical reaction conditions.
An ELISA test developed by the National Institute of Virology (NIV), Pune for detection of rotavirus in stool specimens has been modified and the new rapid test standardized. The test can now be completed in 2-3 hours.
The genesis of a new cholera causing serogroup V.cholerae 0139 formed the impetus to search for V.cholerae O139 phages in and around the country.A comparative study of the phage types of the O139 strains isolated in 1992 and 1993 and between 1996 to 1998 showed that during both periods, phage type 1 was the predominant type. The percentage of O139 strains isolated in 1992 and 1993 belonging to phage type 1 (40.46%) was much higher than the percentage of strains recovered from 1996 to 1998 (34.69%). Molecular studies have shown substantial changes in the organization of the CTX phage module of O139 strains isolated from 1996 to 1998 compared to the organization of the CTX phage module of O139 strain isolated in 1992 and 1993.
The most important finding was the almost complete typability of the O139 strains with a set of five phages. This phage typing scheme would be useful in the study of the epidemiology of cholera caused by V.cholerae O139.
Fig 3 a-c CTX prophage of Vibrio Cholerae 0139 strains
Studies were carried out by NICED , Calcutta on restriction fragment length polymorphism of CTX prophage in view of the drug resistance pattern seen among O139 strains isolated in Calcutta from 1992 to 1998. The results indicated that there is a continuous change in the structure and organization of CTX prophage during the study period along with emergence of a new type of CTX prophage. The 1992-93 strains (Fig.3a) showed two CTX prophages connected by an RS1 element while the 1996 strains (Fig.3b) showed three CTX prophages arranged in tandem. Most of the 1998 strains (Fig.3c) from Calcutta exhibited only one CTX prophage while those isolated from other parts of India are identical to the 1996-97 strains or showed two CTX prophages arranged in tandem. In 1996, O139 strains exhibited two types of CTX prophages with the first of the three prophages being an ElTor-type CTX prophage and the second and third CTX prophages being a new type of CTX prophage, with the difference primarily lying in the rstR gene which codes for the repressor proteins of CTX. In 1998, it was observed that two new clones of O139 have evolved probably from the 1996-97 strains with two epicentres namely Calcutta and Alleppey. Calcutta strains showed only the ElTor-type CTX prophage and not the unique O139 CTX prophage of the 1996 strains while reverse was the case with the Alleppey strains. Therefore, presently, there are two clones of O139 circulating at two locations with different CTX prophages indicating that reassortment in the genome is taking place in the O139 strains. This molecular epidemiological study revealed clonal diversity among the O139 strains and emergence of new epidemic clones, as evidenced by the change in the structure, organization and location of the CTX prophages over a period of seven years.
Continued surveillance of V.parahaemolyticus mediated diarrhoea implicated the predominant association with disease caused by the O3:K6 serovar during 1998-99. Arbitrarily primed PCR (AP-PCR) of these recently emerged O3:K6 strains with those isolated previously from other countries showed that the new strains exhibited a unique profile different from the old strains. Thus, the Calcutta O3:K6 strains and those isolated from different countries were considered to be clonal. Analysis of the toxRS region of the new and old O3:K6 strains was done on the assumption that variation in the toxR sequence may be found in phylogenetically distinct clusters of V. parahaemolyticus. The difference in the sequence between the new and the old O3:K6 strains ranged from 11 to 14 bp within the 1,364 bp region covering 95.4% of the toxRS coding regions, and the sequences differed invariably at 7 base positions (Fig.4). Based on these results a PCR method, referred to as group specific PCR (GS-PCR), was designed to distinguish the new from the old O3:K6 strains. The results indicate that the GS-PCR positive strains belonging to O4:K68 and O1:KUT serovars are genetically very close to the new O3:K6 clone but they also exhibited AP-PCR profiles indistinguishable from that of the new clone. Therefore, GS-PCR positive O4:K68 and O1:KUT strains may have diverged from the existing new O3:K6 clone by alteration of the genes associated with the O:K antigens and followed a spreading pattern similar to the new O3:K6 clone.
Fig 4: Target positions of the PCR primers used to amplify the Vibrio parahaemolyticus tox RS sequences and the essential base difference in the tox RS sequence between the old 03:K6 strain group and the new 03:K6 clone.
A double-blind, randomized, controlled clinical trial was conducted by NICED, Calcutta on malnourished children with acute dehydrating diarrhoea to evaluate the efficacy of oral supplementation of zinc as an adjunct therapy to oral rehydration solution (ORS). Eighty children were randomized to receive zinc sulphate in three divided doses equivalent to 40 mg elemental zinc, in a syrup form and syrup placebo. Clinical parameters and microbiological findings of stool samples were comparable in the two groups at the time of enrolment. All the children (100%) in the zinc supplemented group and 32 (89%) children in the placebo group recovered within 5 days of hospitalization. The zinc supplemented group had a significantly shorter duration of diarrhoea, passed less liquid stool, and consumed less oral rehydration solution and other liquids as compared to the placebo group. These results suggest that zinc supplementation as an adjunct therapy to ORS has beneficial effects on the clinical course of dehydrating acute diarrhoea.
Following the cyclone in Orissa there was a sudden outbreak of gastroenteritis/diarrhoea in the cyclone affected areas. A total of 107 stool samples were collected from different hospitals/PHCs/CHCs of the cyclone affected areas. Rectal swabs were collected from patients with diarrhoea before any antibiotic was administered. Of the 107 samples, 83 (77.6%) were found to be culture positive and 24 (22.4%) were culture negative. Of the 83 culture positive samples, 66 (79.5%) had V.cholerae, 16 (19.3%) E. coli and 1(1.2%) had Shigella flexneri. Of the 66 V. cholerae isolates, 60 were V. cholerae 01 Ogawa and 6 V. cholerae 0139. Clustering of cases of V. cholerae occurred in the worst affected districts of Cuttack and Jagatsinghpur (Erasama, Balikuda, Kujanga, Manijanga) and Astaranga and Kakatpur areas of Puri district.
All isolates were found to be sensitive to tetracycline, ciprofloxacin, furazolidine, strepto-mycin, cotrimoxazole, norfloxacin, gentamicin but resistant to nalidixic acid. The 66 V.cholerae isolates were further analysed at the molecular level with the help of NICED, Calcutta. Multiple PCR assay was done using primer specific for ctx A, tcp A gene for the detection of cholera toxin and toxin co-regulated pilli gene (Classical and ElTor). It was observed that 59 of 60 V.cholerae isolates belong to V.cholerae 01 serogroup and ElTor biotype and were positive for ctx A gene. Of the 6 V. cholerae 0139 strains 5 harboured ctx A gene.
Besides the antibiogram, the strains were randomly selected from different areas for molecular epidemiological study using random amplification of polymorphic DNA (RAPD) analysis of the isolates. For ribotyping, Bgl 1 digested chromosomal DNA was probed for 16S and 23S rRNA. Almost all the strains exhibited ribotype R3 pattern, as it has been reported from different parts of the country after the emergence of 0139 strains of V. cholerae. Primers 1281 and 1283 were used for RAPD to detect clonality, if any. Like the ribotyping results, the V. cholerae 01 strains exhibited similarity with Calcutta strains i.e. V. cholerae 01 that appeared after the V. cholerae 0139 epidemic.
OTHER MICROBIAL DISEASES
A study is being supported at the Vision Research Foundation, Chennai to develop PCR for the detection and genotyping of Chlamydia trachomatis in patients with conjunctivitis. This study envisages molecular amplification of omp-1 gene encoding for major outer membrane protein (MOMP) to help in the epidemiological investigation of C.trachomatis infection, identification of individual genotypes to correlate with symptoms, clinical findings and histopathology in conjunctivitis in the hospital-based population. Fifty five consecutive patients of primary conjunctivitis have been clinically examined and conjunctival scrapings were investigated for bacteria, herpes simplex virus (HSV) and adenovirus and C. trachomatis infections. Almost half the specimens were smear and culture positive for C. trachomatis out of 55 conjunctival scrapings obtained from both eyes. PCR has been standardized to detect omp-1 gene and common endogenous plasmid gene. DNA extraction from the specimens has been done. Molecular amplification of omp-1 gene would now be carried out.
