Ekta Gupta

The changing epidemiology of dengue in Delhi, India.

BackgroundA major DHF outbreak occurred in Delhi in 1996. Following this another outbreak was reported in the year 2003. In the years 2004 and 2005, though no outbreak was reported, a definitely higher number of samples were received in the virology laboratory of A.I.I.M.S. from suspected cases of dengue infection. This study was designed to compare the serological and virological profiles of confirmed dengue cases in the years 2003, 2004 and 2005.ResultsOut of 1820 serum samples received from suspected cases in all three years, 811 (44.56%) were confirmed as dengue infection serologically. Out of these confirmed dengue cases maximum cases, in all three years, were seen in the age group 21–30 years. There was an increase in the number of samples received in the post monsoon period (September to November) with a peak in the second and third week of October. More samples were received from DHF cases in the year 2005 than 2004 and 2003. All four dengue serotypes were seen co-circulating in the year 2003, followed by complete predominance of dengue serotype 3 in 2005.ConclusionEpidemiology of dengue is changing rapidly in Delhi. Dengue infections are seen every year thus making it an endemic disease. After co-circulation of all serotypes in 2003, now dengue serotype 3 is emerging as the predominant serotype.

Controlled attenuation parameter for non‐invasive assessment of hepatic steatosis: Does etiology affect performance?

Hepatic steatosis is an important parameter to assess in chronic liver disease patients. The controlled attenuation parameter (CAP) assesses liver steatosis using transient elastography.

Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting Programmed Cell Death Protein4 (PDCD4) and Phosphatase and Tensin Homologue (PTEN)

Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably) were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01) and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001) in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively) and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression.

Hepatitis B vaccination with or without hepatitis B immunoglobulin at birth to babies born of HBsAg-positive mothers prevents overt HBV transmission but may not prevent occult HBV infection in babies: a randomized controlled trial

Vertical transmission of Hepatitis B virus HBV can result in a state of chronic HBV infection and its complications. HBV vaccination with or without hepatitis B immunoglobulin (HBIG) prevents transmission of overt infection to the babies. However, whether it also prevents occult HBV infection in babies is not known. Consecutive pregnant women of any gestation found to be HBsAg positive were followed till delivery, and their babies were included in the study. Immediately after delivery, babies were randomized to receive either HBIG or placebo in addition to recombinant HBV vaccine (at 0, 6, 10 and 14 weeks). The primary end‐point of the study, assessed at 18 weeks of age, was remaining free of any HBV infection (either overt or occult) plus the development of adequate immune response to vaccine. The babies were further followed up for a median of 2 years of age to determine their eventual outcome. Risk factors for HBV transmission and for poor immune response in babies were studied. Of the 283 eligible babies, 259 were included in the trial and randomized to receive either HBIG (n = 128) or placebo (n = 131) in addition to recombinant HBV vaccine. Of the 222 of 259 (86%) babies who completed 18 weeks of follow‐up, only 62/222 (28%) reached primary end‐point. Of the remaining, 6/222 (3%) developed overt HBV infection, 142/222 (64%) developed occult HBV infection, and 12/222 (5%) had no HBV infection but had poor immune response. All 6 overt infections occurred in the placebo group (P = 0.030), while occult HBV infections were more common in the HBIG group (76/106 [72%] vs. 66/116 [57%]; P = 0.025). This may be due to the immune pressure of HBIG. There was no significant difference between the two groups in frequency of babies developing poor immune response or those achieving primary end‐point. The final outcome of these babies at 24 months of age was as follows: overt HBV infection 4%, occult HBV infection 42%, no HBV infection but poor immune response 8% and no HBV infection with good immune response 28%. Women who were anti‐HBe positive were a low‐risk group, and their babies were most likely to remain free of HBV infection (occult or overt) and had good immune response to the vaccine. Maternal HBeAg‐positive status and negativity for anti‐HBe predicted not only overt but also any infection (both overt and occult) in babies. In addition, high maternal HBV DNA and treatment with vaccine alone were significant factors for overt HBV infection in babies. The current practice of administration of vaccine with HBIG at birth to babies born of HBsAg‐positive mothers is not effective in preventing occult HBV infection in babies, which may be up to 40%. Because the most important risk factors for mother‐to‐baby transmission of HBV infection are the replicative status and high HBV DNA level in mothers; it will be worthwhile investigating the role of antivirals and HBIG administration during pregnancy to prevent mother‐to‐child transmission of HBV infection.

