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Introduction:HIV-1 RNA detection is the most reliable method for monitoring treatment response among people living with HIV. Effective quality control measures that include internal quality control (IQC) are challenging in resource-constrained settings. Methods:We ascertained the utility of the kit low positive control (LPC) as an effective IQC to monitor the reliability of the HIV-1 viral load assay. Variations in LPC values were measured for 390 different runs over 10 years (2011–2021) and compared to in-house IQC data using Levey-Jennings control chart. Results:Overall, the Levey-Jennings analysis showed minimal variation (±0.5 log) for both the LPC and IQC data. The mean LPC value for first 20 runs (20 days) was 2.91. The mean LPC value for the 390 runs comprising 35 different lots was 3.01 ± 0.1 log. Conclusion:Our decadal data reveal that Abbott RealTime HIV-1 assay (Abbott Molecular Inc., IL, USA) LPC exhibited no significant biological variation over 390 runs distributed over 10 years. Hence, assay LPC can supplant the IQC for monitoring assay trends as a stable and commutable material in resource-constrained settings.

The COVID-19 pandemic was unique in the history of outbreaks because of the massive scaling up of resources related to diagnostics, treatment modalities, and vaccines. To understand the impact of the pandemic among laboratory professionals, we aimed to conduct a survey to assess the improvement in the lab capacity post-covid in terms of infrastructure and accreditation status across various levels of hospitals and to determine the changes in the practice of infection control precautions during the pandemic.

Aims:Cervical cancer is one of the leading causes of cancer among women, worldwide. HIV-positive women tend to have persistent infection and infection with multiple human papillomavirus (HPV) types. There is a need for affordable HPV DNA tests as viable alternatives to the existing costly commercial assays. The aim of the study was to establish PGMY-CHUV reverse hybridization assay as a cost-effective tool for HPV genotyping. Study Design:This was a prospective study conducted in a tertiary care centre from March 2011 to July 2012. Subjects and Methods:Fifty cervical brush samples from HIV-infected women and 43 WHO reference samples were tested by both the CHUV assay and linear array (LA). Results:The CHUV assay in comparison to the LA showed a sensitivity of 91%, specificity of 52% and a moderate agreement for all samples that were compared. However, most high-risk HPV types were identified amongst the clinical samples, and the entire range of genotypes in the WHO reference panel was detected. Statistical Analysis:The accuracy indices such as sensitivity, specificity, positive predictive value and negative predictive value were calculated. The level of agreement (kappa value) between the two assays was also calculated. Conclusion:The CHUV assay had an acceptable sensitivity, but it lacked specificity for HPV detection. Despite the lower rates of detection of multiple infections from clinical samples, better results were obtained with the WHO reference samples and the ability of the assay to identify the entire range of genotypes suggests that it can be an efficient tool for genotyping.

Purpose:The sequence variation of human immunodeficiency virus (HIV) capsid region may influence and alter the susceptibility to human tripartite motif 5αprotein (huTRIM5α). Materials and Methods:Molecular docking was carried out with huTRIM5αSPRY domain by the use of ClusPro and Hex docking program for HIV-1 and HIV-2 capsid sequences. Results:The sequence analysis on HIV-1 and HIV-2 capsid gag gene identified 35 (19.7%) single-nucleotide polymorphisms (SNPs) in HIV-1 and 8 (4.5%) SNPs in HIV-2. The variations observed in the HIV-2 capsid region were significantly lower than HIV-1 (P< 0.001). The molecular docking analysis showed that HIV-1 wild type used V1 loop, while HIV-2 used V3 loop of huTRIM5αfor interaction. HIV-1 with A116T SNP and HIV-2 with V81A SNP use V3 and V1 loop of huTRIM5α for interaction respectively. The reduced huTRIM5αinhibition may lead to a faster progression of disease among HIV-1-infected individuals. However, in case of HIV-2, increased inhibition by huTRIM5αslows down the disease progression. Conclusion:Polymorphisms in the capsid protein with both HIV-1- and HIV-2-monoinfected individuals showed the difference in the docking energy from the wild type. This is the first study which documents the difference in the usage of loop between the two HIV types for interaction with huTRIM5α. Variations in the capsid protein result in alteration in the binding to the restriction factor huTRIM5α.

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.

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.

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.