Studies are being conducted at the IOP, New Delhi for development of DNA probe for detection of C. trachomatis infection. During the year under report, PCR has been standardized for detection of DNA in clinical samples using plasmid as well as MOMP primers. So far, 135 samples have been tested by PCR using different primers. The presence of an additional lower sized band apart from the usual 540 base pair band was most probably due to some variation in the particular gene sequence that was amplified showing thereby strain polymorphism on the MOMP gene.
In addition to developing a diagnostic method for C. trachomatis, fluorescent in situ hybridization (FISH) is being used to detect C.trachomatis in McCoy cell culture and in clinical specimens. For this, fluorescein labelled probes targeting rRNA sequences are being examined under a fluorescence microscope after in situ hybridization.
The incidence of cryptococcosis is expected to rise especially in the wake of HIV/AIDS epidemic. Recently Cryptococcus neoformans var. gattii has been isolated from patients with HIV/AIDS both from north and south India. This agent has also been isolated from Eucalyptus camaldulensis trees from border areas of Punjab. Therefore, a project was initiated at PGIMER, Chandigarh to study the natural habitat of C. neoformans var. gattii strains in the environment of Punjab and north Karnataka. This would also help in understanding the epidemiology of the disease. In the first phase, various trees like mango, eucalyptus, bamboo, banyan, pipal, neem evaluated in and around Chandigarh and Belgaum have been found to support the growth of C.neoformans var. gattii. The study is in progress.
To identify the specific immunodominant fractions of various agents of zygomycosis which could be exploited for serological diagnosis of this disease, a study is ongoing at PGIMER, Chandigarh. Hyperimmune sera were raised in outbred healthy New Zealand white rabbits against 12 crude antigens (6 metabolic & 6 homogenate antigens) of 6 medically important agents of zygomycosis. These sera were used in gel diffusion, counter current immunoelectrophoresis (CIEP) and immunoblotting, to find out specific and common immunodominant fractions. Cross reactivity of these antisera was also checked. Several precipitation lines were seen by gel diffusion and CIEP against various antigens. On immunoblotting the cross-reacting and immunodominant antigen was found at 82 kDa fraction. Further characterization of the antigen is being done.
The procedure for the in vivo study of zygomycosis was standardised. Immunosuppression of mice was done with cyclophosphamide given intramuscularly. These immunosuppressed mice were injected intravenously with spores. Four categories of inoculum size were taken i.e. 2x106, 4x106, 8x106 and 1x107 spores/ml of Rhizomucor pussilus. 4x106 spore size was found optimum for development of invasive zygomycosis.
Acinetobacter species are opportunistic pathogens and can lead to invasive diseases in a compromised host. Apart from being important hospital acquired bacterial pathogens, they also commonly colonise the sites of endotracheal tubes, indwelling intravenous cannulae, central venous lines and urinary catheters. The possibility of Acinetobacter species becoming multidrug resistant also exists. Studies were initiated at AIIMS, New Delhi to assess the mode of transmission of this bacteria from the hospital environment to the patients especially in the intensive care units (ICU) by typing various isolates obtained from the colonization sites and environmental sites. Strains (147) of Acinetobacter species isolated from the neurosurgery ICUs (125 from patients and 22 from the environment) were studied. Acinetobacter baummi was the most common isolate (114/147). Antimicrobial resistance studied by MIC (agar dilution method) showed that 59.2% of the isolates were multidrug resistant (to all the newer antibiotics including third generation cepha-losporins, amikacin, ciprofloxacin etc.). However, all the strains have remained sensitive to Imipenem. Whole cell protein analysis was done by SDS-PAGE. AP-PCR and ribotyping are being standardized and these would be compared with the conventional methods.
A study was recently completed to standardise isolation techniques and to develop immuno-diagnostic methods (IFA and ELISA) for Mycoplasma pneumoniae at AIIMS, New Delhi. M.pneumoniae was identified as an important cause of community aquired pneumonia (CAP) both in paediatric (29.80%) and adult age groups (36.6%). Using M.pneumoniae antigen detection in throat swabs by IFA infection could be demonstrated in 19.87% children and 36.6% adults. Incidence was found to be high in immunocompromised (40%) as compared to immunocompetent subjects (17.5%). If the presence of IgM antibodies alone is considered the number of positive patients would be much higher i.e. 29.80% in the paediatric age group. Rapid diagnostic procedures like antigen detection and demonstration of IgM antibodies should be more widely used to detemine the infective etiology early in the course of illness.
Another study was recently completed at PGIMER, Chandigarh on identifying cellular immune resonses in human and experimental cysticercosis. The results revealed that in cysticercosis both humoral and cellular mechanisms may be playing a role in host defence mechanisms. In experimental cysticercosis, studies on kinetics of immune responses showed that the cellular mechanisms were triggered at a late stage compared to humoral responses and may persist longer. In human neurocysticercosis, studies on lymphocyte-proliferative responses indicated that specific antigens might be playing a role in eliciting T cell responses. Immunophenotyping analysis indicated an insignificant increase in B cells and a decrease in total T cells. However, there was a significant decrease in CD8+ cells and no change in CD4+ cells, in contrast to experimental study whereby significant increase in CD8+ T cells was observed. The cytokine profile indicated involvement of TH-I like responses as significantly higher levels of g-IFN and IL-2 were observed. In chronic helminthic infections, shift from TH-I type immune responses in early infection to TH-2 type response in advanced infection has been reported and might occur as well in long standing human cysticercosis. Since the study was cross-sectional, shift could not be assessed. However, the exact role of cell mediated immune responses in human neurocysticercosis and that of cytokines in patients after medical/or surgical therapy needs to be determined to identify any shift in the TH-I type of response after therapy.
The emergence of chloroquin resistance in P.falciparum and vector resistance to commonly used insecticides are the main obstacles in the control of malaria in the country. New technologies are being introduced for malaria control under Enhanced Malaria Control Programme. The roll back malaria programme has been launched simultaneously in all malaria endemic countries. These have thrown new challenges in malaria research. The Council’s institutes viz. Malaria Research Centre (MRC), Vector Control Research Centre (VCRC) and other institutes are making efforts to address these problems through focused research in vector and parasite biology and ecology, development of malaria control tools, drug development, testing and validation of new technologies.
Integrated Vector Control
Under Integrated Disease Vector Control (IDVC) project, research activities at the field stations were concentrated on the evaluation of new antimalarial drugs, diagnostic kits, insecticides, and repellents; transmission dynamics of malaria in different ecosystems, epidemic investigations, development of an action plan for malaria control; and transfer of technology on bioenvironmental methods for malaria control.
Strategies of bioenvironmental interventions using larvivorous fishes have now been well established by MRC, Delhi and currently preparations are on to extend bioenvironmental interventions to the entire Karnataka state including mosquito control in Bangalore city. The technology has also been transferred to Maharashtra and within two years the bioenvironmental methods have spread to the entire state which is being implemented through the PHC system. In Gujarat 174 fish hatcheries have been established for use in the entire state.
In Panaji, Goa, the MRC has used biopesticides and larvivorous fish in controlling Anopheles stephensi breeding and consequently malaria. Bioenvironmental control of malaria has been launched in Panjim (Goa) and Ahmedabad (Gujarat) by the respective city corporations with the technical support of MRC with a marked reduction being seen in mosquito populations and in the transmission of malaria in both the cities. An action plan for the control of malaria for the entire Ahmedabad city has been prepared by MRC. The Goa field station has actively participated during the construction of the Konkan Railway. The technology transfer of the bioenvironmental control project to the State health authorities is being undertaken. Konkan Railway project and Marmugao Port authorities have fully accepted the suggestions made by the Goa field station on malaria control and continue to use their consulting services.
Malaria in Tribal Areas
The VCRC, Pondicherry carried out a study on malaria control in Malkangiri district to assess the feasibility of reducing the morbidity and mortality from malaria in the tribal population by providing early treatment through traditional healers. Disaris were willing to treat malaria cases with chloroquine but their record keeping and reporting was poor, as majority of them had no formal education. Therefore, they were trained to provide treatment with the help of a pictorial guide and pre-packed tablets. A total of 110 disaris from Malkangiri PHC area were trained. Disaris could follow the recording system and in a period of 9 months (April - December, 1999), they treated 27021 patients in all age groups. Most of them administered the correct dosage and reporting of cases was regular. A total of 29 severe/ complicated cases were referred to the PHC. The impact assessment is being done.
