[Epidemiological aspects of gastrointestinal infections]. / Epidemiologische Aspekte gastrointestinaler Infektionen.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate seasonal patterns and age-associated trends of the main bacterial, viral, and parasitic enteric pathogens in Southwest Germany. PATIENTS AND METHODS: From January 2002 through December 2008 a total of 99,057 patients were tested for Norovirus, Rotavirus, bacterial pathogens, Cryptosporidium parvum (C. parvum), and Giardia lamblia (G. lamblia). RESULTS: All these pathogens were detected throughout the whole year. But there were distinctive seasonal patterns of activity of the following pathogens being detected: norovirus was detected mainly from September through April. The highest rotovirus activity was observed from December through June. But bacterial pathogens und C. parvum were found mainly from June to November. The percentage of positive results during the months with the highest activity was 10 - 49% for norovirus, 25% - 41% for rotavirus, 14 - 18% for bacterial infection and 3 - 4 % for C. parvum. G. lamblia and adenovirus were found throughout the year in 7 - 15% and 3 - 10% of samples, respectively. Moreover, the detection rate of different pathogens depended on patient age. In infants younger than one year, rotavirus, norovirus and adenovirus were most frequently isolated pathogenes. Stool samples from kindergarden- and school-age children were positive largely for bacterial pathogens such as Salmonella and Campylobacter particularly in late summer or early autum. In patients older than 60 years, norovirus, rotavirus, and toxin producing Clostridium difficile strains were the most common pathogens. CONCLUSIONS: In view of the age and season related frequency of detection of enteric pathogens, a step-by-step diagnosis of gastrointestinal tract infections is recommended. Considering that most pathogens are detected sporadically over the whole year, the analysis of negative samples should be appropriately expanded. The knowledge of seasonal occurrence can also be applied to improve the application of hygienic measures.

Phylogenetic evidence for a recent spread of two populations of human enterovirus 71 in European countries.

Human enterovirus 71 (EV-71) is a cause of seasonal epidemics of hand, foot and mouth disease, and of less common but severe neurological manifestations. Uncertainty persists regarding the circulation of virus populations in several geographical areas and the timescale of their dissemination. We determined EV-71 sequences at loci 1D (VP1 capsid protein) and 3CD (non-structural proteins) in 86 strains recovered in Austria, France and Germany and performed an evolutionary genetic study of extant virus populations. Phylogenetic analyses positioned 78 of the 86 sequences within two clades among subgenogroups C1 and C2. A minor sequence cluster was assigned to subgenogroup C4. Analyses incorporating the available sequences estimated the substitution rate in genogroup C at 3.66 x 10(-3) and 4.46 x 10(-3) substitutions per site year(-1) for loci 1D and 3CD, respectively, assuming a relaxed molecular-clock model for sequence evolution. Most of the 'European' strains belonged to clades C1b and C2b, which originated in 1994 [95 % confidence interval (CI), 1992.7-1995.8] and 2002 (95 % CI, 2001.6-2003.8), respectively. Estimates of divergence times for locus 3CD were consistent with those measured for locus 1D. Intertwining between clades representing EV-71 subgenogroups and clades corresponding to other enterovirus types (notably early coxsackievirus A prototype strains) in the 3CD phylogeny is highly indicative of ancestral recombination events. Incongruent phylogenetic patterns estimated for loci 1D and 3CD show that a single tree cannot model the epidemic history of circulating EV-71 populations. The evolutionary timescale of genogroup C estimated for both loci was measured only in decades, indicating recent dissemination.

Laboratory-based assessment of influenza in German ambulant patients from 1998 to 2008.