This review is focused at exploring the strengths of modern technology driven data compiled in the areas of virus gene sequencing, virus protein structures and their implication to viral diagnosis and therapy. The information for virome analysis (viromics) is generated by the study of viral genomes (entire nucleotide sequence) and viral genes (coding for protein). Presently, the study of viral infectious diseases in terms of etiopathogenesis and development of newer therapeutics is undergoing rapid changes. Currently, viromics relies on deep sequencing, next generation sequencing (NGS) data and public domain databases like GenBank and unique virus specific databases. Two commonly used NGS platforms: Illumina and Ion Torrent, recommend maximum fragment lengths of about 300 and 400 nucleotides for analysis respectively. Direct detection of viruses in clinical samples is now evolving using these methods. Presently, there are a considerable number of good treatment options for HBV/HIV/HCV. These viruses however show development of drug resistance. The drug susceptibility regions of the genomes are sequenced and the prediction of drug resistance is now possible from 3 public domains available on the web. This has been made possible through advances in the technology with the advent of high throughput sequencing and meta-analysis through sophisticated and easy to use software and the use of high speed computers for bioinformatics. More recently NGS technology has been improved with single-molecule real-time sequencing. Here complete long reads can be obtained with less error overcoming a limitation of the NGS which is inherently prone to software anomalies that arise in the hands of personnel without adequate training. The development in understanding the viruses in terms of their genome, pathobiology, transcriptomics and molecular epidemiology constitutes viromics. It could be stated that these developments will bring about radical changes and advancement especially in the field of antiviral therapy and diagnostic virology.

Purpose:Type III interferon is well known to have diverse antiviral and immunomodulatory activities. Studies describing the association of interleukin (IL)-28 polymorphisms in treatment-experienced HIV participants are limited. This study was aimed to determine the association of IL-28B gene polymorphisms with immunological recovery in HIV patients on 6–9 months of antiretroviral therapy (ART). Methods:Eighty treatment-naive HIV patients were recruited, of which 48 patients were followed up after 6–9 months of ART. Whole blood samples were collected before and after 6–9 months of ART. CD4, CD8 and CD3 counts were enumerated flow cytometry. IL-28B polymorphisms (rs12979860 and rs8099917) were profiled by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The IL-28 mRNA and plasma HIV-1 viral load were estimated using real-time PCR and plasma IL-28 level by ELISA. Results:The CD4, CD4/CD3%, IL-28 mRNA and reversal of CD4/CD8 ratio were significantly increased following 6–9 months of ART (P< 0.01). The rs12979860 CC genotype and rs12979860:rs8099917 (CC: TT) haplotype showed significant association with higher CD4+ T-cell count amongst treatment-naive HIV-infected individuals (P< 0.05). In addition, there was a significant association of rs12979860 CC genotype with increase in CD4/CD3% following 6–9 months of ART. IL-28 mRNA showed correlation with the HIV-1 viral load, and there was a significant increase in the IL-28 mRNA expression following 6–9 months of ART. Conclusion:Our preliminary findings suggest that IL-28 polymorphisms could influence both immunological recovery and therapeutic response in HIV infection. Hence, functional studies are warranted to understand the mechanistic basis of IL-28-mediated host genetic influence on HIV therapeutic response.

Human cytomegalovirus (HCMV) reactivation is a major cause of morbidity and mortality among stem cell transplant recipients post-transplantation.

ABSTRACTThe frequencies of 10 opportunistic DNA viruses were determined by multiplex real-time PCR in paired cerebrospinal fluid (CSF) and brain tissue of HIV-infected individuals. In the CSF, viruses were detectable in 45/55 cases: JC virus (JCV) in 62%, Epstein-Barr virus (EBV) in 44%, cytomegalovirus (CMV) in 25%, varicella-zoster virus (VZV) in 3.6%, herpes simplex virus 1 (HSV-1) in 1.8%, and human herpesvirus 6 (HHV-6) in 1.8% of cases. A single virus was detectable in 20 cases, 19 cases had coinfection with two viruses, and 6 cases were positive for three viruses. JCV was detectable in the CSF of 62% of cases and in 42% of brain tissues, with higher loads in progressive multifocal leukoencephalopathy (PML) (P< 0.05).

ABSTRACTCD4+T cell count estimations are subject to high variations; hence, in this study, the previous day's tested samples were included routinely as the internal quality controls. The percentages of variation of the 2-day values were analyzed for 280 observations and the mean variation for CD4+and CD3+T cell counts ranged from 5.21% to 9.66%. This method is a good internal quality control (IQC) procedure for the estimation of CD3+and CD4+T cell counts in resource-poor settings.