Susceptibility of Anophelines to P. vivax
A study was carried out by MRC, Delhi to assess and compare susceptibility of three recognized malaria vectors i.e. An. stephensi, An. sundaicus and An. fluviatilis species T to Plasmodium vivax sporogony. The laboratory colonized mosquitoes of each species were fed through artificial membrane on P. vivax infected blood drawn from selected patients. The mean number of oocysts, oocyst rate and sporozoite rates were determined for each of these test species. The results have revealed that An. stephensi and An.sundaicus are equally susceptible to P. vivax sporogony whereas in An. fluviatilis species T significantly lower oocyst rate, mean number of oocysts and sporozoite rate were recorded (Fig.5).In field studies, no sprozoite positive specimens were found in An. fluviatilis species T and this species was almost totally zoophagic.
Characterization of An. fluviatilis Complex
An. fluviatilis, a major vector of malaria exists as a species complex comprising 3 sibling species. Being morphologically identical, the incrimination of the vector species is difficult. The VCRC, Pondicherry has developed rDNA-ITS2 based primers for two of the sibling species of the An.fluviatilis complex. These probes were evaluated for their sensitivity and specificity in the differentiation of An. fluviatilis adults collected from different areas of Jeypore and Malkangiri, Orissa.
The VCRC, Pondicherry carried out studies to simplify the PCR technique (both in terms of time and cost). The complete procedure i.e., from DNA extraction to visualizing the product has been brought down from 24 to 5 h. The cost in terms of reagents for DNA extraction has been brought down from Rs. 40 to Rs. 2 per sample. The level of specificity of this PCR assay for the detection of a single An.fluviatilis adult in pools of other mosquitoes was studied. It was found that this PCR assay was able to detect a single An.fluviatilis adult in a pool containing 40-45 Culex quinquefasciatus mosquitoes.
Insecticide treated mosquito nets are known to reduce the man-vector contact, density of the malaria vectors and the incidence of malaria. Therefore, the efficacy of alphacypermethrin, a synthetic pyrethroid, treated mosquito nets was assessed by the VCRC against malaria vectors in Malkangiri district, Orissa. Villages were chosen for evaluation and distribution of alpha-cypermethrin- treated or untreated nets or no-nets before the onset of monsoon season. Indoor resting density of vectors and malaria incidence in the three groups of villages were monitored fortnightly one year before and after the distribution of nets. Re-impregnation of mosquito nets was done in October 1999 to cover the peak transmission season. The reduction in the density of An.fluviatilis was 97.1% after the first treatment and 99.5% after re-impregnation compared to the pre-intervention period and the check area. The corresponding values for untreated nets were 66.1 and 62.4% respectively. The reduction in malaria incidence was 79.2 and 85.7% during the corresponding periods. There was no marked effect on the density of An.culicifacies after the first treatment and the density was low in all the villages during the next season. The use rate of mosquito nets ranged from 20 to 100%, the lowest was during the colder months, December-January.
The indoor residual application of insecticides reduces the density and longevity of malaria vectors and incidence of malaria. Bendiocarb, a carbamate adulticide has not been studied for indoor residual treatment against malaria vectors under Indian conditions. Therefore, bendiocarb (80% WDP) for indoor residual treatment was evaluated by VCRC, Pondicherry for its efficacy against malaria vectors, An.fluviatilis and An. culicifacies in Malkangiri district, Orissa.
The spray coverage was about 90% in human dwellings and 100% in cattle sheds. About 50% of the sprayed houses were mud plastered one month after the spray. There was 94.9 and 86.2% reduction in the resting density of An. fluviatilis and 16.9 and 26.8% in malaria incidence after the spray of 0.4 g/m2 and 0.2 g/m2 respectively. The density of An. culicifacies was low in all the three groups of villages in the season.
Artemisinin, an antimalarial principle isolated from Artemisia annua L. is a sesquiterpene lactone with excellent antimalarial activity. Three formulations viz. Artesunate, Artemether and Arteether are registered in India. A study has been undertaken to evaluate the comparative safety and efficacy of these formulations administered as follows for the treatment of falciparum malaria (i) a-b Arteether 150 mg i.m. O.D. for 3 days, (ii) Arteether 80 mg i.m. B.D., for 3 days; and (iii) Artesunate 120 mg loading dose i.v. and 60 mg i.m. once daily for 5 days.
The results showed fever clearance time of 26.8, 28.09 and 35.65 h and parasite clearance time of 31.2, 31.2 and 28.0 h respectively in groups (i), (ii) and (iii). No significant side effects were seen in any group. Further study to explore the comparative recrudescence rate and gametocytocidal activity is in progress.
To understand the molecular mechanism of development of chloroquin (CQ) resistance, polymorphism of pfmdr 1 gene of CQ sensitive (CQS) and CQ resistant (CQR) strains of P.falciparum was studied at MRC, Delhi using PCR. Twenty two chloroquine resistant and 18 chloroquine sensitive P. falciparum strains were examined for nucleotide changes in pfmdr 1 gene. Results indicate a strong association but incomplete correlation between CQ resistance and allelic variation of pfmdr1 gene in P.falciparum isolates from India. This suggested that the pfmdr1 gene was not solely responsible for CQ resistance and the study of a combination of mutational changes would be required to determine CQ resistance.
The importance of T cells in malaria immunity has been appreciated for a long time. However, T cell epitopes show variation. Two T helper cell epitopes (Th 2R and Th 3R) have been identifed in circumsporozoite protein (CSP). Genetic variation in Th-epitopes of CSP of 40 P.falciparum isolates, collected from Delhi, Jabalpur (M.P.) and Allahabad (U.P.) has been studied. An apparent trend of regionally unbiased restricted polymorphism has been observed among Indian isolates. The nucleotide changes observed have not been reported earlier.
Fig 6 : Distribution of An. dirus in India.
Geographic Information System (GIS)
GIS was used to map the distribution of malaria vector An.dirus in India. Topo sheets published by the Survey of India in the scale of 1: 6,000,000 were digitized to prepare thematic maps of soil, altitude, rainfall, temperature and forest area. The digitization and analysis was done using PC based ARC/Info 7.0 and Arc View 3.1. The altitude map s was taken as the base map, An. dirus has been reported up to 4500 m, these areas were extracted from the altitude map. Rainfall >2000 mm and temperature between >20 to <27.5oC and evergreen forest plus tropical moist forest cover areas were taken as favourable zones. Areas having these ranges were extracted and overlaid on altitude map. Using GIS capabilities intersection of these layers was extracted to map areas favourable for An. dirus (Fig.6).
The distribution of An. dirus mapped by GIS analysis was compared with the reported distribution. The areas where the species has been reported could be located in the predicted GIS map, with many new areas being found in GIS distribution map. The study is in progress.
Management of Malaria Information System
An efficient management and timely flow of information is crucial for any forecasting system and timely implementation of intervention measures. GIS is a powerful tool for management of information system as it allows integration of both spatial and non-spatial information management. A prototype has been developed for maintaining a Malaria Information System.
With the help of this information system one can reach a street and even the houses, and house-wise information such as number of persons, their names, ages, educational status, malaria history, drug resistance level etc. can be attached for instant retrieval. This type of information would allow study of malaria epidemiology in space and time and relate change in the malaria scenario with its determinants for taking timely corrective control measures.
Malaria Parasite Bank
This is a centralized facility at MRC, Delhi to collect human and non-human plasmodial species and strains from different geographical regions. Depending on the species of the parasites, they are cryopreserved in liquid nitrogen or are cultivated in vitro and characterized for various studies using standard techniques. The characterized parasites are also cryopreserved for various collaborative studies. The Parasite Bank also supplies biological materials including non-human and human plasmodia to various research organizations mainly for collaborative studies.
The Council’s Vector Control Research Centre (VCRC) at Pondicherry and RMRC, Bhubaneswar have focused their research in the field of vector biology and control, clinical epidemiology and chemotherapy, applied field research and product development. The Centre has geared its activity towards transferring the technical know-how to the field for optimum and appropriate application for elimination of filariasis so that latest technical knowledge is absorbed into the Programme.
Epidemiological Mapping of Lymphatic Filariasis in India
Rapid mapping methods need to be standardized for prioritisation of areas for intervention. This is an essential step to work towards the elimination of filariasis. The rapid methods for the estimation of prevalence of bancroftian filariasis and its mapping by grid sampling technique are being validated.
