Characteristics of treatment-naïve HBV-infected individuals with HIV-1 coinfection: A cross-sectional study from South India

Purpose: Human immunodeficiency virus-1 (HIV-1) and hepatitis B virus (HBV) coinfection has become a major health problem across the globe. The increased life expectancy of HIV-1 patients due to antiretroviral therapy has led to the emergence of liver disease as a major mortality factor among them. The purpose of the study was to examine the baseline characteristics of HBV in treatment-naïve HBV/HIV coinfection from southern India compared to monoinfected individuals. Materials and Methods: The study was cross sectional in design, and samples were examined from 80 HIV-1, 70 HBV and 35 HBV/HIV-coinfected individuals using chemiluminescent microparticle immunoassay, real-time polymerase chain reaction and flow cytometry assays. Results: There was a significant increase in HBV DNA (P = 0.0001), higher hepatitis B e antigen percentage difference (P = 0.027) and lower CD4 counts (P = 0.01) among the HBV/HIV-coinfected individuals, but no difference in the HIV-1 viral load compared to HIV-1-monoinfected individuals. Also, the aspartate aminotransferase levels, prothrombin time and the international normalised ratio were significantly high among coinfected individuals. Conclusion: These findings conclude that HIV-1 coinfection can have serious implications on the outcome of HBV-related liver disease. To the contrary, HBV infection had no consequence on the progression of HIV-1 disease but distinctly lowered CD4+ T-cells.

BK virus characterisation among HIV-1-Infected individuals and its association with immunosuppression

Purpose: BK virus (BKV) is an opportunistic pathogen which causes significant morbidity and mortality in individuals who are immunodeficient. We aimed to quantitate and characterise BKV and to correlate with the degree of immunosuppression among human immunodeficiency virus (HIV)-1-infected individuals. Methods: BKV DNA detection was carried out using an in-house quantitative real-time polymerase chain reaction on paired whole-blood and urine samples collected from 187 antiretroviral therapy (ART)-naïve HIV-1-infected individuals and 93 healthy individuals who served as controls. Sequencing was performed for a proportion of high BK viral load (VL) samples to observe non-coding control region (NCCR) rearrangements. Results: BKV positivity in urine was 25.6% among HIV-infected individuals and 10.7% in control individuals (P = 0.03). The BK VL showed a significant negative correlation with CD4+ T-cell counts, a positive correlation with WHO clinical staging and no significant correlation with HIV-1 VL. Of 42 BKVs from urine samples sequenced, two showed rearrangements without clinically severe disease or high VL. Their NCCR and VP1 sequence-based genotyping revealed genotype I. In a small subset of individuals (n = 8) on ART who were being followed up, six individuals showed either decrease or complete clearance of virus with ART. Conclusion: There was a higher frequency of BK viruria in HIV-1-infected individuals than among healthy controls and the positivity correlated with the degree of immunosuppression. There was no association of high VL with NCCR rearrangements in urine.

Performance of an in-house real-time PCR assay for detecting Cytomegalovirus infection among transplant patients from a tertiary care centre

Background: Quantitative Cytomegalovirus (CMV) polymerase chain reactions are increasingly being used for monitoring CMV DNAemia in haematopoietic stem cell transplants and solid organ transplants. Objective: In this study, a commercial CMV viral load assay was compared with an in-house viral load assay. Materials and Methods: A total of 176 whole-blood samples were tested for CMV DNAemia using both assays. Results: Our evaluation showed a difference of 1 log10copies/ml between the two assay systems in determining CMV viral loads in the clinical samples. Conclusion: The in-house viral load assay had a better correlation with clinical findings compared to the commercial assay. Quality assessment of these assays was done by the United Kingdom National External Quality Assessment Scheme (UKNEQAS), an external proficiency testing programme, and by the National Institute for Biological Standard and Control (NIBSC) standard. For UKNEQAS and NIBSC standards, the bias between the assays was 0.73 log10and 0.85 log10, respectively. This difference is well within the acceptable range already reported in the literature.

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.

Frequency of Epstein - Barr virus infection as detected by messenger RNA for EBNA 1 in histologically proven gastric adenocarcinoma in patients presenting to a tertiary care center in South India.