Fibrolamellar carcinomas are a unique type of liver carcinoma that arise in non-cirrhotic livers of young individuals. Despite their distinctive appearance, recent studies have demonstrated a lack of consistency in how fibrolamellar carcinomas are diagnosed by pathologists. As a potential aide in diagnosis, we investigated the staining properties of CD68. The CD68 gene encodes for a transmembrane glycoprotein located within lysosomes and endosomes. Macrophages as well as other cell types rich in lysosomes/endosomes are CD68 positive. Cases of fibrolamellar carcinoma were collected from four academic centers. Control groups included hepatocellular carcinomas arising in both non-cirrhotic livers and cirrhotic livers. A group of cholangiocarcinomas were also stained. CD68 immunostaining was scored for both intensity and distribution on a scale of 0 to 3+. Twenty-three primary fibrolamellar carcinomas and 9 metastases (total of 24 individuals) were immunostained and showed a distinctive granular, dot-like or stippled pattern of cytoplasmic staining in nearly all cases (31/32), with a median distribution and intensity score of 3+. In control hepatocellular carcinomas that arose in non-cirrhotic livers, 10/39 showed CD68 staining with a median distribution and intensity score of 2+. In hepatocellular carcinomas arising in cirrhotic livers, 3/27 cases showed CD68 positivity, all with stippled dot-like cytoplasmic staining similar to that of fibrolamellar carcinomas. All five cholangiocarcinomas were negative. Overall, CD68 positivity was strongly associated with fibrolamellar carcinomas, P<0.001 and had a sensitivity of 96%, a specificity of 80%, and a negative predictive value of 98%. In sum, tumor positivity for CD68 staining was highly sensitive for fibrolamellar carcinoma and a lack of CD68 staining should suggest caution in making a diagnosis of fibrolamellar carcinoma.

Fibrolamellar carcinomas are a unique type of liver carcinoma that arise in non-cirrhotic livers of young individuals. Despite their distinctive appearance, recent studies have demonstrated a lack of consistency in how fibrolamellar carcinomas are diagnosed by pathologists. As a potential aide in diagnosis, we investigated the staining properties of CD68. The CD68 gene encodes for a transmembrane glycoprotein located within lysosomes and endosomes. Macrophages as well as other cell types rich in lysosomes/endosomes are CD68 positive. Cases of fibrolamellar carcinoma were collected from four academic centers. Control groups included hepatocellular carcinomas arising in both non-cirrhotic livers and cirrhotic livers. A group of cholangiocarcinomas were also stained. CD68 immunostaining was scored for both intensity and distribution on a scale of 0 to 3+. Twenty-three primary fibrolamellar carcinomas and 9 metastases (total of 24 individuals) were immunostained and showed a distinctive granular, dot-like or stippled pattern of cytoplasmic staining in nearly all cases (31/32), with a median distribution and intensity score of 3+. In control hepatocellular carcinomas that arose in non-cirrhotic livers, 10/39 showed CD68 staining with a median distribution and intensity score of 2+. In hepatocellular carcinomas arising in cirrhotic livers, 3/27 cases showed CD68 positivity, all with stippled dot-like cytoplasmic staining similar to that of fibrolamellar carcinomas. All five cholangiocarcinomas were negative. Overall, CD68 positivity was strongly associated with fibrolamellar carcinomas, P<0.001 and had a sensitivity of 96%, a specificity of 80%, and a negative predictive value of 98%. In sum, tumor positivity for CD68 staining was highly sensitive for fibrolamellar carcinoma and a lack of CD68 staining should suggest caution in making a diagnosis of fibrolamellar carcinoma.