An area covering 41,950 km2 from the eastern coast of India stretching towards the west from Pondicherry, lying between 7809’25’’ and 80013’48’’ East, and 1100’54’’ and 1302’31’’ North was selected for the study. For sampling, a 25 x 25 km grid was overlaid over the study area covering 13 districts. Data on filariasis were collected through questionnaire method; physical examination by health workers (PEHW) for scrotal swelling and immunochromatographic card test (ICT) were done.
Spatial analysis were carried out using a GIS software (GS+) to see whether there was any spatial pattern in the occurrence of filarial disease (scrotal swelling) or antigenaemia in the study area.
For this, variogram models were fitted to data on prevalence of scrotal swelling and filarial antigenaemia. Further advanced analyses with S PLUS after removal of trends (detrending) in the data showed that still there were no spatial differences in the prevalence of antigenaemia. Construction of variograms using the residuals obtained showed that still there was no spatial autocorrelation and that there were large-scale trends against longitude and the pattern was apparently random with latitude. Similar analysis is being carried out for disease prevalence also.
No spatial autocorrelation with respect to antigenaemia prevalence was detected. This could have been because the grid used was too big to capture spatial autocorrelation. The use of smaller grids has been planned for future studies. Therefore, a uniform grid sampling procedure may not be applicable globally.
The VCRC carried out a study to validate the application of epidemiological model LYMFASIM under operational settings for predicting the required duration of interventions for vector control and for mass annual single dose of DEC. The required duration of vector control was determined by simulating the effect of vector control (assuming 80% reduction in monthly vector biting rate, average for 5 yr integrated vector management programme in Pondicherrry) over a period of 5, 10, 11, 12, 13, 14 &15 yr. Simulations were done for about 50 yr since the start of the control programme to predict the trend in prevalence of microfilaria (mf). For chemotherapy programme 4 strategies (5, 10, 15 & 20 annual DEC) were evaluated at 6 different levels of coverage. In simulating these strategies it is assumed that DEC has a microfilaricidal effect of 55% and adulticidal effect of 67% per treatment (based on a VCRC hospital trial). For the treatment programme the required duration of control for each strategy was estimated by calculating the risk of acquiring new infection in children aged less than 5 yr. Each strategy was simulated 50 times. The risk of acquiring new infection is the proportion of simulations that result in a mf prevalence of 0.033% or more (i.e. 1 per 3000 children) over a period of 5 yr after cessation of control. The simulation results suggest that a minimum of 13 yr of vector control with at least 80% reduction in man biting rate of vector mosquitoes is required for elimination of infection and that the minimum coverage required for complete interruption of transmission is 90% and 60% for 5 and 10 annual rounds of DEC in areas with 8.0-10% mf prevalence.
Studies on Mass Chemotherapy
A study is ongoing at the VCRC, Pondicherry to compare the effect of single dose mass drug administration (MDA) with ivermectin and DEC independently and in combination on transmission, microfilaraemia, incidence of acute adeno-lymphangitis (ADL) and prevalence of chronic disease. Four blocks of five villages each were randomly assigned to one of the four arms viz., DEC, ivermectin, DEC with ivermectin and placebo. Initial two rounds of MDA were carried out at 6 monthly intervals for DEC, ivermectin and placebo arms in a double blind fashion. The combination arm with DEC and ivermectin was introduced after the completion of two rounds of MDA. Third and subsequent rounds of mass treatment were conducted in an open manner with the placebo village receiving no drug. The mean coverage during the fifth round of chemotherapy in 1999 was 63 and 61% in ivermectin and DEC blocks respectively and 65% for third round of combination drugs. The rate of adverse reactions ranged between 3.5 and 6.2%. Entomological evaluation showed a reduction in vector infection, infectivity and transmission parameters. Parasitological evaluation after the last round of mass treatment showed a reduction of 48 and 60% in mf rate, and 70-95% in mf density in DEC and ivermectin blocks.
Two alternative methods for delivering mass annual single dose DEC treatment are being evaluated for their operational and economic feasibility as well as impact. The methods include delivery through PHC system and community volunteers (CV). Two rounds of treatment have been completed following the pre-designed implementation process.
Post-treatment survey results on the impact of IEC show that posters and public announcements were the most effective community awareness tools. Community compliance estimated through sample surveys in 10 randomly selected villages for each approach showed that about 76% of the respondents in villages under community approach and 68% under PHC approach consumed the drug. Supervised drug administration was relatively higher under PHC approach (35%) than the CV approach (27%). The CVs visited in the evening, while the PHC staff worked during the day. Consequently, the percentage of people available at the time of distribution was higher in areas under CV approach (69%) when compared to that of PHC approach (39%).
There was a significant reduction in mf prevalence in both the areas after two rounds of mass treatment. The reduction was relatively higher in areas under PHC approach, but it was not statistically significant. Microfilaria density showed a reduction of about 70% in both the areas, after two rounds of mass treatment.
The mass DEC fortified salt programme to control filariasis was launched in 1996 to cover the Kanyakumari district of Tamil Nadu. The total target population was about 17 lakhs, distributed in 4 municipalities and 1885 villages. Fortification of salt with DEC (0.2% w/w) was done by the Tamil Nadu Salt Corporation and supplied to the district civil supply department. Distribution of salt to the community was carried out through Fair Price Shops under the Public Distribution System (PDS). A study was initiated by the VCRC to evaluate the impact of this innovative centralized distribution programme designed to achieve good coverage and community compliance. The disease was reported from 28 of 35 units. The prevalence of disease was found to range between 0.002 and 0.132% in different PHCs/Municipalities. Salt distribution (supply) against monthly demands was analyzed and it was found that there was a steady increase in supply from 3.15% in May 1996 to 34.59% in 1999. Analysis of data on the change in mf prevalence in relation to DEC salt distribution showed that there was a gradual decline in mf rate (Fig.7). Supply against demand was above 30% since July 1998. Microfilaria prevalence reduced to zero after 44 months of DEC salt introduction (August 1999). It is planned to assess antigenaemia prevalence in these seven villages using ICT.
Fig 7: Monthly coverage and mf prevalence in sentinel villages.
A study has been carried out by VCRC in 3 selected filariasis endemic urban localities viz.Pondicherry, Villupuram and Cuddalore following quantitative research methodology.Simple random sampling procedure was followed to select the respondents from the list of patients. A total of 200 respondents were covered in the three areas and the analysis showed that 55% suffered from adenolymphangitis (ADL), of whom 40.18% had utilized government facilities, 24.30% private health facilities and 35.52% opted for self treatment with drugs from the local drug sellers. However, about 2% of the patients did not take treatment for ADL. About 49% of the patients reported to seek treatment for the chronic disease. Accessibility to health care services, cost of treatment, attitude towards the providers and confidence on the treatment are some of the factors that determine the choice of treatment. Average direct cost for treatment of ADL episodes was estimated to be Rs.2.29, 10.43 and 73.12 when treatment was sought from drug sellers, Government facilities and private practitioners respectively.
Development of Molecular Tools
In order to develop infective stage specific DNA probes, 50 genes expressed in the infective stage larvae (L3) of Wuchereria bancrofti were identified by subtractive hybridization and sequenced at the VCRC, Pondicherry. Primers (probes) for six genes were designed and their potential in amplifying the respective genes was tested. Two probes (WbL31 and WbL36) amplified the cDNA of L3 stage larvae but did not amplify cDNA of mf, and L1 and L2 stage larvae. Thus, two infective (L3) stage specific probes developed were evaluated for their usefulness in detecting the infective stage larvae in vectors.
At VCRC, Pondicherry in efforts to develop filarial specific monoclonal antibodies for diagnostic purposes, two clones (B5 and E2) were identified based on their high reactivity with Wuchereria bancrofti (mf) antigens. They were moderately reactive with Brugia malayi (both mf and adult) antigens and poorly with Ascaris lumbricoides and Setaria sp. antigens. Clone B5 was tested for its sensitivity and specificity in detecting filarial specific antigens in the sera of individuals who were non-endemic normals, endemic normals, chronic filariasis patients and mf carriers as determined by membrane filtration technique. The antibody was found to be 91.7% specific and 71.4% sensitive.
