Abstract Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

UNLABELLED ABSTRACT: BACKGROUND Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. FINDINGS Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1)~90% of 100 assemblies with < 10 scaffolds and ~95% of 100 assemblies with < 150 contigs; 2) average contig N50 size is over 331 kb; 3) average single base accuracy is > 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. CONCLUSIONS A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks.

SETTING Patients infected with non-tuberculous mycobacteria usually fail treatment due to erroneous diagnoses. Early detection of mycobacterial species is essential for adequate case management. OBJECTIVE To conduct a multicentre, prospective evaluation of the recently developed biochip test and to determine its sensitivity and specificity in three clinical hospitals in China. RESULTS A total of 1565 clinical isolates obtained from three hospitals were identified as 19 mycobacterial species by 16S sequencing. The same 53 reference strains were detected in all three hospitals as quality control and to evaluate the reproducibility of the assay. The overall accuracy of the species identification assay among all strains was 99.9%(1722/1724). The two unidentified samples, belonging to Mycobacterium parascrofulaceum and M. monacense, were not included among the 17 mycobacterial species. The reference strains demonstrated that the reproducibility of the assay was 100%. CONCLUSION The biochip test provided cost-effective and highly sensitive identification of mycobacterial species in less than 6 h. This will help clinical staff carry out more efficient and personalised clinical treatment without delay.

Paralytic shellfish poisoning (PSP) is one of the most lethal biotoxin-induced diseases worldwide, which may pose serious public health threat and potential devastating economic damage on fisheries industry in the affected region(s). To prevent the importation of PSP contaminated shellfish to a community, detailed documentation on the supply chain and routine surveillance systems are, in principle, crucial measures to protect people from this intoxication. However, difficulties have always been encountered on the traceability of the source/origin of contaminated shellfish. In the present study, we reported the potential application of PSP-toxins profiles with similarity analysis that can be used to identify epidemiological linkage between shellfish samples collected from markets and patients during a PSP outbreak. PSP-toxins were identified and quantified by ion-pair chromatographic separation followed by post-column oxidation to fluorescent imino purine derivatives. Samples from a PSP incident and other surveillance samples collected in our past 7-year record were also compared for their similarity in PSP-toxins profiles patterns. Molar distributions (nmol%) of 10 PSP-toxins were analyzed by Unweighted Pair Group Method with Arithmetric averages (UPGMA). Three prominent clusters emerged with similarity levels reaching over 80% for each, suggesting that each group of samples probably originated from a same source/batch. The PSP-toxins profiles and toxicities determined from surveillance samples could provide premonitory clues on the occurrences of PSP incident and outbreak with corresponding toxin profiles in the later time. Due to species-specific characteristics of PSP-toxins composition and profile in shellfish under varieties of environmental and physiological conditions, PSP-toxins profile can be a specific and useful biochemical indicator for tracing PSP contaminated shellfish provided that spatio-temporal occurrence patterns of toxins profiles are available in a databank for inter-laboratory comparison and standardized methodologies such as consentaneous toxins extraction and identification criteria are used for analysis and comparison.

SETTING Correctional settings and remand prisons. OBJECTIVE To critically discuss calculations for epidemiological indicators of the tuberculosis (TB) burden in prisons and to provide recommendations to improve study comparability. METHODS A hypothetical data set illustrates issues in determining incidence and prevalence. The appropriate calculation of the incidence rate is presented and problems arising from cross-sectional surveys are clarified. RESULTS Cases recognized during the first 3 months should be classified as prevalent at entry and excluded from any incidence rate calculation. The numerator for the incidence rate includes persons detected as having developed TB during a specified period of time subsequent to the initial 3 months. The denominator is person-time at risk from 3 months onward to the end point (TB or end of the observation period). Preferably, entry time, exit time and event time are known for each inmate to determine person-time at risk. Failing that, an approximation consists of the sum of monthly head counts, excluding prevalent cases and those persons no longer at risk from both the numerator and the denominator. CONCLUSIONS The varying durations of inmate incarceration in prisons pose challenges for quantifying the magnitude of the TB problem in the inmate population. Recommendations are made to measure incidence and prevalence.

