Expression of cytokine‑mRNA in peripheral blood mononuclear cell of human immunodeficiency virus‑1 subtype C infected individuals with opportunistic viral infections from India (South).

Human immunodeficiency virus (HIV) disease progression is associated with a marked change in the level of plasma cytokines. The study reported here investigated the level of mRNA expression of different cytokines: Tumour necrosis factor‑alpha (TNF‑α), interferon (INF)‑gamma, interleukin‑10 (IL‑10) and IL‑21 in the peripheral blood mononuclear cell among the antiretroviral therapy naive subtype C HIV‑1 infected individuals and normal healthy controls by real time polymerase chain reaction. The mRNA expressions of all the 4 cytokines in HIV‑1 infected individuals were significantly higher compared to healthy controls (P value range 0.0004–0.01). The mean level of IL‑10, INF‑gamma and TNF‑α were higher in HIV infected individuals with low CD4 counts (<300 cells/μl). The IL‑10 expression showed a significant negative correlation with CD4 counts (r = −0.25, P = 0.04) while IL‑21 showed a positive correlation with CD4 counts (r = 0.26, P = 0.03). There was a significant negative correlation between the cytomegalovirus (CMV) viral load and IL‑21 expression. Cytokine levels by mRNA detection avoids the inherent problem of measuring plasma level and this study also provide information on the cytokine levels and CD4+ T cell level among HIV‑1 subtype C infected individuals with opportunistic viral infections like CMV.

Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals.

Purpose: Opportunistic viral infections are one of the major causes of morbidity and mortality in HIV infection and their molecular detection in the whole blood could be a useful diagnostic tool. Objective: The frequency of opportunistic DNA virus infections among HIV-1-infected individuals using multiplex real-time PCR assays was studied. Materials and Methods: The subjects were in two groups; group 1: Having CD4 counts <100 cells/μl (n = 118) and the group 2: counts >350 cells/μl (n = 173). Individuals were classified by WHO clinical staging system. Samples from 70 healthy individuals were tested as controls. In-house qualitative multiplex real-time PCR was standardised and whole blood samples from 291 were tested, followed by quantitative real-time PCR for positives. In a proportion of samples genotypes of Epstein-Barr virus (EBV) and CMV were determined. Results: The two major viral infections observed were EBV and CMV. The univariate analysis of CMV load showed significant association with cryptococcal meningitis, oral hairy leukoplakia (OHL), CMV retinitis, CD4 counts and WHO staging (P < 0.05) while the multivariate analysis showed an association with OHL (P = 0.02) and WHO staging (P = 0.05). Univariate analysis showed an association of EBV load with CD4 counts and WHO staging (P < 0.05) and multivariate analysis had association only with CD4 counts. The CMV load was significantly associated with elevated SGPT and SGOT level (P < 0.05) while the EBV had only with SGOT. Conclusion: This study showed an association of EBV and CMV load with CD4+ T cell counts, WHO staging and elevated liver enzymes. These viral infections can accelerate HIV disease and multiplex real-time PCR can be used for the early detection. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

Atypical mucocutaneous involvement with Leishmania donovani.

Mucocutaneous leishmaniasis has rarely been reported from India. The usual causative organisms of this infection are Leishmania braziliensis and L. tropica. Another species, L. donovani, which usually causes visceral leishmaniasis, has recently been reported to cause mucocutaneous disease in a few patients from Sri Lanka. We report two patients who had undiagnosed chronic skin lesions for several years. Skin biopsies revealed Leishmania and the species was characterized as L. donovani in both patients. There was considerable improvement in the skin lesions following treatment with liposomal amphotericin B.

Drug resistant mutations detected by genotypic drug resistance testing in patients failing therapy in clade C HIV-1 infected individuals from India.

Purpose: There has been an increase in the number of individuals administered antiretroviral therapy (ART) in India but treatment outcome is hampered by increasing development of drug resistance. Previous reports from India have shown M184V as the commonest mutation in treated individuals. However, there is no evidence for any protease mutations in these reports. This study was done to observe the common/unique mutational patterns observed in reverse transcriptase (RT) and protease (Pr) genes of clade C HIV-1 strains from individuals showing treatment failure in India. Materials and Methods: The assay was done by sequencing the Pr and RT genes of the HIV-1 strains from 18 individuals failing ART. Analysis was carried out using Stanford HIV drug resistance database (SHDB). The sequences were also submitted to the calibrated population resistance tool of SHDB and Rega HIV-1 sub typing tool. Phylogenetic analysis and quality control were performed with Mega 4. Results: Among the 20 strains, 19 showed resistance to both nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), one strain to NNRTIs and five strains showed protease inhibitors (PI) resistance and 3-class resistance. The most common mutation conferring NRTI resistance was M184V (90%) while K103N (45%) was the most common mutation conferring NNRTI resistance. The M46I mutation was seen in 20% of the Pr sequences. Conclusion: Resistance testing to check the prevalence of drug resistance mutations that arise following failure of the first line regimen to establish guidelines for second line regimens in India is a must. Studies are needed to confirm if mutation patterns that arise among clade C following failure of ART are the same as for clade B strains.