Background: Epstein–Barr virus (EBV)‑associated gastric carcinoma is a relatively uncommon entity detected in approximately 10% of gastric adenocarcinoma. Objective: The purpose of this study is to estimate the frequency of EBV‑associated gastric carcinoma and also to assess the nature of presentation, any significant difference between this subgroup and EBV‑negative gastric adenocarcinomas with respect to age and sex predilection, lymph nodal status, site of presentation. Materials and Methods: We prospectively analyzed 100 cases of gastric adenocarcinoma who underwent either a partial or total gastrectomy during the period from March 2010 to August 2011. The tumour and the corresponding normal gastric tissue from the same patient were analyzed for the presence of Epstein–Barr nuclear antigen 1 (EBNA1) messenger ribonucleic acid (mRNA) by real‑time polymerase chain reaction (PCR). Result: EBV was detected in 6% cases of gastric adenocarcinoma. All the positive patients were males. The majority of cases involved the proximal stomach and there was variable lymph nodal involvement. Conclusion: Our study endorses that there is an association between EBV infection and gastric adenocarcinoma in the Indian population. There was no significant difference between this subgroup and EBV‑negative gastric adenocarcinomas with respect to age and sex predilection, lymph nodal status and site of presentation. Short‑term follow‑up of this subgroup of patients seems to indicate a good overall prognosis after appropriate treatment. However, a larger study with long‑term follow‑up is needed to further establish the role of EBV in gastric adenocarcinoma in this study population.

Emergence of HIV‑1 drug‑resistant variants in women following antiretroviral prophylaxis for the prevention of mother to child transmission.

Purpose: Emergence of drug resistance following HIV prophylaxis has an important impact on ART program. Objective: To investigate the emergence of drug resistance in HIV‑1 infected pregnant women. Materials and Methods: Fifty‑three HIV‑1 infected pregnant women who had received 4‑12 weeks of antenatal AZT followed by Nevirapine during delivery and Combivir [AZT + 3TC] for 1 week postpartum (group‑1, n = 48) or who come at the time of delivery and received Nevirapine followed by Combivir for 1 week (group‑2, n = 5) were recruited. Samples were collected prior to the start of the prophylaxis and 5‑8 weeks postpartum. In addition, a third sample was collected between 26‑65 weeks postpartum from 7 women. Amplification of HIV‑1 pol gene and drug resistance analysis was done. Result: Two (3.8%) women in group‑1 showed transmitted drug resistance and they continued to show this even at 6 weeks postpartum. One (2%) woman from group‑1 showed a mutation after 6‑8 weeks of prophylaxis. Among the samples collected between 26‑65 weeks postpartum, 3/7 (43%) showed mutations and all these women belong to group‑1. Women belonging to group‑2 didn’t show mutation prior to or following prophylaxis. Conclusion: In contrast to the available data among pregnant women with ART prophylaxis, our data showed reduced frequency of mutations following 5‑8 weeks of postpartum but an emergence of mutation later (26‑65 weeks). The addition of Combivir with the single dose Nevirapine during delivery and the early stage of the disease with higher CD4 counts could be the reasons for this.

Changing paradigm of Cryptococcal meningitis: An eight-year experience from a tertiary hospital in South India.

Background: Cryptococcal meningitis (CM) is a common opportunistic fungal infection causing sub-acute meningitis with the potential for complications and signifi cant mortality. We conducted this study to describe the difference in presentation and outcome between HIV-infected and HIV-uninfected patients. Materials and Methods: Patients admitted to a tertiary care centre between 2005 and 2013 with confi rmed CM were included in the analysis. Details of the clinical presentation, laboratory fi ndings, treatment details, risk factors for infection and outcome were documented and analysed. Results: During the study period, 102 (87.2%) cases of CM occurred among HIV infected individuals, whereas 15 (12.8%) occurred among HIV-uninfected patients. HIV-infected patients with CM were younger compared with HIV-uninfected patients (38.2 ± 8.5 years vs. 45 ± 11.5 years; P = 0.07). The median duration of symptoms prior to presentation was shorter in the HIV-infected group (20 ± 32 vs. 30 ± 42; P = 0.03). There was no difference between the cerebrospinal fl uid (CSF) lymphocyte counts, CSF protein counts, and CSF sugar levels in both the groups. The diagnostic yield of Cryptococcus was similar with CSF India ink smear (89% vs. 87%), CSF fungal culture (95% vs. 87%), and blood culture (100% vs. 75%) in both the groups. Case fatality rate in the HIV-infected group was 30.6%, whereas there were no deaths in the HIV-uninfected group. Conclusion: HIV-infected patients with CM have a worse outcome compared to HIV-uninfected patients. The overall trend over 3 decades shows increasingly successful rates of treatment and hence early diagnosis and treatment are of paramount importance.

