The relationship of serum mitochondrial creatine kinase and rotavirus gastroenteritis in pediatric patients]

The objective of this study was to investigate the relationship between serum mitochondrial creatine kinase(mCK) and rotavirus gastroenteritis in pediatric patients. Stool and serum specimens were simultaneously collected from 45 patients(25 males and 20 females) with suspected rotavirus gastroenteritis from January to December 1998. Stool specimens were tested by rotavirus latex agglutination assay. Fourteen patients(10 males, 4 females) were proved as positive, and peak season was in winter and early spring(7 positive cases in March). Six of the 14 were younger than one and 7 were between one and two. Total serum CK activity was measured by The Japan Society of Clinical Chemistry (JSCC) recommended method, and mCK activity was calculated from mCK fraction % obtained by CK isoenzyme electrophoresis. Patients' mCK activities were as follows, rotavirus antigen positive patients(n = 14): 60.0 +/- 20.6 U/l and rotavirus antigen negative patients(n = 31): 7.2 +/- 5.5 U/l. Significant difference was observed between rotavirus antigen positive group and rotavirus antigen negative group(p < 0.01), and control children group(n = 105): 7.1 +/- 2.9 U/l, (p < 0.01). The clinical implications and mechanisms of increased serum mCK activity are unclear. It is known that histological study of the small intestine from rotavirus gastroenteritis patients reveal shortened villi and mononuclear cell infiltration of the lamia propria; electron microscopy shows mitochondrial swelling and sparse irregular microvilli. Elevated serum mCK level of rotavirus gastroenteritis patient may therefore reflect diffused intestinal epithelial cell damage.     

Local succession of adenovirus strains in pediatric gastroenteritis

Assessment of the current incidence of different adenovirus types in local gastroenteritis involved the examination of over 1,000 stool specimens annually from 1988–1992. Adenoviruses were detected by electron microscopy and/or cell culture in 32% of the specimens in which a viral pathogen was detected. The identification of every adenovirus isolate to type by neutralization with specific antisera against the first 6 types and by restriction analysis of nonneutralized isolates was started in 1990. Samples from 1988 and 1989 were examined retrospectively. Adenovirus strains were compared to those isolated in a study between 1980-1983. Enumeration of individual adenovirus types revealed a number of trends, demonstrating that rapid changes in the local incidence of several strains were occurring in Manitoba. The incidence of adenovirus type 40 (Ad40) as a cause of gastroenteritis was found to have fallen dramatically in recent years. The predominant cause of gastroenteritis in Manitoba is a variant strain of Ad41, increasing in predominance each year and now responsible for over a third of the symptomatic cases examined since 1990. The majority of restriction site differences of the Ad41 variant strain from the prototype strain Tak were mapped to the hexon and fiber genes, both of which code for the neutralizable external viral epitopes. The probability of the observed pattern of mutations occuring by chance was calculated as P <0.0005, indicating a strong pressure for selection of these immunologically significant alterations to the viral proteins responsible for cell attachment. Novel strains of adenovirus types causing gastroenteritis can arise rapidly in populations, presumably by immunological selection. © 1995 Wiley-Liss, inc.     

Incidence of rotavirus infection in different age groups of pediatric patients with gastroenteritis.

An enzyme immunoassay was used to detect rotavirus in the stools of 176 pediatric patients presenting with gastroenteritis. The highest incidence of rotavirus infection was found among patients less than 1 year of age. In contrast to previously reported studies, 23% of neonates with gastroenteritis had rotavirus in their stools. This relatively easy and rapid method was helpful in the management of pediatric patients with gastroenteritis.     

A combined enzyme immunoassay for rotavirus and adenovirus (EIARA)

A combined enzyme immunoassay for rotavirus and adenovirus (EIARA) was developed as a double-antibody sandwich assay in which test samples are added to plastic wells coated with rotavirus or adenovirus goat antibody. The presence of antigens is detected by mixed-guinea pig antisera to the same viruses followed by rabbit anti-guinea pig IgG conjugated with peroxidase and subsequently by ortho-phenylenediamine substrate. Titrations of rotavirus (SA11) and adenovirus type 2 tissue culture grown viruses in the presence of separate capture sera and mixed detector sera revealed that the two virus-antibody reactions occurred independently and were not affected by each other. Comparison with another enzyme immunoassay for rotavirus, with immunoelectron microscopy and with polyacrylamide gel electrophoresis showed that EIARA is a sensitive and specific method for detecting rotaviruses and adenoviruses in faeces from children with gastroenteritis.     

