Radha Kanta Ratho

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.

Dengue fever related acalculous cholecystitis in a North Indian tertiary care hospital.

Background and Aims:  To document the clinical outcome and prognosis of acalculous cholecystitis in dengue fever.

Measles outbreak in a periurban area of Chandigarh : Need for improving vaccine coverage and strengthening surveillance

Objective : An outbreak of measles was investigated in the periurban areas of Chandigarh Union Territory, during the months of December 1998 to February 1999. Mainly the children below 15 years of age were affected. The children of migrant labourers belonging to the neighbouring states of Uttar Pradesh and Bihar constituted the majority of population in the area under study. They belonged to lower socio economic status with low immunization coverage.Methods : A total of 2968 houses were surveyed for epidemiological investigations in the areas of colony No. 5, Ramdarbar, Palsora and Pandit colony of Kajheri, covering a population of 14,601 and 7.3% (216/2968) of families were affected in the outbreak.Results : Two hundred and eighty three cases of measles were reported with an attack rate of 4.5% and male to female ratio of (M:F) 5.3%:3.6%. Among the measles cases, 48.8% had received measles vaccination. The outbreak was investigated by detecting measles specific IgG/IgM antibodies either in acute or convalescent serum samples or both. Due to inadequate surveillance system and containment measures, the outbreak was in full swing during the winter months. Measles related complications were reported in 31.1% cases (i.e. diarrhoea in 15.2% and Pneumonia is 7.1%).Conclusion : Following smallpox and guinea worm eradication, WHO’S next thrust, is on eradication of poliomyelitis and measles. Hence, strengthening of disease surveillance as well as vaccination policies are mandatory to achieve disease control in these areas.

Pathology and virology findings in cases of fatal influenza A H1N1 virus infection in 2009-2010.

Bal A, Suri V, Mishra B, Bhalla A, Agarwal R, Abrol A, Ratho R K & Joshi K 
(2012) Histopathology 60, 326–335 
Pathology and virology findings in cases of fatal influenza A H1N1 virus infection in 2009–2010

TNF-α promoter polymorphism: a factor contributing to the different immunological and clinical phenotypes in Japanese encephalitis

BackgroundMore than three billion populations are living under the threat of Japanese encephalitis in South East Asian (SEA) countries including India. The pathogenesis of this disease is not clearly understood and is probably attributed to genomic variations in viral strains as well as the host genetic makeup. The present study is to determine the role of polymorphism of TNF-alpha promoter regions at positions -238G/A, -308G/A, -857C/T and -863C/A in the severity of Japanese encephalitis patients.MethodsTotal of 142 patients including 66 encephalitis case (IgM/RT-PCR positive), 16 fever cases (IgM positive) without encephalitis and 60 apparently healthy individuals (IgG positive) were included in the study. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) using site specific restriction enzymes were implemented for polymorphism study of TNF alpha promoter.ResultsFollowing the analysis of the digestion patterns of four polymorphic sites of the TNF- alpha promoter region, a significant association was observed between the allele -308A and -863C with the patients of Japanese encephalitis.ConclusionsTNF- alpha 308 G/A has been shown to be associated with elevated TNF- alpha transcriptional activity. On the other hand, polymorphism at position -863C/A in the promoter region has been reported to be associated with reduced TNF- alpha promoter activity and lower plasma TNF levels. As per the literature search, this is the first study to identify the role of TNF- alpha promoter in JE infection. Our results show that subjects with - 308A and -863C alleles are more vulnerable to the severe form of JE infection.

Utility of multiplex reverse transcriptase-polymerase chain reaction for diagnosis and serotypic characterization of dengue and chikungunya viruses in clinical samples ☆

The reemergence of chikungunya virus (CHIKV) has compounded the already existing dengue problem because of clinical similarities and common vector, demanding the need for a rapid and specific diagnosis. Thus, dengue chikungunya multiplex reverse transcriptase-polymerase chain reaction (DCmRT-PCR) was developed and validated for simultaneous detection of dengue and chikungunya viral infections and its utility in virus serotyping. Blood samples from 97 suspected dengue and chikungunya cases and 10 healthy controls were subjected to dengue and chikungunya conventional RT-PCR and DCmRT-PCR. Thirty-one of 97 samples were positive for dengue or chikungunya viral RNA by RT-PCR and DCmRT-PCR with 100% concordance. DCmRT-PCR products were cycle sequenced. Seven dengue virus strains were clustered within genotype III of DENV-3 and 4 within genotype III of DENV-1, whereas chikungunya sequences were clustered within the Central/East African genotype. DCmRT-PCR was found to be a potential rapid test for simultaneous detection of dengue and CHIKV in clinical samples along with dengue serotyping.

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.

Molecular detection and sequence analysis of hepatitis E virus in patients with viral hepatitis from North India.

Viral hepatitis is a major cause of mortality and morbidity in developing countries. Hepatitis E virus (HEV) is responsible for both sporadic and epidemic outbreaks of viral hepatitis in India. Here a total of 843 samples were collected: 685 from patients with acute viral hepatitis (AVH), 70 from patients with fulminant hepatic failure (FHF), 53 from patients with chronic liver disease (CLD), 11 from patients with antituberculosis therapy (ATT)-induced jaundice, and 24 from pregnant women. When tested for anti-HEV IgM, 58.3% of the pregnant women, 41.4% of the patients with FHF, 38.6% of the patients with AVH, 9.4% of the patients with CLD, and 18.2% of the patients with ATT-induced jaundice tested positive. We found that 34% and 16% of the acute hepatitis patients and fulminant hepatitis patients, respectively, showed no reactivity to the existing viral hepatitis markers and were thus grouped as non A to E. Among the HEV IgM-positive cases, males outnumbered females (62.8% versus 37.1%). HEV RNA was found in 35% of fulminant and 9.4% of acute hepatitis patients. From phylogenetic analysis, we observed that all the isolates were clustered within genotype 1. Critical analysis placed the acute isolates along with strains under subtype Ia, while the fulminant isolates clustered along with the FHF strain (X98292) under subtype Ic. The segregation of HEV isolates from AVH and FHF patients into different subtypes raises interesting questions on the molecular basis of HEV disease severity.

Clinical applicability of single-tube multiplex reverse-transcriptase PCR in dengue virus diagnosis and serotyping

This study has evaluated the clinical applicability of a single‐tube multiplex RT‐ PCR as compared with a two‐step nested RT‐PCR for the diagnosis as well as serotyping of dengue virus in patients samples. Seventy‐six acute phase blood samples collected from clinically suspected dengue patients during the 2008 outbreak were subjected to two‐step nested RT‐PCR and single‐tube multiplex RT‐PCR for dengue diagnosis and serotyping. Of the 76 samples, 17 (22.4%) were positive for dengue viral RNA. Single dengue virus infection was found in 16 cases and 1 had concurrent infection with two serotypes (3&1). Dengue serotype 3 was the predominant serotype (70.5%), followed by serotype 1 (23.5%). Single‐tube multiplex PCR had concordant result with that of two‐step nested RT‐PCR including the one with concomitant infection. This study reveals the predominance of dengue serotype 3 in North India in addition to the co‐circulation of multiple serotypes and concomitant infection. The rapid and accurate diagnostic capability of single‐tube multiplex RT‐PCR used in the study appears to be promising enough to be commonly used for dengue viral detection as well as serotyping. J. Clin. Lab. Anal. 25:76–78, 2011.