Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

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.

Epidemiology, control and prevention of tobacco induced oral mucosal lesions in India.

Oral cancer is one of the leading causes of human morbidity and mortality especially in developing countries like India. Tobacco consumption in smokeless and smoking form along with alcohol is considered as the primary risk factors. Tobacco is a major health challenge with various tobacco products available for use which are known to have deleterious effects on the oral mucosa. The oral lesions caused by tobacco are inclusive of those that are less likely to progress to cancer; lesions with increased tendency to develop into cancer and cancerous lesions. Prevention and control of tobacco induced oral mucosal lesions is the prime requisite currently and mainly involves measures undertaken at primary, secondary and tertiary levels. Primary prevention plays a pivotal role in tobacco induced lesions and steps can be taken at policy level, community as well as individual level. This review paper focuses on the epidemiological data of tobacco induced oral mucosal lesions in India available in the literature with an overview on various strategies for their prevention and control.

Human immunodeficiency virus type-2-A milder, kinder virus: An update.

Human immunodeficiency virus type-2 (HIV-2) belongs to the family retroviridae which is phylogenetically clusters with SIV SM from sooty mangabeys. This virus is morphologically similar to human immunodeficiency virus type-1 (HIV-1) but has got only a 40% homology at the nucleotide level. There is a distinct geographical distribution of HIV-2 unlike HIV-1. There are currently eight subtypes/groups identified with subtype/group A responsible for the majority of infections. HIV-2 shows a considerable difference in the course of the disease. Clinical, haematological and immunological evaluation of individuals infected with HIV-2 has shown the virus to be less pathogenic than HIV-1 although the exact mechanism underlying this difference is not well defined. Similar to HIV-1, the HIV-2 isolates also showed distinct replicative and cytopathic characteristics. The transmission rate for HIV-2 compared to HIV-1 is very low both by heterosexual route and mother to child transmission. The clinical signs and symptoms of immunodeficiency associated with HIV-2 are similar to the ones seen among the HIV-1-infected individuals and they can also progress to AIDS. It is naturally resistant to NNRTI and hence the diagnosis become important as it affects the treatment strategy. Similar to HIV-1, HIV-2 strains of infected individuals also show mutations that can cause drug resistance. The current evidence suggests that there is no protective effective for HIV-2 against HIV-1.

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples.

Purpose : To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods : A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results : Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion : Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).

Could the products of Indian medicinal plants be the next alternative for the treatment of infections.

Indian medicinal plants are now recognized to have great potential for preparing clinically useful drugs that could even be used by allopathic physicians. Traditionally, practitioners of Indian medicine have used plant products in powder, syrup or lotion forms, without identification, quantification and dose regulation, unlike their allopathic counterparts. The present review explores the immense potential of the demonstrated effect of Indian medicinal plants on microbes, viruses and parasites. In the present context, with the available talent in the country like pharmaceutical chemists, microbiologists, biotechnologists and interested allopathic physicians, significant national effort towards identification of an "active principle" of Indian medicinal plants to treat human and animal infections should be a priority.

Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples.

Purpose: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. Material and Methods: A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Results: Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. Conclusions: The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.

Immune responses to Epstein-Barr virus in individuals with systemic and organ specific autoimmune disorders.

Purpose: Autoimmune diseases usually manifest in genetically predisposed individuals following an environmental trigger. There are several viral infections including Epstein-Barr virus (EBV) implicated in the pathogenesis of autoimmune disorders. The aim of this study was to look at the antibody pattern to EBV proteins in the plasma of both systemic and organ specific autoimmune disorders, estimate pro-inflammatory plasma cytokines (IL-8 and TNF-á) among these autoimmune patients and compare the observations with those in normal healthy controls. Materials and Methods: Samples from 44 rheumatoid arthritis patients, 25 Hashimoto's thyroiditis patients, appropriately age and sex matched healthy controls were tested for EBV IgM antibodies by an immunoblot assay and two cytokines (IL-8 and TNF-á) by commercial assays. Results: Among the rheumatoid arthritis patients, 23 (52%) were positive for EBNA1 antibody, while 13 (52%) of the Hashimoto's thyroiditis patients and 12 (30%) of the healthy controls showed similar bands. The intensity of the bands was high in the autoimmune patients when compared to the bands seen in control samples. The difference in the EBNA1 reactivity between rheumatoid arthritis patients and controls were significant (P = 0.038). There was a significant difference in the IgM reactivity to VCAp19 protein between patients and controls (P = 0.011). Conclusion: Our study showed an increased EBV activation among the autoimmune patient groups compared to the normal healthy controls. Further studies are required to delineate the association between the aetiology of autoimmune disorders and EBV.

Frequency of HIV type 2 infections among blood donor population from India: A 10-year experience.

