PulseNet International Survey on the Implementation of Whole Genome Sequencing in Low and Middle-Income Countries for Foodborne Disease Surveillance.

PulseNet International (PNI) is a global network of 88 countries who work together through their regional and national public health laboratories to track foodborne disease around the world. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens to strengthen preparedness and response and lower the burden of disease. Several countries in North America and Europe have experienced significant benefits in disease mitigation after implementing WGS. To broaden the routine use of WGS around the world, challenges and barriers must be overcome. We conducted this study to determine the challenges and barriers countries are encountering in their attempts to implement WGS and to identify how PNI can provide support to improve and become a better integrated system overall. A survey was designed with a set of qualitative questions to capture the status, challenges, barriers, and successes of countries in the implementation of WGS and was administered to laboratories in Africa, Asia-Pacific, Latin America and the Caribbean, and Middle East. One-third of respondents do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for implementation of WGS were lack of funding, gaps in expertise, and training, especially for data analysis and interpretation. Features of an ideal system to facilitate implementation and global surveillance were identified as an all-in-one software that is free, accessible, standardized and validated. This survey highlights the minimal use of WGS for foodborne disease surveillance outside the United States, Canada, and Europe to date. Although funding remains a major barrier to WGS-based surveillance, critical gaps in expertise and availability of tools must be overcome. Opportunities to seek sustainable funding, provide training, and identify solutions for a globally standardized surveillance platform will accelerate implementation of WGS worldwide.

Evolving Epidemiological Characteristics of COVID-19 in Hong Kong From January to August 2020: Retrospective Study.

BACKGROUND: COVID-19 has plagued the globe, with multiple SARS-CoV-2 clusters hinting at its evolving epidemiology. Since the disease course is governed by important epidemiological parameters, including containment delays (time between symptom onset and mandatory isolation) and serial intervals (time between symptom onsets of infector-infectee pairs), understanding their temporal changes helps to guide interventions. OBJECTIVE: This study aims to characterize the epidemiology of the first two epidemic waves of COVID-19 in Hong Kong by doing the following: (1) estimating the containment delays, serial intervals, effective reproductive number (Rt), and proportion of asymptomatic cases; (2) identifying factors associated with the temporal changes of the containment delays and serial intervals; and (3) depicting COVID-19 transmission by age assortativity and types of social settings. METHODS: We retrieved the official case series and the Apple mobility data of Hong Kong from January-August 2020. The empirical containment delays and serial intervals were fitted to theoretical distributions, and factors associated with their temporal changes were quantified in terms of percentage contribution (the percentage change in the predicted outcome from multivariable regression models relative to a predefined comparator). Rt was estimated with the best fitted distribution for serial intervals. RESULTS: The two epidemic waves were characterized by imported cases and clusters of local cases, respectively. Rt peaked at 2.39 (wave 1) and 3.04 (wave 2). The proportion of asymptomatic cases decreased from 34.9% (0-9 years) to 12.9% (≥80 years). Log-normal distribution best fitted the 1574 containment delays (mean 5.18 [SD 3.04] days) and the 558 serial intervals (17 negative; mean 4.74 [SD 4.24] days). Containment delays decreased with involvement in a cluster (percentage contribution: 10.08%-20.73%) and case detection in the public health care sector (percentage contribution: 27.56%, 95% CI 22.52%-32.33%). Serial intervals decreased over time (6.70 days in wave 1 versus 4.35 days in wave 2) and with tertiary transmission or beyond (percentage contribution: -50.75% to -17.31%), but were lengthened by mobility (percentage contribution: 0.83%). Transmission within the same age band was high (18.1%). Households (69.9%) and social settings (20.3%) were where transmission commonly occurred. CONCLUSIONS: First, the factors associated with reduced containment delays suggested government-enacted interventions were useful for achieving outbreak control and should be further encouraged. Second, the shorter serial intervals associated with the composite mobility index calls for empirical surveys to disentangle the role of different contact dimensions in disease transmission. Third, the presymptomatic transmission and asymptomatic cases underscore the importance of remaining vigilant about COVID-19. Fourth, the time-varying epidemiological parameters suggest the need to incorporate their temporal variations when depicting the epidemic trajectory. Fifth, the high proportion of transmission events occurring within the same age group supports the ban on gatherings outside of households, and underscores the need for residence-centered preventive measures.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

A systematic review of transmission dynamic studies of methicillin-resistant Staphylococcus aureus in non-hospital residential facilities.