Role of hepatitis E virus antigen in confirming active viral replication in patients with acute viral hepatitis E infection

BACKGROUND Demonstration of active viral replication is important in serologically confirmed cases of hepatitis E virus (HEV) infection to assess prognosis. Detection of HEV RNA by reverse transcriptase polymerase chain reaction (rtPCR) is the gold standard for demonstration of active viremia. OBJECTIVES The present study was designed to compare the diagnostic utility of HEV antigen (Ag) with HEV IgM and HEV rtPCR in detecting the active viral replication. STUDY DESIGN Blood samples from 156 probable cases of acute viral hepatitis (AVH) were collected. Screening for hepatitis B surface antigen (HBsAg), antibody to hepatitis C virus (anti HCV), IgM antibody to hepatitis A virus (HAV IgM) was done on the Architect platform (Abbott Laboratories, IL, USA). HEV IgM ELISA (Wantai Biological, Beijing, China), HEV-Ag ELISA (Wantai Biological, Beijing, China) and HEV rtPCR were done on all the samples. RESULTS Out of 156 AVH cases in 56 (35.8%) a diagnosis of HEV was confirmed. Positivity being; anti-HEV IgM 44/56, HEV RNA 20/56, and HEV antigen 17/56 in established cases. Male to female ratio was 3:1. The median age was 40 (range 14-71) years. HEV Ag showed a good concordance with HEV RNA (k=0.635, p<0.001). However HEV IgM did not show any concordance with HEV RNA (k=0.14). CONCLUSION HEV antigen assay can be used as an additional diagnostic marker to confirm active viral replication in serologically positive samples.

Hepatitis C virus: Screening, diagnosis, and interpretation of laboratory assays

An estimated 3% of the world population is infected with Hepatitis C virus (HCV), a hepatotropic RNA virus, transmitted primarily via the blood route. The major modes of transmission of the virus include injection drug use, unsafe injection practices, blood transfusion etc. HCV causes chronic hepatitis in about 80% of those infected by it. The mainstay in diagnosing infection with HCV is to initially screen high risk groups for antibodies to HCV (anti-HCV). The inclusion of serum to cut-off ratio (S/CO) in recent guidelines is helpful in deciding the supplemental assay to be used to confirm initially reactive screening results. Nucleic acid amplification tests (NAT) are used as confirmatory tools, and also to determine viral load prior to initiating treatment. Quantitative NAT has replaced qualitative assays. Genotyping is an important tool in clinical management to predict the likelihood of response and determine the optimal duration of therapy. The impact of this infection has begun to emerge in India. The problem of professional blood donation despite an existing law against it, and flourishing unsafe injection practices, are potential sources for the spread of hepatitis C in our country. All health care practitioners need to understand how to establish or exclude a diagnosis of HCV infection and to interpret the tests correctly. In the absence of a preventive or therapeutic vaccine, and also of post-exposure prophylaxis against the virus, it is imperative to diagnose infection by HCV so as to prevent hepatic insult and the ensuing complications that follow, including primary hepatocellular carcinoma (HCC). This review aims to help blood bank staff regarding options for diagnosis and management of donors positive for HCV.

Transfusion-transmitted hepatitis E: is screening warranted?

Hepatitis E virus (HEV) is an emerging infectious threat to blood safety. In recent years, there have been a number of publications delineating this threat by providing evidence of the transmissibility of this virus through transfusions. The extent of transmission and its clinical relevance are issues under debate at present. HEV usually causes a self-limiting illness which subsides in a few weeks barring a few cases where fulminant hepatic failure occurs. The virus poses a risk of higher morbidity and mortality in pregnant females, patients with pre-existing liver disease and solid organ transplant recipients. As these categories of patient often require repeated transfusions or massive transfusions, they are exposed to a greater risk of transmission of HEV. At present, there is little evidence to advocate universal screening for this virus but considering that there is no definitive treatment for HEV induced hepatitis, selective screening should be advocated in blood products for high risk recipients in endemic areas.

Impaired monocyte-macrophage functions and defective toll-like receptor signaling in hepatitis E virus-infected pregnant women with acute liver failure