Five year study of rotavirus gastroenteritis in Bulgaria

We tested by electron microscopy 7,530 samples from children admitted to different hospitals in Sofia between February 1981 and February 1986; from these, rotaviruses were found in 725 (9.6%) faecal samples. Electron microscopic analysis of 264 samples from 181 children admitted to the hospital of infectious diseases in Shumen between December 1984 to February 1986 revealed rotaviruses in 120 (66.6%) of the tested children. A part of the samples positive by electron microscopy was tested by ELISA and polyacrylamide gel electrophoresis for rotavirus RNA segment patterns. Rotaviruses with eight different electropherotypes were found in Bulgaria. The seasonal culmination of the rotavirus gastroenteritis in the winter months has been confirmed. Rotavirus antibodies were found in 73% of the sera from 152 children tested.     

Importance of rotavirus and adenovirus types 40 and 41 in acute gastroenteritis in Korean children.

To examine the role of rotavirus (Rv) and adenovirus types 40 and 41 (Ad40/41) in Korean children with acute gastroenteritis, we evaluated 345 children with acute gastroenteritis and 90 children without acute gastroenteritis in Seoul, Korea, during a 29-month period. Stools were tested for group A Rv antigen and for Ad40/41 by using monoclonal antibody (MAb)-based assays. Rv was found in 68% of the ill children and 19% of the controls (P less than 0.001), whereas Ad40/41 was detected in 9% of the ill children and 2% of the controls (P less than 0.05). Also, 6% of the ill children and 0.01% of the controls excreted Rv and Ad40/41 simultaneously. Among the ill children, 96% of children with Rv and 94% of those with Ad40/41 were younger than 24 months. Although a peak of Rv infection was detected in early winter in both years of the study, there was no apparent seasonal trend with Ad40/41. Diarrhea with more than 10 stools per day, vomiting, or fever was most strongly associated with Rv shedding, whereas the first two manifestations were associated with coinfection of Rv and Ad40/41. To investigate the genetic and serotypic diversity of Rv strains, we tested 195 and 144 fecal Rv specimens isolated from the gastroenteritis cases, respectively, by polyacrylamide gel electrophoresis of the segmented RNA genome and by an enzyme-linked immunosorbent assay with serotype-specific MAbs. Of the 195 specimens, 154 yielded RNA patterns characteristic of group A Rv: 18% had short electrophoretic migration patterns, 81% had long patterns, and 1% had a mixture of short and long patterns. Of the 144 specimens, serotype specificity was determined in 51%: 89% were serotype 1, 10% were serotype 2, and 1% were serotype 3. Analysis of the specimens for which electropherotypes and serotypes were available indicated that a given RNA pattern corresponded to a particular serotype, except in one strain that showed short patterns but serotype 1. We suggest that Rv and Ad40/41 in stools be accepted critically as an important cause of diarrhea among young children in Korea.     

Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus

Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.     

Hospital-acquired rotavirus acute gastroenteritis in 10 consecutive seasons in Umbria (Italy)

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young (aged <5 years) children. Several studies showed that RVA is one of the main cause of nosocomial gastroenteritis in hospitalized pediatric population worldwide, with an incidence ranging from 8 to 33 cases per 100 hospitalized children. Nosocomial infections, in which AGE symptoms develop at least 2 days after admission, may severely affect children already admitted to hospital for other causes. This study aimed to define the trends of the RVA genotypes through statistical analysis of the data obtained by the rotavirus surveillance in Umbria in 10 consecutive seasons, from 2007-2008 to 2016-2017, with update information on hospital-acquired RVA AGE. During RVA gastroenteritis surveillance in Umbria (Italy) in 2007 to 2017, a total of 741 RVA positive faecal samples were collected from children hospitalized with AGE, and RVA strains were genotyped following standard EuroRotaNet protocols. Of the 741 analyzed samples, 75 (10%) were reported to be hospital-acquired. Comparing the distributions of the RVA genotypes circulating in the community or associated with nosocomial infections, we observed a different distribution of genotypes circulating inside the hospital wards, with respect to those observed in the community except in 2010 to 2011, 2011 to 2012, and 2012 to 2013 when G1P[8], G4P[8] and the novel strain G12P[8] caused a large community- and hospital-acquired outbreak. Of the 741 analyzed samples, 75 (10%) were reported to be hospital-acquired. Comparing the distributions of the RVA genotypes circulating in the community or associated with nosocomial infections, we observed a different distribution of genotypes circulating inside the hospital wards, with respect to those observed in the community except in 2010 to 2011, 2011 to 2012, and 2012 to 2013 when G1P[8], G4P[8], and the novel strain G12P[8] caused a large community- and hospital-acquired outbreak. The information from this study will be useful to implement guidelines for preventing nosocomial RVA AGE, which should include an improved management of the hospitalized patients and an increase in vaccination coverage.     

