Enzyme Immunoassay Positive Samples Research Articles

Epidemiology of rotavirus diarrhea among children less than 5 years hospitalized with acute gastroenteritis prior to rotavirus vaccine introduction in India

BackgroundRotavirus is an important cause of severe diarrhea requiring hospitalization, accounting for approximately 78,000 deaths annually in Indian children below 5 years of age. We present epidemiological data on severe rotavirus disease collected during hospital-based surveillance in India before the introduction of the oral rotavirus vaccine into the national immunization schedule. MethodsThe National Rotavirus Surveillance Network was created involving 28 hospital sites and 11 laboratories across the four geographical regions of India. From September 2012 to August 2016 children less than 5 years of age hospitalized for diarrhea for at least 6 h, were enrolled. After recording clinical details, a stool sample was collected from each enrolled child, which was tested for rotavirus antigen using enzyme immunoassay (EIA). Nearly 2/3rd of EIA positive samples were genotyped using reverse transcription polymerase chain reaction to identify the G and P types. ResultsOf the 21,421 children enrolled during the 4 years surveillance, 36.3% were positive for rotavirus. The eastern region had the highest proportion of rotavirus associated diarrhea (39.8%), while the southern region had the lowest (33.8%). Rotavirus detection rates were the highest in children aged 6–23 months (41.8%), and 24.7% in children aged < 6 months. Although rotavirus associated diarrhea was seen throughout the year, the highest positivity was documented between December and February across all the regions. The most common rotavirus genotype was G1P[8] (52.9%), followed by G9P4 (8.7%) and G2P4 (8.4%). ConclusionsThere is high burden of rotavirus gastroenteritis among Indian children below 5 years of age hospitalized for acute diarrhea thereby highlighting the need for introduction of rotavirus vaccine into the national immunization program and also for monitoring circulating genotypes.

Diversity of rotavirus genotypes circulating in children

BackgroundFrom 2016, the Government of India introduced the oral rotavirus vaccine into the national immunization schedule. Currently, two indigenously developed vaccines (ROTAVAC, Bharat Biotech; ROTASIIL, Serum Institute of India) are included in the Indian immunization program. We report the rotavirus disease burden and the diversity of rotavirus genotypes from 2005 to 2016 in a multi-centric surveillance study before the introduction of vaccines.MethodsA total of 29,561 stool samples collected from 2005 to 2016 (7 sites during 2005–2009, 3 sites from 2009 to 2012, and 28 sites during 2012–2016) were included in the analysis. Stools were tested for rotavirus antigen using enzyme immunoassay (EIA). Genotyping was performed on 65.8% of the EIA positive samples using reverse transcription- polymerase chain reaction (RT-PCR) to identify the G (VP7) and P (VP4) types. Multinomial logistic regression was used to quantify the odds of detecting genotypes across the surveillance period and in particular age groups.ResultsOf the 29,561 samples tested, 10,959 (37.1%) were positive for rotavirus. There was a peak in rotavirus positivity during December to February across all sites. Of the 7215 genotyped samples, G1P[8] (38.7%) was the most common, followed by G2P[4] (12.3%), G9P[4] (5.8%), G12P[6] (4.2%), G9P[8] (4%), and G12P[8] (2.4%). Globally, G9P[4] and G12P[6] are less common genotypes, although these genotypes have been reported from India and few other countries. There was a variation in the geographic and temporal distribution of genotypes, and the emergence or re-emergence of new genotypes such as G3P[8] was seen. Over the surveillance period, there was a decline in the proportion of G2P[4], and an increase in the proportion of G9P[4]. A higher proportion of mixed and partially typed/untyped samples was also seen more in the age group 0–11 months.ConclusionsThis 11 years surveillance highlights the high burden of severe rotavirus gastroenteritis in Indian children < 5 years of age before inclusion of rotavirus vaccines in the national programme. Regional variations in rotavirus epidemiology were seen, including the emergence of G3P[8] in the latter part of the surveillance. Having pre-introduction data is important to track changing epidemiology of rotaviruses, particularly following vaccine introduction.

Distribution of rotavirus genotypes in the postvaccine introduction era in Ashaiman, Greater Accra Region, Ghana, 2014-2016.

