Development of a novel molecular assay based on conventional PCR liquid hybridization and ELISA (NAT-ELISA) for the detection of HIV, HCV, and HBV in blood donors.

PURPOSE: Development of a simple, affordable, and sensitive method based on gene amplification by PCR followed by liquid hybridization for detection of HIV-1 & 2, HCV and HBV in plasma samples (NAT-ELISA) for detection of these viruses particularly in their window period. METHODS: Viral nucleic acid extracted from WHO International Standards for HIV1, HIV2, HCV and HBV and 199 blood donor plasma samples were amplified by reverse transcription -PCR with forward and biotinylated reverse primers designed from the conserved regions of p-24 gene of HIV-1 and HIV-2, 5' UTR of HCV and middle part of S gene of HBV respectively. The biotinylated amplicons were immobilized on streptavidin coated ELISA plates, denatured chemically, and were hybridized with digoxigenin labelled specific oligonucleotide probes of HIV-1, HIV2, HCV and HBV. Hybridization signals were measured colorimetrically by indirect ELISA using anti-digoxigenin HRP conjugate and TMB substrate at 450 â€‹nm. Analytical Sensitivity or lower limit of detection (LOD) of this assay was determined with WHO standards. RESULTS: PCR amplified products of WHO standards were 321bp of HIV-1, 291bp of HIV- 2, 229bp of HBV, and 105bp of HCV. The LOD95 and LOD99.7 endpoints of HIV1, HIV2, HCV and HBV were 13, 6, 15 and 11 IU/ml and 15, 7, 17 and 12 IU/ml respectively. Diagnostic sensitivity and specificity of NAT-ELISA assay were calculated on 199 samples and was 97.4% and 99.4% respectively. CONCLUSIONS: The results suggest that this assay is highly suitable for the blood banks which do not have facilities for exorbitantly expensive NAT test for the detection of these viruses during their window period particularly in the blood donors who test negative by antibody/antigen screening tests.

Genetic diversity of HIV type 1 subtype C env gene sequences from India.

Considering the severity of the HIV-1 subtype C epidemic, data on the epidemiology and distribution of HIV subtypes in India are relatively sparse. Keeping this in view, 28 env gene sequences from patients were sequenced and analyzed. The samples were collected over a period of 10 years from 1995 to 2004. Assessment of the interisolate genetic distances of the study isolates, which were all subtype C, showed interisolate distances varying from 2 to 19% (mean: 14%) with the maximum diversity observed in the samples collected in 2003-2004. Analysis of the phylogenetic relationships among subtype C env sequences from six different countries and our study isolates revealed an overall star-like phylogeny with almost all sequences from India forming a monophyletic lineage. A lower diversity within the immunodominant epitopes was found. The data generated from this study should prove valuable for the production of vaccine against subtype C.

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 subtype C infected patients in Northern India.

BACKGROUND: Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. OBJECTIVES: The objective of this study was to develop and validate an affordable one step real-time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. RESULTS: We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. CONCLUSIONS: This RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India.

rpoB gene sequencing and spoligotyping of multidrug-resistant Mycobacterium tuberculosis isolates from India.

Multi drug-resistant Mycobacterium tuberculosis (MDR TB) has been well studied in outbreaks in settings of low endemicity in developed countries. However, the characteristics of MDR TB in the community with high endemicity such as India have not been well investigated. Mutations in the 81-bp rifampicin resistance-determining region of the rpoB gene were analyzed by DNA sequencing of 187 M. tuberculosis clinical isolates (149 resistant and 38 sensitive) from different parts of India. 146-Point mutations and two insertions were found in 146 of 149 resistant isolates in seven codons. The most common mutations were in codons 531 (59%), 526 (22%), and 516 (11.5%). Mutations were not found in three (2%) of the resistant isolates. N-terminal sequencing in these isolates showed no mutation at codon V176. None of the drug-susceptible isolates showed any mutation in the 437-bp rpoB gene segment sequenced. Genotypic analysis revealed a total of 80 different spoligotypes. A unique pattern was found in 65 (43.6%) isolates, whereas 84 (56.4%) were in 15 clusters. Comparison with an international spoligotype database showed ST26, Delhi type (18.1%), ST1, Beijing type (9.4%), and ST11 (5.4%), as the most common. The majority of isolates in the Beijing genotype (13/14) were associated with mutation 531TTG and similar drug-resistance patterns while other major clusters showed that the nature and frequency of occurrence of mutations in the rpoB gene were independent of spoligopatterns.