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.

Antiviral efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B subjects from Indian subcontinent.

Adefovir is one of the therapeutic options for the treatment of chronic hepatitis B. A total of 30 adefovir-experienced subjects with the median treatment duration of 12 (interquartile range (IQR) 6-18) months were studied. Virological response was measured by hepatitis B virus deoxyribonucleic acid (HBV DNA) levels. HBV reverse transcriptase (rt) domains were sequenced for the identifi cation of resistance mutations. Among the 30 subjects, two (7%) showed virological response and 19 (63%) were non-responders. The virological response for the remaining nine (30%) subjects was not determined. On sequence analysis, two subjects were identifi ed with rtI169L and rtA181V mutation after 9 months and 18 months of adefovir treatment, respectively. Though the frequencies of adefovir resistance mutations are low, a large majority of subjects showed non-response. Therefore, adefovir in the management of HBV should be used judiciously.

Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South).

Purpose: The use of dried blood spots (DBS) for HIV-1 viral load determination could greatly enhance the management of HIV infected individuals in resource-limited countries. Objective: To compare the HIV-1 viral load values obtained between parallel collected plasma and DBS. Materials and Methods: DBS and plasma samples were collected from 62 HIV-1 infected individuals and were used for determination of HIV-1 RNA concentrations using the Abbot real-time HIV-1 PCR. Result: Mean of the log difference of viral load values between plasma and DBS was -0.41 log. DBS viral load values significantly correlated with plasma viral load (r = 0.9818, P < 0.0001). Conclusion: These results suggest that DBS samples can be used as an alternative to plasma for the estimation of HIV-1 viral load if samples are appropriately stored.

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.

Evaluation of a dry format reagent for CD4+ and CD8+ T-cell enumeration with FACSCount and Guava polymerase chain reaction.

Purpose: In all CD4+/CD8+ T-cell estimation systems, the reagents used are liquid in nature and have to be transported and stored at 2°-8°C. This causes problems in countries where the ambient temperature is high for most parts of the year or where the laboratories are at remote places. Materials and Methods: We evaluated a dry format of CD4/CD8 reagents from ReaMetrix (Bangalore, India) against the existing liquid reagents from Becton Dickinson (San Jose, CA, USA) and Guava PCA system (Guava Technologies, Hayward, CA, USA). Blood samples collected during March 2009 through May 2009 from 102 HIV-infected individuals and 31 normal healthy individuals in a tertiary care centre in India (south) were tested by Guava; EasyCD4™ System (PCA) and FACSCount using the respective reagents and the corresponding ReaMetrix reagents. Results: Overall, the correlation (r) of the new Rea T Count and FACSCount reagents for the CD4+ T-cell estimation was 0.98, while with ReaPan 3 4 G reagent in the Guava PCA system with the Guava reagent was 0.97. The mean bias for CD4+ T-cell measurements between Rea T count and BD reagent was -6 cells/ml, while the same with ReaPan 3 4 G reagent in the Guava PCA system was 78 cells/ml. The mean bias for the Rea T count and the ReaPan 3 4 G reagent tested in the FACSCount and Guava PCA system was 17 cells. Conclusions: The dry reagents were found to be reliable and cheaper compared to the existing liquid reagents. This allows the transportation of reagents in the absence of cold chain and will facilitate a more user-friendly CD4+ T-cell testing system.

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%).

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.

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).

An unusual seroconversion profile in a pregnant woman infected with the human immunodeficiency virus-1: need for using later generations HIV screening assays.

The first HIV-1 marker that appears in blood following infection is HIV-1 RNA and usually the load is in millions of copies/ ml preceding seroconversion. A 24-year-old pregnant woman, gravida 2, parity 1 was tested for HIV as part of antenatal screening. Three samples were collected and tested from this individual over a period 70 days. The HIV-1 RNA level during seroconversion phase was very low, contrary to the well understood natural history of HIV infection. The reactivity rate in the ELISA and the Western Blot profile showed a gradual increase over the 70 days with a weak reactivity in a second generation assay (detects IgG only) for the third sample. This case illustrates the uncertainties regarding the serological window period in HIV infection and the need to use at least a third generation assay in testing centres for early detection of HIV infection.

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.