Group A Rotaviruses (RVAs) are the most important etiological agents of acute gastroenteritis (AGE) in children less than 5 years of age. Mortality resulting from RVA gastroenteritis is higher in developing countries than in developed ones, causing a huge public health burden in global regions like Africa and South-East Asia. This study reports RVA genotypes detected in Ashaiman, Greater Accra Region, Ghana, in the postvaccine introduction era for the period 2014-2016. Stool samples were collected from children less than 5 years of age who visited Ashaiman Polyclinic with AGE from November 2014 to May 2015 and from December 2015 to June 2016. The samples were tested by enzyme immunoassay (EIA), and one-step multiplex reverse transcription polymerase chain reaction was performed on the EIA positive samples for gel-based binomial genotyping. Of the 369 stool samples collected from children with AGE, 145 (39%) tested positive by EIA. Five VP7 (G1, G3, G9, G10, and G12) and three VP4 (P[4], P[6] and P[8]) genotypes were detected. Eight G/P combinations were identified of which, G3P[6], G12P[8], G1P[8], and G9P[4] were the most prevalent and responsible for 93 (68%) of the AGE cases, and seven mixed-types were detected which represented 8% of the RVA cases. High prevalence, diversity, and mixed-types of RVAs were detected from Ashaiman with the emergence of unusual genotypes.

Rotavirus gastroenteritis in Indian children\u2009

BackgroundIn 2016, the Government of India introduced the oral rotavirus vaccine (ROTAVAC, Bharat Biotech, India) in 4 states of India as part of the Universal Immunization Programme, and expanded to 5 more states in 2017. We report four years of data on rotavirus gastroenteritis in hospitalized children < 5 years of age prior to vaccine introduction.MethodsChildren from 7 sites in southern and northern India hospitalized for diarrhoea were recruited between July 2012 and June 2016. Stool samples were screened for rotavirus using enzyme immunoassay (EIA). The EIA positive samples were genotyped by reverse-transcription polymerase chain reaction.ResultsOf the 5834 samples from the 7 sites, 2069 (35.5%) were positive for rotavirus by EIA. Genotyping was performed for 2010 (97.1%) samples. G1P[8](56.3%), G2P[4](9.1%), G9P[4](7.6%), G9P[8](4.2%), and G12P[6](3.7%) were the common genotypes in southern India and G1P[8](36%), G9P[4](11.4%), G2P[4](11.2%), G12P[6](8.4%), and G3P[8](5.9%) in northern India.ConclusionsThe study highlights the high prevalence of rotavirus gastroenteritis in India and the diversity of rotavirus genotypes across different geographical regions. Pre- vaccine surveillance data is necessary to evaluate the potential change in admission rates for gastroenteritis and circulating rotavirus genotypes after vaccine introduction, thus assessing impact.

Molecular epidemiology of rotaviruses in the south-east Asian region from 2009 to 2015

BackgroundIn Asia, rotavirus accounts for approximately 45% of admissions due to acute gastroenteritis in children <5 years, and causes about 145,000 deaths every year. We studied the distribution of rotavirus strains from Myanmar, Sri Lanka, and Nepal during 2009–2015. MethodsStool samples collected from children <5 years of age hospitalized with acute diarrhea in the three sites and positive for rotavirus antigen by enzyme immunoassay (EIA) were sent to the Christian Medical College, Vellore from 2009 to 2015. G and P typing of rotavirus strains were performed using reverse-transcription polymerase chain reaction (RT-PCR). Result: Of the 2354 EIA positive samples tested, G12P[8] (36.8%), G1P[8] (30.1%), and G12P[6] (41.3%) were the most common strains isolated from Myanmar, Sri Lanka, and Nepal respectively. ConclusionThere was substantial diversity of rotavirus genotypes, and continued surveillance in developing countries of Asia will help in understanding the epidemiology of rotavirus before and after introduction of vaccines.

Sentinel hospital-based surveillance for assessment of burden of rotavirus gastroenteritis in children in Pakistan.