Utility of polymerase chain reaction in diagnosis of tuberculosis from samples of bone marrow aspirate.

Fever of unknown origin (FUO) poses a diagnostic challenge to the clinicians, with a differential diagnosis as varied as neoplastic and infectious diseases. In developing countries, the infectious causes are responsible for more cases of FUO, with tuberculosis as one of the main causes of classic FUO. Disseminated tuberculosis with negative pulmonary findings is a diagnostic problem. This study examines the diagnostic utility of the polymerase chain reaction (PCR) in samples of bone marrow aspirate in 85 patients presenting with diverse clinical symptoms. Using primers specific for Mycobacterium tuberculosis, tubercular etiology was detected in 33% of patients clinically suspected of tuberculosis while culture on Lowenstein-Jensen medium grew M. tuberculosis in only one patient (2.5%). None of these patients had been diagnosed by microscopy. Clinical improvement with ATT was observed in 85% of the patients with positive PCR. PCR demonstrated much higher sensitivity and specificity, thereby facilitating early therapeutic decisions for suspected extrapulmonary tuberculosis.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis by in-house, reverse line blot assay.

Drug resistance in tuberculosis is a significant problem in countries endemic for tuberculosis. A sensitive, specific, and high-throughput reverse line blot assay (RLBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. DNA sequencing done for 72 resistant isolates from Delhi, for baseline data, showed mutations within the rpoB core region in all RIF-resistant strains. The RLBA includes oligonucleotide probes specific for wild-type and mutant sequences, allowing sensitive detection of both genotypes in a single assay. The assay based on reverse hybridization principle simultaneously detects 13 different mutations affecting 6 independent codons, including the most prevalent mutations at positions 531 and 526. Application of the method to a panel of 292 MDR TB isolates and susceptible strains from 5 different cities in India showed 98% concordance with the sequencing results. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in Mycobacterium tuberculosis.

Adjuvant action of murine IL-2/Ig plasmid after intramuscular immunization with Indian HIV-1 subtype C recombinant env.gp 120 construct.

The human immunodeficiency virus (HIV) epidemic is probably the greatest scourge to affect mankind in the 20th century. Containment of the acquired immunodeficiency syndrome (AIDS) epidemic will require an effective vaccine. Of various vaccine approaches, immunization with DNA plasmids containing HIV-1 structural genes is the most popular approach. However, an important limitation of DNA immunization is that these responses are relatively weak and are often only transient in their nature. The use of immunologic adjuvants together with DNA vaccines is a promising way to enhance and to optimize DNA-derived immunity. Cytokines have been widely used to enhance the immune responses of DNA vaccines. In the present investigation, we studied the in vivo immunomodulation of HIV-1 Indian subtype C plasmid construct (pJWSK3, encoding envgp120 gene) by plasmid-based murine IL-2/Ig construct. Subcloning of mIL-2/Ig gene from pVRCmIL-2/Ig construct into pJW4304 vector was done followed by its in vitro expression study on the COS-7 cell line. Co-immunization of the recombinant HIV-1 env-gp120 construct with the IL-2/Ig construct in the female Balb/c mice by the intramuscular route resulted in induction of significantly higher levels of both HIV-1-specific antibody response and cell mediated immune response than by DNA plasmid construct alone (p < 0.001 and p < 0.05, respectively). The induced HIV-1-specific murine IFN-gamma response was robust, broad based, and seen even at the end of 6 months after immunization. Taken together these results indicate that the strategy of using IL-2/Ig plasmid can be highly effective when used along with recombinant DNA constructs and serve as the potential tool for the development of more rationally designed vaccines against HIV-1.

Augmentation of HIV-1 subtype C vaccine constructs induced immune response in mice by CpG motif 1826-ODN.