Epidermal growth factor receptor (EGFR) is overexpressed in a significant proportion of hepatocellular carcinomas. Recent studies of EGFR inhibitors to treat hepatocellular carcinoma have been encouraging and better understanding of EGFR signaling may lead to more effective strategies for inhibiting this key pathway. The EGFR can be phosphorylated at different tyrosine sites, leading to subsequent activation of different pathways. Cell line and animal studies have shown that MAPK and STAT-3 are important mediators of the EGFR signal in liver cells. However, little is known about EGFR phosphorylation and subsequent signaling in primary hepatocellular carcinoma. We investigated the site of EGFR phosphorylation by Western blot in 18 hepatocellular carcinomas. Fourteen of 18 hepatocellular carcinomas had detectable EGFR by Western blotting and 13 of 14 showed phosphorylation at tyrosine 845. In contrast, no EGFR phosphorylation was detected at tyrosine 998, tyrosine 1045, or tyrosine 1068, which signal through other pathways including STAT-3 and MAPK. These findings were further explored by examination of EGFR expression and signaling pathway activation in tissue arrays comprised of 73 hepatocellular carcinomas using antibodies that recognize phosphorylated (or activated) proteins. Tissue array studies also found no correlation between EGFR expression (29% of cases) and STAT-3 nuclear positivity (16%), AKT (4%), MAPK (3%), or STAT-5 (3%) positivity, all P>0.05. EGFR expression was correlated with hepatitis B infection, but not with tumor size, nuclear grade, or proliferative rate. We conclude that EGFR is phosphorylated at tyrosine 845 in most hepatocellular carcinomas and that EGFR expression by immunohistochemistry does not correlate well with STAT-3, STAT-5, MAPK, or AKT immunostaining.Modern Pathology (2006) 19, 1456–1461. doi:10.1038/modpathol.3800665; published online 25 August 2006

Hedgehog (Hh) pathway activation promotes tumors in several endodermally derived tissues, but its role in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. Although normal hepatocytes lack Hh signaling, activation of the Hh pathway in endodermal progenitors is required for liver development. Thus, we hypothesized that hepatocarcinogenesis may involve regulation of Hh signaling. This pathway is activated when Hh ligand binds to its receptor, Patched (PTC). In an unoccupied state, PTC normally functions as a tumor suppressor that inhibits Smoothened (SMO), a proto-oncoprotein, from activating downstream components and transcription of target genes. Here we show that in HCCs, overexpression of the Smo proto-oncogene, as well as an increase in the stoichiometric ratio of Smo to Ptc mRNA levels, correlated with tumor size, a prognostic indicator in HCC biology. In one tumor we identified a novel Smo mutation in an evolutionarily conserved residue. We also demonstrated that HCC cell lines (HepG2 and Hep3B) expressed Hh pathway components and activated Hh transcriptional targets. In Hep3B cells, cyclopamine, an inhibitor of wild-type SMO, had no effect, but KAAD-cyclopamine, a blocker of oncogenic SMO, inhibited Hh signaling activity by 50%, decreased expression of the hepatocarcinogenic oncogene, c-myc, by 8-fold, and inhibited the growth rate of Hep3B cells by 94%. These data support our hypothesis that Hh signaling is dysregulated in human hepatocarcinogenesis. We demonstrate that overexpression and/or tumorigenic activation of the Smo proto-oncogene mediates c-myc overexpression which plays a critical role in hepatocarcinogenesis and suggests that Smo is a prognostic factor in HCC tumorigenesis.

ABSTRACTThe performance characteristics of a rapid immunochromatographic-screening test, SD Bioline HIV-1/2 3.0 (Standard Diagnostics Inc., Kyonggi-do, South Korea) on 23,754 sera and 30 plasma samples are reported. The sensitivity and specificity for the assay on serum samples are 100% and 99.4%, respectively. The assay detected antibodies in individuals infected with human immunodeficiency virus type 1 (HIV-1) genotypes A and C and HIV-2. This straightforward assay is a reliable diagnostic tool for screening HIV in resource-poor settings.

ABSTRACTTwo rapid human immunodeficiency virus (HIV) screening assays, HIV TRI-DOT and HIV-SPOT were compared with standard enzyme-linked immunosorbent assays according to a testing algorithm. Sensitivities and specificities in the real-time evaluation were 99.5 and 99.9% for TRI-DOT and 98.2 and 99.7% for HIV-SPOT, respectively. These two tests are suitable for use where facilities and laboratory expertise are limited.

ABSTRACTThe performance of a rapid particle agglutination test for human immunodeficiency virus (HIV) (Capillus HIV type 1 [HIV-1]/HIV-2) on hospital samples is compared with enzyme-linked immunosorbent assays. The test had a sensitivity and specificity of 99 and 98.9%, respectively. In addition, the test was reactive on plasma samples from all individuals infected with HIV-1 subtype C. This test can safely be used for voluntary counseling and testing in India.