Acute viral hepatitis resulting due to hepatitis E viral infection (AVH‐E) is often serious in pregnancy and could result in acute liver failure (ALF). The role of monocytes and macrophages (mono‐macs) in the pathogenesis of AVH‐E and development of ALF‐E in pregnancy is unclear. We investigated the functions of mono‐macs in pregnant (P), AVH‐E (n = 44), ALF‐E (n = 12), healthy controls (HC; n = 20) and compared with nonpregnant (NP) AVH‐E (n = 10), ALF‐E (n = 5), and HC (n = 10). We also recruited non‐hepatitis E virus‐related pregnant (P), ALF‐NE (n = 5) and non‐pregnant (NP), ALF‐NE (n = 12) patients with ALF. Mono‐macs, dendritic cell (DC) phenotypes, and Toll‐like receptor (TLR) expressions were studied by flow cytometry and reverse‐transcriptase polymerase chain reaction. Mono‐macs functionality was determined by analyzing their phagocytic activity and reactive oxygen species (ROS) generation by using flow cytometry. Frequency of mono‐macs and DCs was increased during HEV infection compared to HC (P < 0.001). Macrophages were increased (P < 0.002) in ALF‐E(P) compared to ALF‐NE(P). The macrophage phagocytic activity and Escherichia coli‐induced ROS production was significantly impaired in ALF‐E(P) compared to AVH‐E(P) (P < 0.001), ALF‐E(NP), and ALF‐NE(P) patients (P < 0.02). TLR3 and TLR9 expression and downstream MYD88 signalling molecules IRF3 and IRF7 were significantly down‐regulated in ALF‐E(P) (P < 0.00) compared to AVH‐E(P) and ALF‐NE(P). Conclusion: Functionality of mono‐macs is impaired in pregnant ALF‐E patients compared to AVH‐E(P). Reduced TLR3 and TLR7 expression and TLR downstream‐signaling molecules in pregnant ALF‐E patients suggests inadequate triggers for the innate immune responses contributing to development and severity of ALF‐E. Studies using TLR agonists to activate mono‐macs may be of use and in vitro studies should be undertaken using patient samples.(Hepatology 2015;62:1683–1696)

Histopathology for the diagnosis of infectious diseases.

Histopathological examination of tissue biopsies for the identification of infectious organisms is a very important diagnostic tool. Conventional culture confirmation of tissue biopsies often fail to identify any pathogen as, first of all, invariably most of the tissue samples that are collected and sent for culture isolation are inappropriately collected in formalin, which prevents pathogen growth in culture media. Inadequate processing like grinding, etc. further hinders isolation. Presence of inhibitors like dead tissue debris, fibers, etc. also delays isolation. Microbiologists often lack expertise in identifying infectious pathogens directly from tissue biopsies by microscopic visualization. This review therefore acquaints microbiologists with the various methods available for detecting infectious agents by using histological stains. On histopathological examination of the tissue biopsy once, it is determined that a disease is likely to be due to an infection and has characterized the inflammatory response and hence associated microorganisms should be thoroughly looked for. Although some microorganisms or their cytopathic effects may be clearly visible on routine haematoxylin- and eosin-stained sections, additional histochemical stains are often needed for their complete characterization. Highly specific molecular techniques, such as immunohistochemistry, in situ hybridization and nucleic acid amplification, may be needed in certain instances to establish the diagnosis of infection. Through appropriate morphologic diagnoses and interlaboratory communication and collaboration, direct microscopic visualization of tissue samples can thus be very helpful in reaching a correct and rapid diagnosis.

Point -of -care testing (POCT) in molecular diagnostics: Performance evaluation of GeneXpert HCV RNA test in diagnosing and monitoring of HCV infection

BACKGROUND Molecular testing at the point-of-care may turn out to be game changer for HCV diagnosis and treatment monitoring, through increased sensitivity, reduced turnaround time, and ease of performance. One such assay GeneXpert® has recently been released. OBJECTIVES Comparative analysis between performances of GeneXpert® and Abbott HCV-RNA was done. STUDY DESIGN 174 HCV infected patients were recruited and, one time plasma samples from 154 patients and repeated samples from 20 patients, obtained at specific treatment time-points (0, 4, 12 and 24) weeks were serially re-tested on Xpert®. RESULTS Genotype 3 was the commonest, seen in 80 (66%) of the cases, genotype 1 in 34 (28.3%), genotype 4 in 4 (3.3%) and genotypes 2 and 5 in 1 (0.8%) each. Median HCV RNA load was 4.69 log10 (range: 0-6.98log10) IU/ml. Overall a very good correlation was seen between the two assays (R2=0.985), concordance of the results between the assays was seen in 138 samples (89.6%). High and low positive standards were tested ten times on Xpert® to evaluate the precision and the coefficient of variation was 0.01 for HPC and 0.07 for the LPC. Monitoring of patients on two different regimes of treatment, pegylated interferon plus ribavirin and sofosbuvir plus ribavirin was done by both the systems at baseline, 4, 12 and 24 weeks. Perfect correlation between the assays in the course of therapy at different treatment time- point in genotypes 3 and 1 was seen. CONCLUSION The study demonstrates excellent performance of the Xpert® HCV assay in viral load assessment and in treatment course monitoring consistency.