The greatest biomedical challenge of this century is to develop a preventive vaccine against Human Immunodeficiency Virus (HIV-1). For an HIV vaccine to be effective, it appears logical to develop new strategies that enhance the level of the immune response as well as steer it towards the desirable cellular type. In view of this, there is a need for rational inclusion of biological adjuvants into the HIV-1 vaccination strategies that could potentiate the immune responses both qualitatively and quantitatively. The adjuvant may include the immunostimulatory oligonucleotides containing CpG motifs, whose immunomodulatory characters are well established and represent the basis for an effective vaccine adjuvant. In our study, we investigated the use of an immunostimulatory oligonucleotide (or CpG motif), 1826-ODN to augment the immune response elicited by plasmid DNA vaccine constructs containing Indian subtype C HIV-1 envelope gp120 and gag-protease genes in Balb/c mouse model system. A dose of 2-microg CpG motifs/mouse was found to be optimum when co-administered with the DNA vaccine constructs with the peak level of humoral and cell mediated immune responses at 6 weeks post immunization. Murine IFN-gamma ELISpot assay demonstrated that the use of 1826-ODN led to a broad based and long term recognition of the subtype C envelope and gag peptides. The use of CpG motifs has been effective in augmenting the immune responses generated by the DNA constructs. Taken together, these results are an important advancement towards the design of future preclinical and clinical trials of these vaccine constructs.

Analysis of HIV type 1 subtype C full-length gag gene sequences from India: novel observations and plausible implications.

Twenty-four HIV-1 gag genes from patients in India were sequenced and analyzed. All measured 1476-1491 nucleotides with an average of 1483 nucleotides in length. Phylogenetic analysis revealed a homogeneous epidemic of HIV-1 subtype C. Intragenotypic divergence of up to 6.6% was present. Fourteen novel conserved signature pattern residues were delineated for HIV-1 subtype C strains. Each of the 15 nucleocapsid (NC) basic residues was highly conserved p6 gag LXSLFG and PT/SAPP motifs were highly conserved except for PTVPT in three and PTAPT in two strains. Zinc finger motifs were conserved in all. Documented HIV-1 subtype C gag immunodominant CTL epitopes were conserved. The evolving predominance and the change in nature of the epidemic from Thai B to that of subtype C in the Northeastern regions of India were observed. Tracking the evolution of the Indian epidemic has implications for developing a vaccine.

Improved diagnostic value of PCR in the diagnosis of female genital tuberculosis leading to infertility.

Histopathological and mycobacteriological examinations have limited utility in the diagnosis of genital tuberculosis. In this double-blind study, 61 samples, consisting of endometrial aspirates (EAs), endometrial biopsies (EBs) and fluid from the pouch of Douglas (POD), from 25 women suffering from infertility were investigated for the presence of the mpt64 gene of Mycobacterium tuberculosis by PCR and correlated with laparoscopic findings. PCR demonstrated M. tuberculosis DNA in 14 out of 25 patients (56.0 %), compared to one smear with acid-fast bacilli (1.6 %) and two culture-positive samples (3.2 %). The presence of M. tuberculosis DNA was observed in 53.3 % of EBs, 47.6 % of EAs and 16.0 % of POD fluid samples. All patients with laparoscopy suggestive of tuberculosis, 60 % of those with a probable diagnosis and 33 % of those with incidental findings were positive by PCR. However, one EA sample from an infertile patient with normal laparoscopy was also positive. Multiple sampling from different sites and amplification of the mpt64 gene segment by PCR offered increased sensitivity in determining tuberculous aetiology in female infertility.

Immunogenicity of recombinant Modified Vaccinia Ankara Viruses (rMVA) expressing HIV-1 Indian subtype C gag-protease and env-gp120 genes in mice.

India has currently an estimated 5.1 million HIV-infected individuals, and almost 95%of them are infected with subtype C strain. Therefore, it is imperative that a vaccine from locally circulating Indian HIV-1 subtype C be developed which would induce a robust immune response in the recipients. In this study, recombinant Modified Vaccinia Ankara Viruses (rMVA) expressing Indian HIV-1 subtype C envelope and core proteins were tested for their immunogenicity in the Balb/C murine model. Mice were immunized with two doses (10(7) pfu/dose) of recombinant MVA constructs intradermally. Both the constructs produced high levels of antibodies against gag and envelope gp120 and also elicited broad based as well as robust cell mediated immune response as evaluated by IFN-gamma ELISpot assay. These epitopes were distributed through out the length of gag and envelope gp120 proteins.