Purpose: In India, HIV-2 epidemic is alongside with HIV-1. Blood banks are introducing nucleic acid testing (NAT) for screening. The limitation of NAT systems is the inability to detect HIV-2. Materials and Method : An analysis of HIV screening of a blood bank at a tertiary care center from 1998 to 2007 was carried out. Results : A total of 175026 donors were screened by serological assays and 789 were reactive for HIV antibody. Only 478 (61%) were confirmed positive by Western blot/immunoblot. There were 465 (97.2%) donations positive for HIV-1, 6 (1.3%) for HIV-2 (monotypic infection) and 7 (1.5%) for HIV-1 and HIV-2 (dual infection). Conclusion : We show the presence of HIV-2 infection among the blood donors and the need for incorporating HIV-2 detection also in the NAT systems.

Nanotechnology tools for single-virus particle detection.

The development and potential application of nanotechnology tools for single-virus particle detection by emergent nanotechnology is likely to revolutionise diagnosis and determining treatment endpoints for life threatening virus infections. Direct detection of biological macromolecules using semiconducting nanowires or carbon nanotubes for electrical field change measurements is a milestone application in this field. The promise of selective detection at a single particle level (stochastic sensing) with nanowire or nanotube field-effect transistor-based devices is a major breakthrough for outbreak situations, where a rapid and specific detection of the viral agent allows intervention at public health level. The same technology would be eminently suitable for bedside diagnosis and therapeutic intervention.

Hantavirus species in India : A retrospective study.

Hantaviruses cause hemorrhagic fever with renal syndrome in Europe and Asia. There are about 20 documented hantavirus species and newer species are being described worldwide, especially in non-rodent reservoirs, i.e shrews. Focus reduction neutralization test is the classical serotyping technique for hantavirus. However, this study employs a previously established serotyping ELISA, to retrospectively analyze known hantavirus IgG reactive samples for infecting serotypes. The result suggests presence of Thailand virus- like and Hantaan virus -like strains in India.

Hantavirus infection: A case report from India.

The clinical presentation of hantavirus infections in India is unclear. We report here a case of hantavirus infection in a 46 year old quarry worker presenting with fever, abdominal pain, jaundice, thrombocytopenia and renal dysfunction. Seroconversion and rising anti-hantavirus IgG titers were taken as evidence of hantavirus infection. Clinicians should consider hantavirus infections in the differential diagnosis of acute febrile illness along with scrub typhus, leptospirosis and dengue.

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).

A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods.

BACKGROUND: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. OBJECTIVES: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. STUDY DESIGN: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol ) gene of HSV isolates. RESULTS: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). CONCLUSION: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.

Hantaviruses: an emerging public health threat in India? A review.

The emerging viral diseases haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) are a cause of global concern as they are increasingly reported from newer regions of the world. The hantavirus species causing HFRS include Hantaan virus,Seoul virus, Puumala virus, and Dobrava-Belgrade virus while Sin Nombre virus was responsible for the 1993 outbreak of HCPS in the Four Corners Region of the US. Humans are accidental hosts and get infected by aerosols generated from contaminated urine,feces and saliva of infected rodents. Rodents are the natural hosts of these viruses and develop persistent infection. Human to human infections are rare and the evolution of the virus depends largely on that of the rodent host. The first hantavirus isolate to be cultured, Thottapalayam virus,is the only indigenous isolate from India,isolated from an insectivore in 1964 in Vellore, South India. Research on hantaviruses in India has been slow but steady since 2005. Serological investigation of patients with pyrexic illness revealed presence of anti-hantavirus IgM antibodies in 14.7% of them. The seropositivity of hantavirus infections in the general population is about 4% and people who live and work in close proximity with rodents have a greater risk of acquiring hantavirus infections. Molecular and serological evidence of hantavirus infections in rodents and man has also been documented in this country. The present review on hantaviruses is to increase awareness of these emerging pathogens and the threats they pose to the public health system.

Nanotechnology: a new frontier in virus detection in clinical practice.

Researchers are expanding the applications of nanotechnology in the field of medicine since mid-2000. These technologies include nanoarrays, protein arrays, nanopore technology, nanoparticles as a contrivance in immunoassays and nanosensors, among others. Nanobiotechnologies are clinically applicable and possess the potential to be useful in laboratory diagnosis of infections in general and viral infections in particular. Nanotechnology is a significant advance in molecular diagnostics. The technology strengthens and expands the DNA and protein microarray methods. In particular, the waveguide technology is an emergent area with many diagnostic applications. Nanosensors are the new contrivance for detection of bioterrorism agents. All these new technologies would have to be evaluated in clinical settings before their full import is appreciated and accepted.

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.