BACKGROUND: In Germany, the cost for PCR diagnosis of influenza in ambulant patients was not covered by the national statutory health insurance system until 2008. Therefore, cell culture was the standard method applied for routine diagnosis. We have prospectively compared a 1-day rapid cell culture assay (RCA) with conventional cell culture (CCC) during the influenza seasons from 1997/1998 to 2007/2008 and with real-time PCR analysis during the influenza seasons 2003/2004 and 2006/2007. PATIENTS AND METHODS: This study is based on 4,262 respiratory samples obtained from ambulant patients between January 1998 and May 2008. The RCA was performed in microtiter plates that were stained with monoclonal antibodies to influenza virus A and B 16 h after inoculation. RESULTS: A total of 1,221 specimens were found to be positive by the cell culture methods - 1,143 (93.6%) by the RCA and 1,012 (82.9%) by the CCC. The sensitivity of the RCA and CCC versus PCR was 75.4% (221/293) and 58% (170/293), respectively. The specificity of both cell culture assays versus PCR was 100%. Influenza A represented 79.3% of the cases diagnosed. An increased activity of influenza was observed between January and March, with the rate of influenza-positive cases being highest for kindergarten and school-aged children. CONCLUSION: While PCR is the most sensitive assay for the diagnosis of influenza, the RCA can still be used for diagnosis and surveillance of this disease. Based on our findings and given the known fact that influenza antibodies reach a plateau 2-4 weeks after immunization, the optimal time for vaccination in Germany is from October through November. Kindergarten and school-aged children represent an important reservoir of infection. Consequently, routine immunization should be considered for this age group to prevent the spread of influenza.

Laboratory approaches to the diagnosis of adenovirus infection depending on clinical manifestations.

BACKGROUND: While commercial enzyme immunoassays (EIA) intended for the detection of adenovirus in fecal specimens are widely used, there are no rapid, convenient, and sensitive commercial tests available for the detection of adenoviruses in respiratory and conjunctival specimens. The applicability of EIA for the detection of adenovirus in stool and throat samples was investigated. One-day rapid culture assay (RCA) for the detection of adenovirus in respiratory and conjunctival specimens was developed and evaluated. PATIENTS AND METHODS: Stool samples from patients with gastroenteritis were tested by adenovirus EIA and by cell culture using human embryonic lung cells (HEL) and Graham 293 cells. Blood and stool samples from two BMT patients were also tested for adenovirus by PCR for at least 6 months. Throat specimens from patients with respiratory infections and conjunctival specimens were used for the evaluation of 1-day RCA compared with conventional adenovirus isolation in Graham 293 cells. RESULTS: A total of 3,860 stool samples were tested by EIA Ridascreen, 8,169 by Novitec, and 2,218 by ProSpectT yielding 135 (3.5%), 308 (3.7%), and 77 (3.5%) positive results, respectively. From 305 Ridascreen- and 340 Novitec-negative stool samples, adenoviruses were isolated in three (0.9%) and eight (2.4%) cases, respectively, including two patients undergoing BMT. Multiple sequential stool samples from one BMT patient were repeatedly negative by EIA, but positive by PCR and cell culture. Graham 293 cells were better suited for isolation of adenovirus than HEL. EIA proved unreliable for detecting adenovirus in throat swabs. The sensitivity and specificity of RCA in throat swabs were 90% (37/41) and 100% (64/64), respectively, and 76% (16/21) and 100% (132/132) in conjunctival specimens, respectively. CONCLUSIONS: Generally, EIA is sufficiently sensitive for the diagnosis of adenovirus-associated diarrhea. However, it may not be sensitive enough to detect adenovirus in immunocompromised patients undergoing BMT and shedding very few viral particles in stools. Thus, in such cases, a more sensitive assay, such as PCR, is recommended. Furthermore, EIA is not sufficiently sensitive for the reliable detection of adenoviruses in throat swabs. One-day RCA may be useful for the detection of adenoviruses in respiratory and conjunctival specimens.

[Rational and efficient enterovirus diagnosis]. / Rationale und rationelle Enterovirus-Diagnostik.