In separate studies, in order to develop a simple and rapid method for the extraction of DNA of filarial parasite Brugia malayi, suitable for PCR, four different methods (A, B, C & D) were tried. Method D was found to be simple and efficient for the extraction of DNA from a single mf in pools of 25 mosquitoes and the DNA was suitable for PCR amplification yielding a band of 322 bp with primers specific for B. malayi. Dot-blot hybridization confirmed the specificity and sequence similarity of the PCR amplified fragment. On blind testing, 50 pools of infected mosquitoes tested positive and 50 uninfected tested negative. The DNA extracted by this method was stable for about one year. The cost of a single PCR reaction using DNA extracted by this method worked out to be Rs. 63.19 which is half that of the standard method and the hands-on time was minimized by five times. The new method besides being specific and sensitive as the standard method is also rapid, safe and cost effective in assessing the B.malayi infection in pools of vector mosquitoes.
Development and Testing of New Product
Preliminary characterization of the mosquito pupicidal metabolites of the bacterium, VCRC B426 showed that the active principle is a ~80 kDa protein. The active ingredient was formulated as a flowable concentrate. The LC50 and LC90 of this formulation against pupae of four vector species were determined. Pupae of Cx.tritaeniorhynchus were the most susceptible followed by Cx.quinquefasciatus, An. stephensi and Aedes aegypti. The shelf life of the formulation studied indicates that it is quite stable for at least 4 months.
Kala-azar is a major public health problem in the states of Bihar, West Bengal and Uttar Pradesh. Cases have also been reported in Delhi. Research on various aspects of kala-azar is being conducted mainly through the Council’s Rajendra Memorial Research Institute of Medical Sciences (RMRI) at Patna and various extramural projects. New research areas such as development of DNA/RNA diagnostic probes and their use in mapping of the distribution of the parasite in kala-azar endemic areas and epidemiological modelling have been identified recently. A Leishmania parasite bank has been established. Clinical trials of different combinations of existing drugs for the treatment of kala-azar are being undertaken to optimize the drug dosages and minimize the associated side reactions. Disease epidemiology is being studied to assess the infection dynamics in the population, and prediction of epidemics through application of remote sensing technique.
A longitudinal study for the estimation of infection dynamics of Leishmania donovani in a kala-azar endemic population in Bihar was undertaken at RMRI, Patna. A total of 433 (80%) individuals of all age groups and both sexes from a defined endemic village was examined for their clinical status, and response to direct agglutination test (DAT) and leishmanin skin test (LST). Two rounds of vector surveillance in each household were carried out to assess the morphology, density and infection status of the vectors.
Of the 433 subjects screened, 88 (20%) were found LST positive. No significant difference in leishmanin positivity was observed between males and females. The frequency of leishmanin positives increased with advancement of age reaching a peak in the age group of 35-44 yr. Seropositivity (DAT) was found in 42.75% of the population screened being highest in the age group of 26-34 yr. No significant difference in seropositivity was noticed between males and females. The cohort is being followed up every six months to assess the infection dynamics.
Molecular Biological Studies
Studies were carried out at RMRI, Patna to develop nucleic acid probes and PCR primers for diagnosis and detection of kala-azar, to study strain variation and to develop prototype kit for easy field application. Genomic nuclear DNA from the promastigotes of 17 different isolates was restriction digested using EcoR1, HindIII, Hinf l and HaeIII enzymes. HaeIII pattern was similar at one and different in one restriction site of DNA in KA and post kala-azar dermal leishmaniasis (PKDL) isolates. In a comparative study, the kala-azar isolates reflected that the SAG responsive and unresponsive cases differed at EcoR1 and HaeIII restriction sites. A PCR-RFLP system has been standardized targeting the spacer sequence with rRNA gene. DNA of 17 isolates, 15 visceral leishmaniasis (VL) and 2 PKDL along with one reference strain was amplified by PCR using the same primers. Differences were observed at different sites of 5 VL (4 unresponsive and 1 strain responsive to SAG) and 2 PKDL isolates as evidenced from different banding patterns of nucleotide base pairs. Two SAG responsive and 8 unresponsive strains showed similarity. These strains belong to Patna, Vaishali, Muzaffarpur, Samastipur, Begusarai, Katihar, Madhubani and Buxer districts.
Field trials were carried out by RMRI, Patna to establish DAT as a diagnostic tool for kala-azar and for mass screening of the population for epidemiological studies. Sensitivity and specificity were found to be 91.6% and 100% at a dilution of 1:800 (cut-off titre). Positive and negative predictive values were 100% and 98% respectively at this dilution.
Validation studies carried out after 6 months in an endemic area, indicated that 18.6% of the subjects were DAT positive during the first survey, of whom 1.9% continued to be DAT positive and developed clinical symptoms of kala-azar during the second survey. None of the subjects DAT negative in the first survey was found positive in the second survey. Periodic evaluation for validity is being monitored at 6th, 9th and 12th months in the same population group.
A study was carried out at RMRI, Patna to assess the efficacy of immunohistochemical (IHC) staining technique in the diagnosis of PKDL in comparison to the conventional methods like tissue smear and culture. The results of the study indicated that IHC technique gave better yield of sensitivity (83%) as compared to biopsy smear (66.6%) and culture (33.3%). More samples are being studied for confirmation and at different stages of treatment.
PCR primers based on kinetoplast DNA were designed at the Institute of Pathology (IOP), New Delhi in order to develop a PCR assay for kala-azar and PKDL. The assay was found to be highly sensitive with culture isolates as well as samples isolated from patients.
A study was carried out at RMRI, Patna to assess the frequency of unresponsiveness to SAG (20 mg/kg body wt for 30 days) in 64 previously untreated, confirmed patients of kala-azar and to evaluate whether any factor(s) related to disease are associated with unresponsiveness. Sixty two patients completed the full course of treatment and two developed adverse reactions. The apparently cured subjects (clinical and parasitological cure at the end of treatment) were 30 (48%) and unresponsive at the end of the treatment were 32 (52%). It was observed that unresponsiveness was not associated with age, sex, duration of illness, existence of other associated diseases or Hb levels in the patients. Distribution of patients from different endemic zones was observed to be significantly associated with frequency of SAG unresponsiveness.
A pilot study to assess the efficacy of SAG in the treatment of PKDL was carried out at RMRI, Patna. Thirteen patients with different clinical forms of PKDL were treated with SAG 10 mg/kg body wt. continuously for 90 days. The patients had macular (7), nodular (5) and papular (1) dermal changes. Only 10 had a past history of kala-azar (2-12 yr prior to the onset of PKDL). Patients(8) who completed the treatment were parasite negative at the end of 3 months and had reduction of dermal lesions, but did not show complete clearance at 2 months post treatment. These patients are being followed up till complete disappearance of lesion or relapse.
A study was carried out to find an association, if any, between differentially expressed CD2+T cells with immunological profile (IL-2 and MIF) in kala-azar patients during treatment with SAG.
Leishmania antigen failed to induce significant CD2+T cells in the peripheral blood (35.07%) and bone marrow (49.85%) of kala-azar patients during active infection presenting with low immunological profile. On the other hand, leishmanial antigen induced significant number of CD2+T cells in vitro in the sera of cured kala-azar patients (78.26%) and healthy controls (65%). This significant association between CD2+T cells in protected subjects was associated with significant MIF (36.85%) and IL-2 release. CD2 induction of T cells in SAG unresponsive cases was 43.64% in the peripheral blood and 46.87% in bone marrow which was associated with low MIF (22.33%) and IL-2 level by the T cells of such patients. The results indicate that CD2+T cell proliferation in response to Leishmania antigen is indicative of drug response leading to T cell activation and is possibly involved in influencing immunological profile in kala-azar patients.
A study was undertaken at RMRI, Patna to identify protein antigen fractions recovered from different Leishmania isolates from various geographical regions of Bihar by SDS-PAGE, and their diagnostic ability was examined by Western blot. A 8kDa antigen was frequently observed in isolates obtained from untreated and SAG unresponsive patients in kala-azar endemic and non-endemic areas. Sera from all kala-azar patients examined recognised the 8kDa antigen of L.donovani. However, sera from cured patients, normal individuals, tuberculosis and leprosy patients did not show any reactvity with 8kDa antigen indicating the specificity of 8kDa antigen to L.donovani only. This could be a potential candidate antigen for development of diagnostic tests. Isolation and gene sequencing of 8kDa protein fraction is being carried out.
Studies carried out at the IOP, New Delhi on Western blot analysis of 35 kala-azar positive sera revealed that leishmania antigens of 40, 55, 65, 70 and 82 kDa were recognized most frequently.