Detection of opportunistic DNA viral infections by multiplex PCR among HIV infected individuals receiving care at a tertiary care hospital in South India.

Purpose: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention. Materials and Methods: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naοve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV -1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells. Results: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/µL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/µL was 3.88 log 10 while among those with greater than 200 CD4+ T cells it was 3.75 log 10 . The mean CMV load was 6.98 log 10. Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses. Conclusions: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/μL).

Investigation of apoptotic markers among human immunodeficiency virus (HIV-1) infected individuals.

BACKGROUND & OBJECTIVES: Apoptosis causes a decline in the counts of uninfected bystander CD4+ T cells in HIV infection. The rate of disease progression of HIV infection is considered to be faster in the developing countries. The present study was carried out to investigate certain markers for apoptosis in immunopathogensis of disease in HIV infected south Indian population. METHODS: Soluble Fas (sFas) antigen and Fas ligand levels in plasma samples from 39 antiretroviral treatment naïve patients was estimated and compared with T cell subsets and HIV-1 viral load. RESULTS: The mean sFas antigen levels among controls and the CDC A, B and C clinical stages were 2.77, 3.08, 3.26 and 3.28 ng /ml respectively, higher though not significantly among HIV-1 infected individuals compared to controls. The mean sFas ligand levels in CDC A, B and C stages were 0.138, 0.125 and 0.117 ng/ml respectively were higher (P<0.001) than controls (0.073 ng/ml) and positively correlated with total lymphocyte % (r=0.43, P =0.007). sFas antigen levels were negatively correlated with total WBC count (r=-0.34, P=0.04), CD4% (r=-0.4, P=0.01) and CD4:CD8 ratio (r=-0.37, P=0.02). There was an increase in plasma levels of sFas antigen and Fas ligand over time in asymptomatics. INTERPRETATION & CONCLUSION: The high levels of sFas antigen and Fas ligand seen in HIV infected individuals suggest increased activation and apoptosis of T cells, due to constant stimulation of the immune system by inter-current infections of HIV infected individuals in south India.

Evidence for lower CD4+ T cell and higher viral load in asymptomatic HIV-1 infected individuals of India: implications for therapy initiation.

PURPOSE: We have earlier documented that the south Indian population had lower CD4 counts. The aim of this study was to investigate a previous suggestion on a new CD4+ T cell cut off and association with HIV-1 RNA levels for decision on anti retroviral therapy in India (south). METHODS: We evaluated a new methodology i.e., artus real-time PCR and CD4+ T cell count by Guava EasyCD4 system. From 146 HIV infected individuals seen at a tertiary care centre, blood was collected for CD4+ T cell and HIV-1 RNA estimation. RESULTS: The receiver operating characteristic curve cut off value for the CD4 counts to distinguish between CDC clinical categories A and B was 243 cells/microL, and to distinguish B and C was 153 cells/microL. The RNA level that differentiated CDC A and B was 327473 RNA copies/mL, while for CDC B and C was 688543 copies/mL. There was a significant negative correlation (r = -0.55, P + T cell counts in HIV infected individuals. CONCLUSIONS: A majority with CD4 counts of 201-350 cells/microL in our population had higher viral load than the treatment threshold suggested by the International AIDS society and the above two methodologies are useful in monitoring HIV infections.

Genotypic resistance profile of HIV-1 protease gene: a preliminary report from Vellore, south India.

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Chemokine profile among human immunodeficiency virus-1 (HIV-1) infected individuals from southern India.

BACKGROUND & OBJECTIVE: Individuals infected with HIV-1 have higher levels of chemokine producing cells compared to uninfected individuals. It is important to know the changes in chemokine levels associated with rate of progression of disease. There is a paucity of information on the plasma chemokines in HIV-1 infected individuals from India. We therefore carried out this study to estimate the levels of three chemokines namely macrophage inflammatory protein alpha (MIP1alpha), MIP1beta and RANTES, in relation to disease status in HIV-1 infected individuals and compared with uninfected individuals. METHODS: RANTES and MIP1alpha were estimated using ELISA in 114 HIV-1 infected and 30 controls, whereas MIP1beta was estimated in 101 HIV infected individuals only and 30 controls. The values were compared to the T cell subsets, HIV-1 viral loads and plasma cytokines (interferon gamma and interleukin-10). RESULTS: Compared to controls the mean MIP1alpha and RANTES level among the HIV-1 infected individuals was higher while MIP1beta level was lower in HIV infected individuals except CDC C groups. There was a significant positive correlation for MIP1á with HIV-1 viral load and IFNgamma, for MIP1alpha with viral load and IL10. There was a significant negative correlation between MIP1alpha with CD4 count and CD4: CD8 ratio and MIP1beta with CD4 count and CD8 count. There was a negativecorrelation between RANTES values and CD8 per cent. INTERPRETATION & CONCLUSION: In conclusion, our study showed a significantly higher level of beta chemokines in south Indian HIV-1 infected individuals compared to controls. These beta chemokines may have the inhibitory effect on HIV-1 only during the initial period and with the progression of disease this inhibitory effect wanes as shown by the positive correlation of beta chemokines with HIV-1 viral load.