BACKGROUND AND OBJECTIVE: Demonstration of the causative pathogen by isolating the virus in cell culture is taken as the standard in the diagnosis of diseases caused by enterovirus. When diagnosing the virus in cerebrospinal fluid (CSF), isolation of the virus has been largely replaced by the rapid demonstration of the virus using the reverse transcriptase polymerase chain reaction (RT-PCR), because of its greater sensitivity. The serological diagnosis is mostly made with the complement binding reaction (CBR). A new enzyme immunoassay for demonstrating anti-enterovirus IGM antibodies (IgM-EIA) allows a more rapid diagnosis from a single serum sample. It was the aim of this study to compare the diagnostic value of these various tests. METHODS: Several methods for demonstrating virus from faeces, swabs and CSF (virus isolation in cell culture and RT-PCR) and of antibodies in serum (IgM-EIA and CBR) were compared. The clinical material was obtained largely from children under the age of 10 years, many of whom had serous meningitis, flu-like symptoms or enteritis. In one cohort (C1), only stool or throat swabs were available for each of 154 patients. In the other cohort (C2) of 164 patients, CSF and at least one serum sample were available in addition to occasional stool samples. RESULTS: From C1 enteroviruses were isolated from 32 patients. rotavirus twice from stool or throat swab and rotavirus once from stool or throat swab, and herpes simplex once from throat swab. RT-PCR was positive 55 times for enterovirus, five times false-negative when the virus had been isolated. In C2 enterovirus nucleic acid was demonstrated in 43 patients from CSF. Parallel serological tests gave positive IgM values for 15 patients, while CBR titres were raised in nine. CONCLUSIONS: Complementary tests of CSF, stool, swabs and serum samples by all possible combinations of viral isolation, RT-PCR and IgM-EIA improve the diagnosis of enterovirus-associated diseases. RT-PCR is the method of choice. The serological diagnosis should be confirmed by the demonstration of virus in stool or swab.

Evaluation of enterovirus serological tests IgM-EIA and complement fixation in patients with meningitis, confirmed by detection of enteroviral RNA by RT-PCR in cerebrospinal fluid.

An enzyme immunoassay (EIA) for detection of anti-enterovirus IgM antibodies was compared with complement fixation test in 43 patients with confirmed enterovirus meningitis by RT-PCR of cerebrospinal fluids (CSF). In 34% of patients with enterovirus meningitis, IgM antibodies could be found, whereas complement fixation tests were positive in only 20%. The specificity was determined with sera of 105 patients with non-enterovirus meningitis. Specificity of IgM EIA and of complement fixation was 94% and 85%, respectively. In four patients with meningitis but without enterovirus detection in CSF, RT-PCR and virus isolation from stools were positive. In three of these patients, IgM antibodies were detected, giving a strong indication of an enterovirus-associated disease. Because of the high specificity of IgM EIA, diagnosis of enterovirus-associated diseases can be carried out in a single serum sample, whereas by complement fixation tests, only fourfold increases in antibody titres in paired sera indicate an acute infection. The application of IgM EIA is especially important in cases of meningitis when CSF samples are not available and for diagnosis of enterovirus diseases with other clinical symptoms such as fever, enteritis, and hand-foot-and-mouth disease.

A new enzyme immunoassay for the detection of enteroviruses in faecal specimens.

A new enzyme immunoassay (EIA) for direct detection of enteroviruses based on a group-specific monoclonal antibody was evaluated using stool samples from patients with suspected enteroviral infection. The EIA was compared with polymerase chain reaction (PCR) and virus isolation in cell culture. Of 204 samples tested, 20 were positive by EIA, 34 by PCR, and 18 by cell culture. Compared with PCR, the most sensitive method, the sensitivity of EIA was 58% (20/34); the sensitivity of cell culture isolation was 52% (18/34). The results of both assays correlated in only 60% of cases. The combination of EIA and cell culture isolation detected 76% of PCR-positive stool samples. Enterovirus EIA provides results within 3-4 hr and requires only standard EIA equipment. It represents a rapid, reliable, and cost-effective diagnostic tool for enterovirus diagnosis from faecal samples. Negative results must be confirmed by other techniques, such as PCR or virus isolation in cell culture.