First detection of group C rotavirus in patients with gastroenteritis in Slovenia

Group C rotaviruses are associated with sporadic gastroenteritis and outbreaks of diarrhea in children and adults worldwide. Three cases with group C rotavirus infection are described, and the molecular characterization of the gene for the major capsid protein VP6 is reported. Patients described in this report were 10 years old or more and had mild to moderate clinical symptoms. A high nucleotide (>98%) and amino acid (100%) identity was observed among all three isolated Slovenian group C rotavirus strains. The similar identity is confirmed of Slovenian strains with other human group C rotavirus isolates, which were seen to cluster separately from the animal group C rotavirus isolates by a phylogenetic analysis. This is the first report of group C rotavirus detection in Slovenia.     

Comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children.

The development of mucosal immunity is presumed to be the most important marker of rotavirus infection. The practical difficulties of obtaining small-bowel secretions stimulated this study of the antibody response to acute rotavirus infection at other sites. Forty-four infants admitted to the hospital with rotavirus gastroenteritis had serum, saliva, and feces collected at the acute phase (median, 5.5 days), during convalescence (median, 33.5 days), and 4 months later (median, 12.2 weeks). A subgroup of 19 children also had duodenal juice collected in parallel. Rotavirus-specific immunoglobulin G (IgG), IgA, secretory immunoglobulin, and IgM were measured and compared in all samples. The results showed that the estimation of antirotavirus serum IgM, serum IgG, duodenal juice IgA, and duodenal juice IgM by an enzyme immunoassay indicated an immune response to severe primary rotavirus infection in all children. Four months later, the levels of serum IgG and IgA served as the most sensitive markers of the preceding rotavirus infection. The predictive accuracies of immune responses at different sites in relation to a positive IgA immune response in the duodenum were calculated. Fecal IgA predicted duodenal IgA rotavirus antibodies with accuracies of 86% at 1 month and 92% at 4 months. The high sensitivity of serum IgM and IgG in detecting rotavirus infection and the high predictive accuracy of fecal IgA as an indicator of duodenal IgA abrogates the need for duodenal intubation to detect (or monitor) an immune response to rotavirus infection. This finding has important practical implications for epidemiological studies of acute diarrhea in children and in rotavirus vaccine trials.     

Clinical utility of nested multiplex RT-PCR for group F adenovirus, rotavirus and norwalk-like viruses in acute viral gastroenteritis in children and adults.

BACKGROUND: The diagnosis of viral gastroenteritis can be carried out by non-molecular techniques such as electron microscopy (EM), enzyme-immunoassay and latex agglutination tests and various molecular techniques. Normally molecular detection requires the use of three separate protocols to detect the three main causes of viral gastroenteritis, adenoviruses, rotaviruses and norwalk-like viruses (NLV) which have different types of nucleic acid. The development of a sensitive and specific assay which could detect these targets would have major advantages for the clinical virology laboratory. OBJECTIVES: The aims of the present study were to develop a sensitive and specific multiplex molecular assay and to apply it to the detection of viral agents in clinical cases of acute gastroenteritis. STUDY DESIGN: The multiplex assay was designed using Access RT-PCR (Promega). Primers were researched and selected for their specificity and broad range detection of the viral agents across the various genotypes of group A rotaviruses, NLV and group F adenoviruses. RESULTS: From September 2000 to August 2001 we tested 1945 clinical specimens. Rotavirus infections were detected in 190 with an age range from 12 days to 8 years old. Group F adenovirus was detected in 96 patients ranging from 15 days to 10 years old. A further single case of group F adenovirus was detected in an adult of 75 years old. NLVs were detected in 132 patients. There were 55 infections in children less than 7 years old. In 10 different outbreaks involving 130 adult patients there were 57 NLV positives. Sporadic NLV infection was detected in 11 of 600 adult patients. There were 4 patients with dual infections. CONCLUSIONS: The assay detailed here has proved an invaluable tool for the investigation of acute gastroenteritis in specimens from patients of all ages. We found it convenient to use a single mastermix with a single protocol to test all specimens from patients of all ages. NLV in children is often overlooked and/or under reported, particularly where less sensitive assays such as EM are being employed for diagnosis.