Systematic Literature Review of Role of Noroviruses in Sporadic Gastroenteritis

Noroviruses accounted for 12% of severe gastroenteritis cases among children <5 years of age

Enteroviral infection in Greek AIDS patients

Objective: Prolonged intestinal replication of polioviruses has not previously been studied in Greek AIDS patients. The objective of our study was to estimate the prevalence of enteroviral infections in this population. Methods: Nineteen stool samples were investigated from 19 different patients. Collection took place at the Hellenic Red Cross Hospital, Athens, Greece, between August and October 2002. Samples were processed as follows: virus isolation was attempted by cell culture using three different cell lines (human epidermoid carcinoma [Hep]-2, rabdomyosarcoma [RD], and mouse cells genetically modified in order to express the polio virus receptor in their cell surface [L20B]). An enterovirus-specific reverse transcription (RT)-PCR was then applied. Finally, seroneutralization tests were performed on 11 blood samples taken from a number of the patients who had supplied stool samples. Results: Samples were negative for enterovirus detection of any serotype on all cell lines. No cytopathic effect was observed. Enterovirus-specific RT-PCR assays were also negative for the detection of enteroviral RNA. Seroneutralization revealed relatively high antibody titers against poliovirus 1 and 2 in three of the eleven blood samples. Conclusions: Greek AIDS patients are not vulnerable to enteroviral infections and do not constitute a potential reservoir of poliovirus-prolonged excretion in Greece

Whole genome sequencing of M. tuberculosis for detection of drug resistance: a systematic review

OBJECTIVES: We conducted a systematic review to determine the diagnostic accuracy of Whole Genome Sequencing (WGS) of M. tuberculosis for the detection of resistance to first and second line anti-tuberculosis (TB) drugs. METHODS: The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, PPV and NPV of WGS using phenotypic Drug Susceptibility Testing (DST) methods as a gold standard were determined. RESULTS: Anti-TB agents tested included all first line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first line drugs were 0.98 (95% CI, 0.93 to 0.98) and 0.98 (95% CI, 0.98 to 1.00) for rifampicin and 0.97 (95% CI, 0.94 to 0.99) and 0.93 (95% CI 0.91 to 0.96) for isoniazid, respectively. Due to high heterogeneity in studies' design, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. CONCLUSIONS: WGS could be considered a promising alternative to existing phenotypic and molecular DST methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes

Whole genome sequencing of M. tuberculosis for detection of drug resistance: a systematic review

OBJECTIVES: We conducted a systematic review to determine the diagnostic accuracy of Whole Genome Sequencing (WGS) of M. tuberculosis for the detection of resistance to first and second line anti-tuberculosis (TB) drugs. METHODS: The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, PPV and NPV of WGS using phenotypic Drug Susceptibility Testing (DST) methods as a gold standard were determined. RESULTS: Anti-TB agents tested included all first line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first line drugs were 0.98 (95% CI, 0.93 to 0.98) and 0.98 (95% CI, 0.98 to 1.00) for rifampicin and 0.97 (95% CI, 0.94 to 0.99) and 0.93 (95% CI 0.91 to 0.96) for isoniazid, respectively. Due to high heterogeneity in studies' design, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. CONCLUSIONS: WGS could be considered a promising alternative to existing phenotypic and molecular DST methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes

Concentration, Detection and Identification of Infectious Enteroviruses in Sewage

This work presents a new approach for the molecular 'serotyping' of enteroviruses concentrated and isolated from sewage. Viruses were adsorbed to cellulose nitrate membrane filters, isolated from the membrane filter using the VIRADEN method and identified by RT-PCR, followed by 57-UTR RFLP analysis and partial sequencing of the VP1 protein coding region. All 42 isolates belonged to the same genetic sub-cluster of non-polio enteroviruses. Partial VP1 sequencing revealed that isolates belonged to serotypes CBV4 (33 isolates, 79%), CBV1 (8 isolates, 19%) and Echovirus7 (1 isolate, 2%). The methodology presented here is highly promising for environmental surveillance of enterovirus circulation and epidemiology. 57-UTR RFLP analysis with Hpall is useful for initial subclassification, while partial VP1 sequencing is efficient for molecular 'serotyping'. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA

Environmental surveillance of circulation of enteroviruses-Public health effects

Objective: This study presents a new approach for the molecular "serotyping" of enteroviruses concentrated and isolated from urban sewage. Method: Samples were collected from the Nicosia Sewage Treatment Plant in Cyprus. Viruses were adsorbed on cellulose nitrate membrane filters, isolated from the membrane filter using the VIRADEN method and identified by RT-PCR, followed by 5-UTR RFLP analysis and partial sequencing of the VP1 protein coding region. Results: In total, 42 enteroviruses were isolated. Initial sub-grouping based on HpaII restriction profile showed that all isolates belonged to the same genetic subcluster of non-polio enteroviruses. Partial VP1 sequencing revealed that isolates belonged to serotypes CBV4 (33 isolates, 79%), CBV1 (8 isolates, 19%) and Echovirus 7 (1 isolate, 2%). All isolates of the same serotype had highly similar VP1 sequences. The HpaII digests predicted the genetic sub-cluster in all cases. Finally, phylogenetic analysis revealed that the isolates correlate with strains of the same serotype isolated during the last four decades in Europe, Asia, Northern Africa and the Middle East, implying a possible epidemiological relationship. Conclusions: In conclusion, the results confirm that this methodology is highly promising for environmental surveillance of enterovirus circulation and epidemiology. 5-UTR RFLP analysis with HpaII is useful for initial sub-classification, while partial VP1sequencing is efficient for molecular serotyping

Novel molecular techniques for the identification of the members of Brucellaceae family

An account is presented of an attempt to identify a multiresistant, gram-negative bacterium that was isolated from raw sewage in Cyprus. The API 2ONE system was initially used to identify the strain on the basis of its biochemical profile. In addition, a 16S rRNA-specific PCR was applied on genetic material extracted directly from BGM cell culture material after which the sequence of the largest part of the 16S rRNA gene (approximately 1000 nucleotides) was obtained BLAST software was used to compare the sequences obtained with corresponding sequences of other strains belonging to members of the family Brucellaceae, which were available in the GenBank database. The isolated bacterium was identified as Ochrobactrum anthropi by both its biochemical profile and its 16S rRNA sequences, which showed a 98% similarity with other O. anthropi strains. It was found to be resistant to several antibiotics, including b-lactams and aminoglycosides. Phylogenetic analysis showed that 16S rRNA gene sequencing should be used with caution for the identification of family Brucellaceae members that are genetically closely related. In conclusion, this study recorded the first isolation of an O. anthropi strain from environmental samples in Cyprus. This strain was found to be highly resistant to antibiotics. Sequencing of the 16S rRNA gene was useful for the verification of the identity of the strain. However, phylogenetic comparisons with other related bacterial strains showed that this method is perhaps not the most decisive for identification of members of the family Brucellaceae, due to the consonance between different species and genera

Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5′ untranslated region (5′-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Α9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5′-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates