Mini P. Singh

Usefulness of RT-PCR for the diagnosis of Japanese encephalitis in clinical samples

The present study was carried out between July 2003 and December 2005 in PGIMER, Chandigarh, India and aimed to compare IgM capture ELISA and nested RT-PCR for the diagnosis of Japanese encephalitis (JE). The samples collected were cerebrospinal fluid and blood from 40 febrile patients with encephalitis (n=40, group I) and blood samples from febrile patients without encephalitis residing in JE endemic areas (n=45, group II). Overall, in CSF samples JE specific RNA was detected in 9/40 (22.5%), while 7/28 (25%) patients showed the presence of specific IgM antibodies. Only 28 CSF samples could be subjected to both RT-PCR and IgM and, among these, 13 cases were found to be confirmed JE based on IgM and/or RT-PCR positivity. Among the confirmed cases, 6 (6/13, 46.5%) could be detected by RT-PCR alone, 4 (4/13, 30.7%) by IgM capture ELISA and 3 (3/13, 23.1%) patients were positive by both the methods. All the RT-PCR positive cases had presented within 5 d of onset of illness. The serum samples of only 16 patients in group I could be tested for IgM antibodies and 5 (31.25%) were found to be positive, while in group II, 11.1% (5/45) positivity was observed. JE specific RNA could not be detected in serum samples of either group of patients. This study highlights the need for carrying out RT-PCR in CSF samples, compared to IgM antibody detection, for the early detection of JEV.

NS1 antigen as an early diagnostic marker in dengue: report from India

Detection of specific IgM antibodies by ELISA forms the mainstay for diagnosis of dengue infection. However, IgM antibodies develop after 4 to 5 days of infection. The methods for early diagnosis include virus isolation and reverse transcriptase polymerase chain reaction (RT-PCR) which need a sophisticated laboratory. Another alternative that has recently come up is NS1 antigen detection. The present study compared IgM antibody detection with NS1 antigen for the diagnosis of acute dengue in 87 samples. NS1 antigen could be detected with good sensitivity (71-100%) till day 3 of fever, whereas IgM had a sensitivity of 0% to 50% at this time. On day 4 of illness, both the tests had comparative sensitivity. Beyond day 4, IgM antibody detection was superior to NS1. Both these diagnostic modalities were also compared with RT-PCR in 40 acute samples. NS1 detected additional 15 samples, which were missed by PCR. NS1 antigen is an early diagnostic marker that is feasible in a routine diagnostic laboratory.

Hepatitis E virus antigen detection as an early diagnostic marker: report from India.

Hepatitis E virus (HEV) is implicated in many outbreaks of viral hepatitis in the Indian subcontinent. The conventional diagnosis of such outbreaks rests on the detection of anti‐HEV IgM antibodies. However, IgM antibodies develop after 4–5 days of infection. An early‐diagnostic marker is imperative for timely diagnosis of the outbreak and also initiation of control measures. This study aimed to determine the use of hepatitis E virus antigen detection as an early diagnostic marker in an outbreak in comparison to anti‐HEV IgM and RT‐PCR analyses. Forty samples were collected during a suspected outbreak of viral hepatitis due to HEV. A total of 36 samples were positive for one or more HEV markers. The positivity for anti‐HEV IgM, HEV antigen, and RT‐PCR was 91.6%, 69.4%, and 47.2% respectively. RT‐PCR and HEV antigen detection gave the highest positive results (100%) in the first 3 days of illness. Positive HEV PCR declined to 54% by Days 4–7, whereas HEV antigen and IgM detection were 88% and 100%, respectively. Sequencing of representative HEV samples indicated that the strains responsible for this outbreak belonged to genotype I, subtype 1a. HEV antigen was found to be an early diagnostic marker of acute infection. HEV antigen was detected in three additional cases in the early phase (1–3 days), and they had no detectable anti‐HEV IgM antibodies. These three samples were also positive for HEV RNA. After Day 7, anti‐HEV IgM was the main diagnostic indicator of infection. J. Med. Virol. 85:823–827, 2013.

Detection of Mycobacterium tuberculosis genome in vitreous fluid of eyes with multifocal serpiginoid choroiditis.

PURPOSE To compare 3 different molecular techniques to detect the Mycobacterium tuberculosis genome in vitreous fluid of eyes with multifocal serpiginoid choroiditis (MSC). DESIGN Prospective, interventional case series. PARTICIPANTS Eleven patients (11 eyes) with active MSC in at least 1 eye underwent diagnostic pars plana vitrectomy (PPV) between October 2012 and December 2013. METHODS Vitreous fluid samples were subjected to multitargeted polymerase chain reaction (PCR) for a M. tuberculosis assay, the Gene Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), and a line probe assay (GenoType MTBDRplus; Hain Lifescience, GmbH, Nehren, Germany). The samples with positive results were subjected to rpoB gene sequencing to demonstrate rifampicin resistance. The clinical details, digital fundus imaging, and treatment details and outcomes also were noted. MAIN OUTCOME MEASURES Detection of the M. tuberculosis genome and rifampicin resistance in the vitreous samples. RESULTS Of the 11 eyes subjected to PPV, the multitargeted PCR results for tuberculosis were positive for 10 eyes, the MTBDRplus assay results were positive in 6 eyes, and the Gene Xpert MTB/RIF assay results were positive in 4 eyes. Rifampicin resistance was detected in 3 eyes by rpoB gene sequencing, in 3 eyes by the MTBDRplus assay, and in 1 eye by the Gene Xpert MTB/RIF assay. CONCLUSIONS We detected the M. tuberculosis genome in the vitreous fluid of eyes with MSC using 3 different molecular techniques. Rifampicin resistance was detected for the first time in eyes with MSC.

High levels of circulating HMGB1 as a biomarker of acute liver failure in patients with viral hepatitis E

Viral hepatitis E clinically ranges between acute self‐limiting hepatitis (AVH) and acute liver failure (ALF). The varied clinical course of the disease possibly thought to be immune‐mediated. High‐mobility group box 1 (HMGB1) is a non‐histone chromosomal nuclear protein with recently discovered pro‐inflammatory and immunomodulatory action. Its presence in abundance within hepatocytes is thought provoking in patients with hepatitis.

Congenital rubella and cytomegalovirus infections in and around Chandigarh

AIMS This study has analyzed the role of rubella and cytomegalovirus (CMV) in infections of children and pregnant women. SETTINGS AND DESIGN The study was carried out in a tertiary care hospital. Data from blood samples from pregnant women (asymptomatic and also women with obstetric problems) and children (suspected of intrauterine infections) that were received in the laboratory over a period of 8 years were analysed. MATERIALS AND METHODS The samples were tested for rubella- and CMV-specific IgM antibodies by capture enzyme linked immunosorbent assay. RESULTS In children, the overall positivity for rubella- and CMV-specific IgM antibodies was 2.8% and 12.5%, respectively. In asymptomatic pregnant females, rubella positivity was 0.7% while in women with obstetric complications it was 3.4%. IgM antibody positivity in cases of CMV was 7.8% in both asymptomatic pregnant women and also in women with obstetric complications. CONCLUSIONS The study indicated that infection with CMV is more common than the rubella virus. The incidence of rubella has reduced over the past few years. Hence, screening for rubella infection may be reserved for women with obstetric complications only. The routine screening for CMV among all antenatal cases is a debatable issue.

Human papillomavirus-associated cancers: A growing global problem

Human papillomavirus (HPV) infection is linked with several cancers such as cancer cervix, vagina, vulva, head and neck, anal, and penile carcinomas. Although there is a proven association of HPV with these cancers, questions regarding HPV testing, vaccination, and treatment of HPV-related cancers continue to remain unanswered. The present article provides an overview of the HPV-associated cancers.

Institutional outbreak of rubella in a healthcare center in Chandigarh, North India.

Rubella is traditionally considered a childhood disease but it has the potential to cause outbreaks in closed communities when a susceptible population accumulates. The present study reports an outbreak of rubella among healthcare workers in the pediatric center of a tertiary care North Indian hospital. The cases of rubella were identified by clinical features and confirmed by the detection of anti‐rubella IgM antibodies in blood by enzyme linked immunosorbent assay. A total of 23 cases of rubella occurred over a period of one and a half month, out of which 9 (39%) were males. All the patients were in the age group of 21–35 years. None of the patients gave a history of rubella vaccination. This outbreak of rubella occurred due to the accumulation of a susceptible population in a closed hospital environment. There is need for the introduction of rubella vaccination in healthcare workers to prevent outbreaks at work place. J. Med. Virol. 82:341–344, 2010.

Cost‐effectiveness of human papillomavirus vaccination for adolescent girls in Punjab state: Implications for India's universal immunization program

Introduction of human papillomavirus (HPV) vaccination for adolescent girls is being considered in the Punjab state of India. However, evidence regarding cost‐effectiveness is sought by policy makers when making this decision. The current study was undertaken to evaluate the incremental cost per quality‐adjusted life‐years (QALYs) gained with introduction of the HPV vaccine compared with a no‐vaccination scenario.

Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay

Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [3 H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher ( P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.