Majority (83%) of the sera from kala-azar patients recognized at least four of these five antigens. The 70 kDa antigen, which may include a member of the heat shock protein 70, produced a positive reaction in 94% of patients. Further studies are ongoing.
Studies are being carried out at the Indian Institute of Chemical Biology (IICB), Calcutta to study the role of host protein kinase C (PKC) in the uptake and multiplication of L.donovani promastigotes by mouse macrophages. Results showed that staurosporine, an inhibitor of PKC, inhibited phorbol myrsitate acetate (PMA) dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. The study has relevance on membrane fluidity and leishmanial infection.
Study has been taken up at RMRI, Patna to find out different species of Phlebotomus argentipes population in order to incriminate the vector responsible for transmission of kala-azar. Survey has been conducted in different kala-azar endemic districts of Bihar (Patna, Vaishali and Samastipur). Sandflies of different species – P. argentipes, P. papatasi and Sergentomyia species were collected in different seasons. Besides these, two new species i.e.P.sergenti and P.major were also identified using the key of Lewis. Morphological variations were observed in specimens of P.argentipes in the measurement of genital filament (0.124-0.294 mm), aedeagus (0.017-0.139 mm), coxite (0.077 – 0.124 mm) and style (0.093 – 1.7 mm).
DNA from pooled wild caught sandflies and total DNA probe prepared from reference strain of L. donovani were used for dot blot hybridisation assay. The result revealed that out of 92 pools, 59.78% showed the presence of parasite (Leishmania) and P. argentipes showed 65.8% positivity. High parasite positivity indicates high transmission and is the reason for endemicity.
Establishment of Leishmania Bank
The leishmania parasites isolated from kala-azar/PKDL patients manifesting different clinical pictures/severity of disease, from different geographical regions of Bihar, need characterization on aspects of their growth kinetics pattern, virulence, isoenzyme type and RFLP. So far 45 isolates (KA-36 and PKDL-9) of Leishmania promastigotes are being maintained in vitro, 33 are under adaptation and a few have been cryopreserved. The isolates are being utilized in various studies and are provided to other institutes on request for research purposes.
Application of Remote Sensing for Prediction of Kala-azar Epidemics
Study has been initiated to evaluate the feasibility of application of remote sensing by use of satellite data for monitoring the macro-ecosystem, specific vegetation cover and human settlements and to relate them with the changing sandflygenic conditions. It has applicability in epidemic prediction in the hyper-endemic Vaishali district and non-endemic Lohardagga district. Five villages were selected randomly in one block of each district. Vector density in both study sites observed in different seasons for the last two years showed consistent pattern. Satellite data of selected foci are being collected to compare with the digitized map of the same foci to generate data on the association of sandflygenic conditions with environmental determinants to assist prediction.
Viral diseases are major public health problems. Research in the area of viral diseases is being undertaken at the Council’s National Institute of Virology (NIV), Pune, Enterovirus Research Centre (EVRC), Mumbai, National AIDS Research Institute (NARI), Pune, Centre for Research in Medical Entomology (CRME), Madurai and Virus Unit, Calcutta. Studies for developing vaccine against Japanese encephalitis (JE), surveillance of poliomyelitis, molecular epidemiology of wild polioviruses, molecular biological studies in hepatitis and AIDS are some of the important areas of research which have been addressed during the year under report.
At NIV, Pune B and T cell epitopes have been identified on the JE virus envelope protein. A chimeric peptide has been synthesized which contains one B and one T cell epitope. The chimeric peptide when used as an immunogen was only partially protective against live virus challenge. Continuing this work further, 14 new T cell epitopes were identified, some on NS1, a nonstructural protein coded by the JE virus. Some of the epitopes were found to have a T helper cell response suggesting their immunogenic potential. These studies will lead to development of a cocktail vaccine representing additional cellular and humoral immune responsive epitopes which would offer complete protection against JE virus infection.
Studies employing monoclonal antibodies suggested that some of the epitopes on the JE virus envelope protein have molecular mimicry with the host’s histone proteins. During natural JE infection antibodies generated against such epitopes react with cellular histones as auto-antibodies. Their role in JE virus pathogenesis is under study.
The role of Anopheles subpictus in JE virus transmission in South Arcot district, Tamil Nadu has now been elucidated. Vector abundance coincided with the time of paddy cultivation but virus isolations were made throughout the year. Generally there were two peaks of both vector abundance and virus isolations – a wet season (August-December) and a dry season (March-June) peak (Fig.8). Being mainly zoophagic, An.subpictus appears to play a role in zoonotic cycle of JE transmission.
Longitudinal study of ecology and JE virus infection of mosquitoes in Kuttanadu (Kerala) seems to have provided a clue to the role of Mansonia sp. in JE transmission. The results of the study suggest that Culex tritaeniorhynchus and Mansonia sp. served as vectors for JE virus transmission in a sequential way – mansonioides maintaining the zoonotic cycle during the period when JE infection in humans is not apparent (April-November) followed by virus amplification in Cx.tritaeniorhynchus in the period (December- March) when encephalitis cases are reported (Fig.9). Thus Cx.tritaeniorhynchus appears to act as the primary vector and mansonioides as the secondary vector in Kerala.
Acute Flaccid Paralysis Surveillance
The four metro cities (Delhi, Mumbai, Calcutta and Chennai) have a high incidence of paralytic poliomyelitis. Urban slums are the main breeding ground of the poliovirus. Eradication of poliomyelitis from these cities is considered difficult because of the high population density, large slum population and also because these cities attract patients from all over the country for treatment.
During 1999, 194 patients of acute flaccid paralysis (AFP) were recorded in Mumbai, of these 116 were residents of Mumbai (a decline of 31% over the previous year) and 78 were non-residents. AFP cases were reported from 15 out of 16 municipal wards. Most cases occurred in slums. The four wards (F,G,M and R) historically known to contribute high numbers of poliomyelitis cases had 52.4% of all reported cases of the city. Faecal samples from 109 (94%) cases were studied for enterovirus detection; 69 (63.3%) were found to be virologically negative. Wild poliovirus was isolated only from three cases, vaccine strains from 12 and non-polio enteroviruses were isolated from 25 (22.9%) cases. These results showed that the historical hot spots of wild poliovirus transmission have been completely eliminated by the OPV immunisation.
Four doses of OPV were administered to children in Mumbai during mass immunisation campaigns (October 1999-January 2000) which also included house-to-house mopping up. From January to March 2000, 16 AFP cases were reported. No wild poliovirus has as yet been detected among these cases. Mumbai, therefore, appears to be reaching towards a stage of polio control.
During 1999, 663 AFP cases from Maharashtra were investigated for poliovirus infection. Wild polioviruses were isolated from 18 cases in ten districts, 12 had poliovirus type 1 and 6 had poliovirus type 3. Wild poliovirus type 2 has not been isolated from Maharashtra since 1996. The results indicate widespread distribution of wild virus in the State. However, there was a significant decline (87%) in the total number of wild virus isolates during 1998. In 1999, vaccine poliovirus strains were isolated from 65 cases.
From January to March 2000, 105 AFP cases have been reported. Only one case of wild poliovirus type 3 infection has so far been detected.
In 1999, 16 wild poliovirus positive cases of paralytic poliomyelitis were detected in 637 AFP cases studied virologically. These included 8 cases each of wild poliovirus type 1 and 3. Wild poliovirus type 2 was not detected. The 16 wild virus isolates were isolated from cases from 12 districts of MP.
From January to March 2000, of the 139 AFP cases investigated, 1 wild poliovirus has been detected in MP.
During 1999, the EVRC tested 1849 poliovirus isolates from 1840 cases of AFP from all over India by ELISA and nucleic acid probe hybridization for defining vaccine or wild viruses.
Wild polioviruses were detected in cases from almost all larger states of the country except Kerala, Orissa, Himachal Pradesh, Jammu & Kashmir and north-eastern states. Uttar Pradesh and Bihar together had about 90% of all wild polioviruses isolated in the country. Wild poliovirus type 3 predominated throughout north India. Wild poliovirus type 2 was isolated from Uttar Pradesh (9 cases) and West Bengal (one case) in early 1999.
Wild poliovirus type 1 (8 cases) and poliovirus type 3 (26 cases) have been detected among the poliovirus isolates (113 cases) during the first 3 months of 2000. Majority of these wild virus isolates are poliovirus type 3 indicating the continued transmission of the virus in Uttar Pradesh. However, wild viruses appear to be under control in the southern states of the country.