Induction of broad-based immune response against HIV-1 subtype C gag DNA vaccine in mice.

Prevention of HIV infection through an effective vaccine is need of the hour as per the AIDS pandemic scene, particularly in the developing world. Here we report the work done with gag gene construct pJWgagprotease49587 from HIV-1subtype C Indian strain. The construct pJWgagprotease49587 was tested positive for expression in COS-7 cells by p24 antigen capture ELISA, immunoblotting and by transmission electron microscopy that revealed virus like particle formation. Immunogenicity studies showed induction of good lymphoproliferative and cytotoxic (CTL) responses in Balb/c mice. The cytokine repertoire elicited showed a TH1 type of immune response. In an epitope mapping study, IFNgamma secretion by spleen cells from immunized mice was observed to seven peptides from different regions of gag. Recognition of multiple epitopes demonstrates elicitation of a broad based immune response against gag following immunization with the construct. In view of the high propensity of escape mutant induction during the course of HIV infection, it is encouraging to use immunogens eliciting viable immune responses to a broad spectrum of epitopes. Hence the construct pJWgagprotease49587 is a good candidate for immunogenecity testing in nonhuman primates as a probable vaccine candidate.

Suspected small-scale interpersonal transmission of Mycobacterium tuberculosis in wards of an urban hospital in Delhi, India.

Genotypes of Mycobacterium tuberculosis causing disease were investigated in pulmonary tuberculosis patients admitted to two adjacent wards of a tuberculosis hospital in Delhi, India. Genetic markers, the insertion sequence IS6110, a direct repeat sequence, and a polymorphic GC-rich sequence supported the circumstantial epidemiologic link between eight strains of M. tuberculosis, suggesting their possible involvement in small-scale, interpersonal transmission of both drug-sensitive and drug-resistant tuberculosis. This is the first report of a suspected acquisition of M. tuberculosis among hospitalized patients in India. The use of multiple molecular typing markers and techniques unequivocally identified the exact clonality of strains isolated from the hospital. The result of this study emphasizes the need for more comprehensive investigation of high-risk situations for tuberculosis transmission and long-term follow-up analysis for identifying such instances of unsuspected transmission.

Two mycobacterium fortuitum strains isolated from pulmonary tuberculosis patients in Delhi harbour IS6110 homologue.

We report 2 isolates of Mycobacterium fortuitum from patients with pulmonary tuberculosis lesions hybridizing to IS6110 probe in restriction fragment length polymorphism (RFLP) typing. Results of polymerase chain reaction-hybridization formats using the non-specific region of IS6110 for the molecular detection of mycobacteria in clinical material should be interpreted with caution.

Tuberculosis infection in HIV-infected Indian patients.

Individuals with HIV infection are at increased risk for tuberculosis (TB). The altered CD4 T-cell homeostasis induced by HIV infection may play a key role in the development of tuberculosis in HIV-infected patients. In this retrospective analysis, lymphocyte profiles (CD4 and CD8 count) of subjects infected with HIV, with or without TB, were evaluated. The influence of tuberculosis treatment on the CD4 count in dually infected patients was analyzed in a subset of patients available for follow-up. Of 421 subjects with HIV infection studied, 105 (24.9%) were positive for TB (HIV+TB+). A statistically significant difference (p = 0.0001) was found in the median CD4+ counts between the HIV+TB- (297.5 per microliter) and HIV+TB+ (181 per microliter) groups. TB was found to be the indicator disease for HIV infection in 36 (34.2%). In 65.7% of HIV-infected patients, TB was the first AIDS-defining disease. Of 72 patients who were receiving TB treatment, 33 (45.9%) showed an increase in CD4 counts, but this was statistically not significant. None of these patients was undergoing antiretroviral therapy prior to TB treatment. We conclude from this retrospective study that TB, a common HIV-related opportunistic infection in Indian subjects, is associated with lower CD4+ counts. The influence of TB therapy on CD4 counts in the patients needs to be further investigated.