HASH(0x15a5d5a0)

BACKGROUND: Reliable DST against second-line anti-tuberculosis drugs (SLDs) is crucial for the management of the increasing burden of patients affected by multidrug- and extensively drug-resistant TB. METHODS: This study utilizes 252 clinical isolates of Mycobacterium tuberculosis from five countries (Hong Kong Special Administrative Region, Korea, Latvia, Peru, Philippines) with documented treatment histories to establish clinically and microbiologically relevant critical concentrations (CCs) of six SLDs for three routine testing methods: the absolute concentration method using Löwenstein-Jensen (LJ) medium, the 1% proportion method using Middlebrook 7H10 agar medium, and the radiometric BACTEC 460 system. FINDINGS: In LJ medium, CCs of capreomycin, ethionamide, kanamycin, ofloxacin, rho-aminosalicylic acid and cycloserine (CS) were respectively 40.0, 40.0, 30.0, 3.0, 1.0 and 30.0 mg/l. In 7H10 agar medium, the respective CCs for the first five antibiotics (except CS) were 8.0, 2.0-3.0, 3.0-5.0, 1.0-1.5 and 0.5-1.0 mg/l. In BACTEC 460 broth, the respective CCs were 1.5-2.0, 1.0-1.5, 2.0-3.0, 0.5-1.0 and 0.5-1.0 mg/l. Precautions in DST interpretation was also discussed. INTERPRETATION: By adopting this set of CCs as a global standard to define second-line drug susceptibility and resistance, as well as precautions in result interpretation, the screening, diagnosis and management of patients with drug-resistant TB can be greatly improved.

We evaluated the performance of two immunoblot assays: the INNO-LIA Syphilis Score (LIA) and the MarDx T. pallidum IgG Marblot Test (TWB), as compared with that of the Murex ICE Syphilis enzyme immunoassay (EIA), the Serodia Treponema pallidum particle agglutination (TPPA) assay and the fluorescent treponemal antibody-absorption (FTA-abs) assay, for the serological diagnosis of syphilis using serum samples of 135 attendees of the social hygiene clinics of the Department of Health in Hong Kong newly diagnosed with syphilis and provided with clinical stages (39 in primary, 20 in secondary, 18 in early latent and 58 in latent of unknown duration) and of 43 normal healthy subjects between October and December 2004. The differences in the overall sensitivities of the LIA assay and the EIA/TPPA/FTA-abs assays were not statistically significant (P > 0.05) whereas the overall sensitivity of the TWB assay was significantly lower (P < 0.05) than the overall sensitivities of the EIA, the TPPA and the FTA-abs assays. The LIA assay had an overall sensitivity of 94.1%(95% CI 88.7-97.0%) whereas the TWB assay 65.2%(95% CI 56.8-72.7%). Both the LIA and the TWB assays have a specificity of 100%. When consensus results were derived from the most predominant results of the EIA, the TPPA and the FTA-abs assays, the LIA assay had a positive agreement with the consensus results of 98.5%(95% CI 94.5-99.6%) whereas the TWB assay 68.2%(95% CI 59.8-75.6%). Therefore, the LIA assay performed significantly better (P < 0.05) than the TWB assay. The LIA assay can be considered to be a valid alternative confirmatory test for the serological diagnosis of syphilis.

Setting: the tuberculosis Supra-National Reference Laboratory (SRL) Network Objectives: to investigate the origin of highly discordant rifampin (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing Design: nine SRLs tested RMP susceptibility of 19 selected Mycobacterium tuberculosis strains, using standard culture-based methods. Strains were classified as definitely resistant (R, n=6) or susceptible (S, n=2), and probably resistant (PR, n=8) or susceptible (PS, n=3), based on rpoB mutations and treatment outcome. Results: all methods yielded a susceptible result for the two S and three PS strains lacking a rpoB mutation, and a resistant result for one R strain with a Ser531Leu and one PR strain with a double mutation. Although the remaining twelve R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric BACTEC(TM) 460TB or BACTEC(TM) 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen, and two on Middlebrook 7H10 agar. Conclusions: low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level resistant isolates, and to identify technical improvements that may identify such strains.