A large number of wild virus isolates from all over the country have now been sequenced to track wild poliovirus transmission routes. The database contains sequences of more than 150 type 1, 70 type 3 and recent type 2 poliovirus isolates. The data show tight clustering of genetically related viruses in parts of UP and Bihar. At least 9 lineages have been identified during 1999 in these two states.
In October 1999, a case of paralytic poliomyelitis due to wild poliovirus type 1 occurred in China after more than a 3 yr gap. The database available with the EVRC helped the Global Polio Eradication Programme in defining genetic relationship of the virus. The virus isolates detected in China during 1999 belonged to the lineage currently (1998-99) circulating in north India. The closest virus was identified from Madhya Pradesh.
Vaccine Derived Poliovirus Isolates
A large number of AFP cases were found to excrete vaccine derived poliovirus strains (observed in all states). Many of these cases may be true vaccine associated paralytic poliomyelitis (VAPP) thus requiring further characterization of the virus isolates. Vaccine-derived poliovirus type 2 isolates were studied by using site-specific PCR and 5¢UTR sequencing to detect mutation. Among the 29 isolates so far characterized, 16 revertants at major attenuation site (No. 481), 7 revertants at minor attenuation site (No. 398) and 4 recombinants in 5¢UTR region have been found. Further characterization is in progress.
OPV Potency Testing
The most important aspect of the polio eradication programme is administration of potent live oral poliovirus vaccine (OPV) to children. Monitoring the potency of field samples of the vaccine provided information on conditions during storage and transportation. During 1999, 3067 OPV field samples were tested of which 2630 (86%) were found to have satisfactory quality.
Since 1998, OPV vials have a thermo-sensitive label which indicates the cumulative effect of exposure to unfavourable temperatures. In a study to check the correlation between colour change of the vaccine vial monitor (VVM) and thermal exposure, the vaccine vial monitoring system was found to work satisfactorily. However, discrepant results in VVM colour reading and vaccine potency tests have been detected in some field samples. This calls for a large-scale evaluation of VVM.
A study was carried out at AIIMS and Kalawati Saran Hospital, New Delhi to monitor the poliovirus strains for inter- and intratypic variations by RFLP and sequencing. In this study polioviruses were isolated and characterized by RT-PCR for the regions coding for VP1-2A and also the 3D polymerase gene. The positive samples obtained by RT-PCR of 3D region were further characterized by RFLP. Two poliovirus type 1 isolates were obtained from 66 samples.
RT-PCR for 3D region and VP1 region was standardized and both these regions were amplified using separate PCR protocols for all the 40 poliovirus isolates. Fifteen PCR products of 3D region were further characterized by RFLP using Rsa I, HaeIII and Dde I enzymes. The RFLP pattern of 12 of these were similar to poliovirus type 1, three isolates had patterns of poliovirus type 2. Of these 12 RFLP patterns, 7 strains were characterized as wild type poliovirus 1 and five as Sabin type poliovirus 1. All three poliovirus type 2 strains had RFLP pattern of Sabin type poliovirus 2. It was also observed that one strain of poliovirus type 1 had an extra restriction enzyme site with HaeIII.
The diagnosis of hepatitis A virus (HAV) is primarily based on detection of virus specific IgM antibodies in blood. An IgM capture ELISA has been standardized for urine samples and blood samples collected on filter paper by finger prick. The sensitivity and specificity of the test is comparable to ELISA performed on conventional blood sample. Since HAV patients are children, collection of urine or finger prick blood would be most ideal in field or clinic settings.
Studies were undertaken at AIIMS, New Delhi to detect and characterize hepatitis B virus (HBV) mutants circulating in acute and chronic hepatitis patients who were serologically negative for all markers of known hepatitis viruses. About 500 sera were screened for the presence of HBV genome using PCR for ‘S’ and core region. Of these, 23 of the 59 fulminant hepatic failure patients, 24 of the 279 chronic hepatitis patients, 34 of the 137 blood donors positive for IgG anti-HBc and one of the 25 donors positive for antibodies to core and surface antigens were found to carry HBV genome. Approximately 16% of the total cases examined carried HBV genome.
Efforts are also being directed to generate meaningful data on the hepatitis B prevalence among the primitive tribes of the Andaman and Nicobar islands. In order to create awareness about hepatitis B, its modes of transmission and the possibility of eradication through vaccination, a health education campaign involving tribal chiefs, religious leaders and medical and paramedical staff was carried out in Car Nicobar island. Two villages namely Tamaloo and Lapathy having a total population of 3016 were selected randomly from Car Nicobar island for this study. The entire population of these villages was enumerated and information about different risk factors such as family history of jaundice, blood transfusion, major operations, sickness, multiple injections, dental treatment, tattooing, promiscuous behaviour etc. was collected. Till date, 900 serum samples have been collected from one of the selected villages. Serological testing of these samples for various markers of hepatitis B infection is in progress. Individuals negative for HBsAg and anti-HBs will be selected for hepatitis B vaccination.
In a study conducted at NIV, Pune, the presence of a new transfusion transmitted virus (TTV) has been detected in voluntary blood donors and individuals at high risk for parenteral transmission. Non parenteral transmission also plays an important role in the transmission of the virus in our setting. The phylogenetic analysis suggests that the majority of them belong to group 1a. Public health importance of TTV infections is being worked out.
Insect Cell Lines
Development of a cell line from lepidopteran insects was reported earlier. During the year under report cell lines were further characterized and it was shown that these could be cryopreserved and revived. The Spodoptera litura cell line was used for expression of cloned genes from JE and hepatitis C viruses. These cell lines can be used for large scale growth of baculovirus. This virus has potential application for biological control of pests.
HIV INFECTION / ACQUIRED IMMUNE DEFICIENCY SYNDROME
The human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS) is increasing in India. ICMR’s research activities have helped to understand the problem of HIV/AIDS and have contributed in formulating strategies for the control and management of the disease. A prospective cohort of HIV seronegative persons attending STD clinics gave the first reliable estimates of incidence of HIV infection in this highrisk population and identified the behavioural and biological risk factors associated with acquiring HIV infection. Presently this cohort is being used for studies on acute pathogenesis and intervention of HIV/AIDS.
Basic research including developmental research has been given special importance. High in the priorities are vaccine related virological and immunological studies, development of diagnostic tests for opportunistic infections, testing of drugs for anti-retroviral and immunopotentiating activity and generation of reagent and virus repositories for use in HIV research. HIV infection is unique due to its social aspects. Studies in the social and behavioural sciences have been continuing specially in areas related to women and education, awareness, reproductive health and treatment seeking behaviour.
HIV Prevalence Associated with High Risk Factors
Studies on a cohort of HIV seronegative STD patients established in 1993 have been continued at the National AIDS Research Institute (NARI), Pune. The longitudinal study has enabled detection of HIV seroconverters and persons with p24 antigenaemia who were enrolled for the study of acute pathogenesis of HIV infection. The focus of the study has now gradually shifted from descriptive epidemiology to behavioural studies and intervention trials. Analysis of trends in various categories in the cohort has shown no significant change.
Analysis of results in 5763 referred samples at NARI showed prevalence of HIV in 78.6% sexually promiscuous persons, 48% patients with STD, 70% commercial sex workers and 55% recipients of blood or blood products. As these patients were referred to NARI for confirmation of the HIV diagnosis, the percentages of reactive patients may not reflect the actual prevalence in these risk groups.
There is an association between HIV and tuberculosis infection in terms of predisposition. In studies carried out at the NARI clinic, 27% of the TB patients (1565) were found to be co-infected with HIV. In the Sasson General Hospital, HIV prevalence was 23.0% in TB patients and 3.50% in pregnant women. Sentinel surveillance in the antenatal clinic at Bhosari, and Talera Hospitals, Pune showed 1.25 and 2% HIV seroprevalence respectively. In yet another study carried out by the NIV, Pune at the Sasson General Hospital and TB wards of the Naidu Hospital, a HIV prevalence of 21.35% (112/520) and 34.06% (140/411) respectively, was seen. Some of the samples (7/441) were seroreactive for both HIV-1 and HIV-2 and only 3 samples were reactive with HIV-2.