PCR diagnosis of tuberculosis--experience in India.

Rapid and accurate diagnosis of tuberculosis is the cornerstone of global tuberculosis control programmes. With increasing incidence of tuberculosis epidemics, the low sensitivity and the length of time taken by traditional diagnostic modalities have hampered the efforts to interrupt disease transmission. Introduction of Polymerase Chain Reaction has enhanced the diagnostic predictability of the disease especially in the extrapulmonary, paucibacillary samples. High specificity and sensitivity have been reported in different samples. The technique is capable of picking as few as ten to fifty tubercle bacilli. When PCR technique is performed under quality controlled conditions, false negatives (due to underfined polymerase inhibitors) and false positives (due to cross contamination during sample collection or in the laboratory) can easily by avoided. Samples from sites with a possible latent infection focus or DNA from dead bacilli may give a positive reaction. The use of PCR with traditional diagnostic tools along with clinical presentation can prove helpful in patients presenting with a diagnostic dilemma.

Evolution of HIV-1 in India.

Nearly 25 years after the discovery of the human immunodeficiency virus type 1 (HIV-1) effective control of the AIDS pandemic remains elusive. At the root of this challenge is the evolution of this virus to elude immune control. Error-prone nature of replication and retro-transcription is the hallmark of this virus. This fidelity of replication in HIV-1 is due to the absence of proof-reading/repair and post-replicative error correction mechanisms that normally operate during replication of DNA viruses. Advances in sequencing technology and expanded disease surveillance have allowed researchers to characterize the variation in HIV-1 around the world and within individual patient overtime. Although HIV-1 has been classified into distinct subtypes, the classification does not reflect dynamic genetic evolution of HIV-1 through which new strains are constantly emerging. The resultant viral diversity has implications for differential rates of disease progression in different geographical areas, differential responses to antiretroviral therapy (including the development of resistance), and vaccine development. In this review evolution of HIV-1 in India is discussed.

Correlation of T-lymphocyte subpopulations with immunological markers in HIV-1-infected Indian patients.

Progressive HIV disease is characterized by CD4 T cell decline and activation of the immune system. We aimed to study the quantitative alterations in the naive (CD45RA+CD62L+), memory/effector (CD45RO+) and activated (HLA-DR+CD38+) T-lymphocyte subpopulations in antiretroviral treatment naive, HIV-1 infected Indian patients by three-color multi-parametric flow cytometry. The association of different CD4+ and CD8+ T cell subsets with the immunological markers- CD4+ and CD8+ T cell percentages was examined by calculating the partial correlation coefficients. We also observed significant differences in the expression of different CD4+ and CD8+ T-cell subsets among the two groups of patients formed using the median CD4+ T cell percentage value (15%) of the study population. The correlations of different CD4+ and CD8+ T cell subsets reflected the quantitative alterations in the T-lymphocyte subpopulations and activation of the immune system during HIV-infection. The study outcome also emphasizes the significance of the CD38+CD8+ T-lymphocyte subset as a prognostic marker for HIV management and ART monitoring in resource-limited settings of developing countries like India.

Development of a candidate DNA/MVA HIV-1 subtype C vaccine for India.

Development of a vaccine against human immunodeficiency virus type-1 (HIV-1) is the mainstay for controlling the AIDS pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell-mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. The present study focuses on the stimulation cell-mediated and humoral immune responses by recombinant DNA-MVA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development. Candidate recombinant DNA-MVA vaccine formulations expressing the human immunodeficiency virus-1 env and gagprotease genes from HIV-1 Indian subtype C were constructed and characterized. A high level of expression of the respective recombinant MVA (rMVA) constructs was demonstrated in BHK-21 cells followed by the robust humoral as well as cell mediated immune (CMI) responses in terms of magnitude and breadth. The response to a single inoculation of the rDNA vaccine was boosted efficiently by rMVA in BALB/c mice. This is the first reported candidate HIV-1 DNA/MVA vaccine employing the Indian subtype C sequences and constitutes a part of a vaccine scheduled to enter a preclinical non-human primate evaluation in India.