ObjectivesTo determine the burden and molecular epidemiology of rotavirus gastroenteritis in children hospitalized with severe acute watery diarrhea in Pakistan prior to introduction of rotavirus vaccine.MethodsA cross-sectional study was carried out over a period of two years from 2006 – 2008 at five sentinel hospitals in the cities of Karachi, Lahore, Rawalpindi, and Peshawar. Stool samples collected from children under five years of age hospitalized with severe acute watery diarrhea were tested for rotavirus antigen via enzyme immunoassay (EIA) (IDEA REF K6020 Oxoid Ltd (Ely), Cambridge, United Kingdom). A subset of EIA positive stool samples were further processed for genotyping.Results6679 children were enrolled and stool specimens of 2039 (30.5%) were positive for rotavirus. Rotavirus positivity ranged from 16.3% to 39.4% in the 5 hospitals with highest positivity in Lahore. 1241 (61%) of all rotavirus cases were in infants under one year of age. Among the strains examined for G-serotypes, the occurrence of G1, G2, G9 and G4 strains was found to be 28%, 24%, 14% and 13%, respectively. Among P-types, the most commonly occurring strains were P6 (31.5%) followed by P8 (20%) and P4 (12%). Prevalent rotavirus genotype in hospitalized children of severe diarrhea were G1P[8] 11.6% (69/593), followed by G2P[4] 10.4% (62/593), and G4P[6] 10.1% (60/593).ConclusionsApproximately one third of children hospitalized with severe gastroenteritis in urban centers in Pakistan have rotavirus. Introduction of rotavirus vaccine in Pakistan's national immunization program could prevent many severe episodes and diarrheal deaths.

Molecular surveillance of rotavirus strains circulating in Yaoundé, Cameroon, September 2007–December 2012

Rotavirus is the most common cause of severe diarrheal disease in children under 5years of age worldwide. The World Health Organization (WHO) estimated that 453,000 rotavirus-attributable deaths occur annually. Through the WHO, the Rotavirus Sentinel Surveillance Program was established in Cameroon in September 2007 with the Mother and Child Center (MCC) in Yaoundé playing the role of sentinel site and national laboratory for this program. The objectives of this surveillance were to assess the rotavirus disease burden and collect baseline information on rotavirus strains circulating in Cameroon. Diarrheal stool samples were collected in a pediatric hospital from children under 5, using the WHO case definition for rotavirus diarrhea. Antigen detection of rotavirus was performed by using an enzyme immunoassay (EIA). The genotypic characterization was performed using multiplexed semi-nested reverse transcription-polymerase chain reaction (RT-PCR) assays. Between September 2007 and December 2012, 2444 stool samples were received at the MCC laboratory for rotavirus antigen detection, of which 999 (41%) were EIA positive. Among EIA positive samples 898 were genotyped. Genotype prevalence varied each year. Genotype G9P[8] was the dominant type during 2007 (32%) and 2008 (24%), genotype G3P[6] predominated in 2010 (36%) and 2011 (25%), and G1P[8] was predominant in 2012 (44%). The findings showed that the rotavirus disease burden is high and there is a broad range of rotavirus strains circulating in Yaoundé. These data will help measure the impact of vaccination in the future.

Rotavirus shedding in premature infants following first immunization

There is limited data regarding rotavirus vaccine shedding in premature infants. We describe the natural history of rotavirus shedding in premature infants in the 2-week period following first immunization with RotaTeq(®), the pentavalent rotavirus vaccine (RV5), and the risk for symptomatic transmission to household contacts (HHC). A prospective pilot study of 15 premature infants of gestational ages 26-34 weeks immunized with RV5 between 6 and 14 weeks chronological age on discharge from the NICU was conducted. Stool samples collected in the following 2 weeks and analyzed for rotavirus antigen by enzyme immunoassay (EIA), cell culture, and RT-PCR. Solicited adverse events were collected on study subjects and any symptoms of fever, vomiting and diarrhea in HHC. Rotavirus antigen shedding after immunization was detected, with positive rotavirus EIA results in 53.3% of premature infants and in 22.1% of 86 stool samples collected. Shedding rates by RT-PCR were higher with 86.7% of infants and 76.7% of samples being positive. Only 42% of EIA positive samples were positive by cell culture (8/86 total samples, 9.3%). None of 53 HHC reported symptoms of rotavirus infection during the 4 weeks following immunization of the infants. The findings of this study demonstrate that premature infants have positive stools by EIA, viral culture, and RT-PCR at varying time points during 2 weeks following first-dose immunization with RV5. RT-PCR shedding rates need to be clinically evaluated in the context of virus quantification by cell culture, which was low. No symptomatic transmission to HHC was detected in this study, supporting low transmissibility of vaccine virus shed by these infants born prematurely.

Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA qualitative assay: experience of a transplant center

Hepatitis C virus (HCV) is frequently a silent infection in hemodialysis (HD) patients with a prevalence of 8-10%. Improving HCV detection in this population prior to transplantation is critical both for infection control and optimal patient care. To assess the current HCV testing practice of the National Institute for Transplantation (PCR testing of enzyme immunoassay (EIA) positive HD patients) by evaluating a subset of EIA positive and EIA negative samples with the VERSANT HCV RNA Qualitative Assay based on transcription mediated amplification (HCV Qual (TMA)) (sensitivity < or = 9.6 IU/ml) and in-house PCR (HCV Qual (PCR)) (sensitivity approximately 149 IU/ml). 2321 HD patients were screened by Abbott HCV EIA 2.0. A subset of 80/169 E IA positive samples and 100/2152 EIA negative samples were tested by both assays. TMA/PCR discordant samples were genotyped. PCR and TMA gave concordant results in 67/80 (83.8%) of EIA positive samples. 11/80 (14.7%) were reactive by HCV Qual (TMA), but not by HCV Qual (PCR); 2/80 (2.7%) were reactive by HCV Qual (PCR), but not by HCV Qual (TMA). 2/100 (2%) EIA negative samples were reactive and 95/100 (95%) were non-reactive by both assays. Three (3%) were only HCV Qual (TMA) reactive. 11/14 TMA+/PCR-samples with sufficient volume were genotyped. HCV Qual (TMA) identified active HCV infection in more EIA positive and EIA negative patients than HCV Qual (PCR) and should be part of our testing algorithm.

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A.

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false...

Evaluation of Commercial Enzyme Immunoassay (EIA) and Immunofluorescent Antibody (IFA) Test Kits for Detection of Cryptosporidium Oocysts of Species Other than Cryptosporidium parvum

A commercial enzyme immunoassay (EIA) (ProSpect Rapid Assay), a direct immunofluorescence antibody (IFA) test for stool testing (MERIFLUOR Cryptosporidium/Giardia), and an indirect IFA test for environmental testing (Hydrofluor-Combo Cryptosporidium/Giardia) were evaluated for detection of low public health risk Cryptosporidium oocyst isolates, and for C. parvum oocyst isolates from human and bovine feces that represent a high public health risk. There was no cross-reactivity of EIA with ova of eight medically important helminths, three Eimeria species oocysts, Sarcocystis cruzi sporocysts, and two Candida sp. isolates. All nine snake oocyst isolates (C. serpentis), two of seven lizard oocyst isolates, one turtle oocyst isolate, two avian oocyst isolates (turkey, C. meleagridis), one C. wrairi oocyst isolate from guinea pigs, one C. muris oocyst isolate from hyrax, one heifer C. muris isolate, and two C. muris-like oocyst isolates from a camel were positive by both IFA tests; six of these 19 oocyst isolates were EIA-positive. There was no difference in the sensitivity and specificity between direct and indirect IFA tests. The sensitivity of the EIA and both IFA tests to the C. parvum oocysts was 100%. The EIA showed less cross-reactivity with the non-C. parvum oocysts (24%) than direct or indirect IFA (76%), and was less sensitive to those isolates (20%) than both IFA tests (63%). A simulated sampling model for high and low public health risk Cryptosporidium oocysts showed that the low risk oocyst isolates may constitute up to 35% of all positive environmental samples by direct or indirect IFA determination, and up to 12% of all EIA positive samples. This study indicates a superiority of direct and indirect IFA and EIA for screening of human-or-bovine-origin fecal specimens, whereas testing of environmental samples may lead to misidentification of medically important isolates. The results demonstrated that the EIA kit can more accurately identify environmental samples containing oocytes pathogenic for humans than both IFA tests. The specificity of commercially available diagnostic kits to C. parvum should be critically examined for cross-species identification before they are recommended or adopted for use in testing environmental samples.

Multi‐residue enzyme immunoassays for screening for β‐agonists in faeces and feeds

Multi‐analyte screening methods for β‐agonists in faeces and feeds based on enzyme immunoassay (EIA) were developed. Commercially available EIA kits were used, and the results presented. The extraction for both matrices was performed under acid conditions. The addition of an organic solvent to the extradant, resulting in higher recoveries, was only retained for feeds as the background absorption for faeces increased substantially. Clean‐up for faecal extracts was achieved in a one‐step extraction with isobutyl alcohol. Detection limits in faeces were slightly different for the two types of kits used. They ranged from 0.8ng g−1 for clenbuterol to 10ng g−1 for terbutaline. In feeds, corresponding detection limits for both compounds were 5 ng g−1 and 110 ng g−1. Confirmation of EIA positives was established by liquid chromatography with post‐column diazotation and gas chromatography‐mass spectrometry. In the screening of faeces over 1 year about 70% of the EIA positive samples were confirmed as positive.