Studies have been carried out at the All India Institute of Hygiene and Public Health, Calcutta to understand the prevalence of HIV and other STD infections among the commercial sex workers in Calcutta. A total of 584 samples were tested in 1996-97, 79 samples in 1997-98 and 204 samples in 1998-99. HIV seropositivity rate was 12, 7.6 and 17% respectively during these periods. Syphilis had a 45.3, 20.1 and 26.4% prevalence in these groups. Smear positivity for gonococcal urethritis varied from 33% in the first year to 41.8% in the third year. Sera from patients of gonococcal urethritis tested for IgM antibody for chlamydiae showed that 45% were positive indicating the possibility of new infection.
Prevalence of Hepatitis in STD Patients
In a retrospective study to estimate hepatitis B virus (HBV) incidence in STD clinic attendees, 497 subjects who attended the NARI clinics and had returned for follow up, were investigated and the data analysed. Three hundred and eighty six (77.7%) were males and 111 (22.3%) females. Seventy three were HIV infected at entry, whereas 33 seroconverted for HIV during the study period. Interim analysis shows that the incidence of HBV infection as determined by seroconversion to core antibodies may be as high as 13.05%. Further analysis is in progress.
Prevalence of Hepatitis B and C Viruses Amongst HIV Seropositive Intravenous Drug Users
The seroprevalence of HCV among healthy Indians (voluntary blood donors) is estimated to be 0.12-4.0%. HbsAg carrier rate in the Indian population is between 2-7%. Some information on HIV and HBV prevalence among intravenous drug users (IDUs) of Calcutta has been reported. Although IDUs are at much greater risk of HCV infection (70-90%), reports to this effect are hardly available from India. Study was undertaken of the prevalence of HBV and HCV infections among 77 Manipuri couples where the husbands were intravenous drug users and HIV positive. A high prevalence of HCV (92%) and HBV (100%) infection was found in the IUDs from Manipur.
Since HCV and HBV are also transmitted through the sharing of injection equipment amongst IDUs, it is expected that the prevalence of these infections would be high. This study showed that almost all (92%) of the HIV infected IDUs were positive for HCV. Anti-HBc was present in 100% of the IDUs and in 95% of the wives. One wife, however, was positive for HbsAg suggesting recent infection. It is also probable that heterosexual transmission of HCV (11%) took place from male IDUs to their non-injecting wives . Stringent control measures to prevent the transmission of hepatitis viruses (B and C) are urgently required in Manipur.
HIV-1 specific cytotoxic T lymphocyte (CTL) response is being studied in HIV seropositives, especially in recent seroconverters and long-term non-progressors. A standard 51Cr release assay using autologous B cell lines infected with HIV expressing vaccinia as target cells measured the CTL response.
Fourteen individuals were assessed for the HIV-1 specific cross-clade CTL activity. Eight of them showed CTL response against env B and/or env C antigens (7 showed cross-clade CTL response against env of subtype B and C while one individual showed CTL response only against subtype C env). This finding has implication on the development of indigenous HIV vaccine in India.
Immunopathogenesis of Acute Primary HIV Infection in Pune
Patients reporting at STD clinics and HIV referral clinics at NARI were screened for anti-HIV antibodies. Those found negative were tested for the presence of p24 antigen to detect acute primary HIV infection before inclusion in the study. Out of the 1547 patients screened, 12 were found to be eligible as p24 antigen positive; 7 of the p24 antigenaemic and 6 seroconverters were enrolled for the study. Five patients with acute HIV-1 infection were studied for proliferative responses against mitogen(PHA), recall antigens (PPD and tetanus toxoid) and HIV antigens (p24, p55 and gp120). All 5 patients showed good proliferative response against PHA and PPD. None showed response against tetanus toxoid and any of the HIV antigens.
Normal Ranges of Lymphocyte Sub-populations in Indian Population
In view of the large variation observed in human CD lymphocyte counts in different studies conducted in India and the non availability of the range of normal values of CD lymphocytes in the Indian population, a multicentric study was carried out to establish ranges of the important lymphocyte sub-populations in the normal Indian population.
Mean T cell count (CD3+) was slightly lower in Indians (68.55%) as compared to the Western average (73.0%), whereas B cell counts (CD19) were similar (14.5% vs. 14.0%). The most remarkable feature appears to be a significantly lower CD4 count in Indians (36.9% as compared to the Western average of 44.0%). CD8 counts were, however, similar which resulted in a lower CD4/CD8 ratio in Indians (1.17 as compared to the Western value of 1.40). Interestingly, CD4/CD8 ratios differed significantly within different states of India. These findings are important for clinical research, drug and vaccine trials and immunological monitoring of patients including HIV/AIDS patients. Further analysis is being done to understand the factors /parameters including the regional differences influencing the CD counts.
Subtype Identification of HIV-1 in Western India
Identification of recombinant strains is very important in HIV control strategies, especially vaccine development. In view of the importance of HIV subtype prevalence and its analysis in the country, NARI has continued subtyping of HIV-1 isolates. During the year under report 134 blood samples were collected from different parts of western India. The commonest subtype prevalent throughout this region is subtype C. In addition a few subtype A samples were also obtained.
Full length amplification and sequencing of 6 HIV-1 strains from seroconverters was carried out. Indian samples collected from Pune by NARI upon heteroduplex mobility assay (HMA) analysis also showed that subtype C is the most predominant subtype. This could be further divided into C2 and C3 genotypes. Additionally, subtypes A and B were also detected suggesting that Pune is experiencing an HIV epidemic where multiple subtypes are involved. Recently 6 HIV-1 subtype C strains isolated by NARI have been completely sequenced. One of the strains was found to be a recombinant between subtype C and subtype A. It is for the first time that a recombinant virus has been detected in India. The majority of the mosaic virus genome was subtype C but the envelope gene and portion of LTR (long terminal repeat) were from subtype A.
In addition to CD4 molecule, HIV requires chemokine co-receptors (mainly CCR-5 or CXCR-4) for internalization of virus into the cell. For HIV-1 subtype B viruses (which are most common in the developed world) utilization of CXCR-4 correlates with advanced disease condition and poor prognosis. Studies were carried out in samples collected from different parts of the country from individuals presenting with HIV disease ranging from asymptomatic to AIDS defining conditions. HIV (40 strains) was isolated and studied for co-receptor utilization. It was observed that 39 of 40 viruses were CCR-5 dependent suggesting that subtype C viruses, predominant in India, were CCR-5 dependent, irrespective of disease stage. Only one virus, a strain of HIV-2, was found to be dual tropic but predominantly using CXCR-4 co-receptor.
HIV-1 and HIV-2 Repository
A total of 71 HIV-1 and 6 HIV-2 isolates from samples obtained from different parts of India were characterized. Subtyping of 71 HIV-1 isolates by HMA revealed predominance of HIV-1 with subtype C. Further analysis is ongoing. Phenotypic characterization of 35 HIV isolates was carried out using MT2 cell lines and the results indicated that 34 isolates were non-syncytium inducing and syncytium induction was seen in only 1 isolate.
Until now Simian retrovirus (SRV) has not been reported in Hanuman langur monkeys in India. At the NIV, Pune a strain of SRV has been isolated from langur PBMCs. This is the first report from India. Nucleotide sequencing of amplified envelope region suggests this to be phylogenetically a unique SRV-6. Further studies are going on.
Knowledge, Attitude and Behavioural Studies
Studies were carried out by NARI to determine the HIV seroprevalence and awareness about AIDS among rural pregnant women. A comprehensive, community based seroepidemiologic study was conducted in the area covered by three PHCs (Chakan, Kadus and Khed Shivapur) in Pune district of Maharashtra .
Out of the 1251 blood samples tested, 15 were seropositive, indicating an overall prevalence of 1.2% among pregnant women in rural areas of Pune district. HIV seroprevalence was significantly higher among villages situated within 1 km of a national or state highway. A majority of the participating women were housewives and 70% of them were aware of the existence of the disease and had heard about AIDS. One third of the respondents were aware of all the main modes of HIV transmission. Among those who were aware of the existence of the disease, almost a third was not aware about the possibility of HIV transmission through blood transfusion or from HIV infected pregnant women to the baby during delivery or through breast milk. Majority also had misconceptions about the mode of transmission of HIV.
Studies were carried out at National Institute of Epidemiology, Chennai to understand AIDS awareness among college students. The results of the study revealed that only about 20% of the Chennai college students had fairly good knowledge (a score of 90% or more) about HIV/AIDS. A small percentage of the students had misconception about the modes of transmission. There is a need to improve the level of awareness among them on various aspects of HIV/AIDS