BACKGROUND: Non-hospital residential facilities are important reservoirs for MRSA transmission. However, conclusions and public health implications drawn from the many mathematical models depicting nosocomial MRSA transmission may not be applicable to these settings. Therefore, we reviewed the MRSA transmission dynamics studies in defined non-hospital residential facilities to: (1) provide an overview of basic epidemiology which has been addressed; (2) identify future research direction; and (3) improve future model implementation. METHODS: A review was conducted by searching related keywords in PUBMED without time restriction as well as internet searches via Google search engine. We included only articles describing the epidemiological transmission pathways of MRSA/community-associated MRSA within and between defined non-hospital residential settings. RESULTS: Among the 10 included articles, nursing homes (NHs) and correctional facilities (CFs) were two settings considered most frequently. Importation of colonized residents was a plausible reason for MRSA outbreaks in NHs, where MRSA was endemic without strict infection control interventions. The importance of NHs over hospitals in increasing nosocomial MRSA prevalence was highlighted. Suggested interventions in NHs included: appropriate staffing level, screening and decolonizing, and hand hygiene. On the other hand, the small population amongst inmates in CFs has no effect on MRSA community transmission. Included models ranged from system-level compartmental models to agent-based models. There was no consensus over the course of disease progression in these models, which were mainly featured with NH residents /CF inmates/ hospital patients as transmission pathways. Some parameters used by these models were outdated or unfit. CONCLUSIONS: Importance of NHs has been highlighted from these current studies addressing scattered aspects of MRSA epidemiology. However, the wide variety of non-hospital residential settings suggest that more work is needed before robust conclusions can be drawn. Learning from existing work for hospitals, we identified critical future research direction in this area from infection control, ecological and economic perspectives. From current model deficiencies, we suggest more transmission pathways be specified to depict MRSA transmission, and further empirical studies be stressed to support evidence-based mathematical models of MRSA in non-hospital facilities. Future models should be ready to cope with the aging population structure.

Next generation genome sequencing reveals phylogenetic clades with different level of virulence among Salmonella Typhimurium clinical human isolates in Hong Kong.

BACKGROUND: Salmonella Typhimurium is frequently isolated from foodborne infection cases in Hong Kong, but the lack of genome sequences has hindered in-depth epidemiological and phylogenetic studies. In this study, we sought to reconstruct the phylogenetic relationship and investigate the distribution and mutation patterns of virulence determinants among local S. Typhimurium clinical isolates using their genome sequences. RESULTS: We obtained genome sequences of 20 S. Typhimurium clinical isolates from a local hospital cluster using a 454 GS FLX Titanium sequencing platform. Phylogenetic analysis was performed based on single nucleotide polymorphism positions of the core genome against the reference strain LT2. Antimicrobial susceptibility was determined using minimal inhibitory concentration for five antimicrobial agents and analyses of virulence determinants were performed through referencing to various databases. Through phylogenetic analysis, we revealed two distinct clades of S. Typhimurium isolates and three outliers in Hong Kong, which differ remarkably in antimicrobial susceptibility and presentation and mutations of virulence determinants. The local isolates were not closely related to many of the previously sequenced S. Typhimurium isolates, except LT2. As the isolates in the two clades spanned over 10 years of isolation, they probably represent endemic strains. The outliers are possibly introduced from outside of Hong Kong. The close relatedness of members in one of the clades to LT2 and the Japanese stool isolate T000240 suggests the potential reemergence of LT2 progeny in regions nearby. CONCLUSIONS: Our study demonstrated the utility of next-generation sequencing coupled to traditional microbiological testing method in a retrospective epidemiological study involving multiple clinical isolates. The evolution of multidrug- and ciprofloxacin-resistant strains among the more virulent clade is also an increasing concern.

Multicenter Evaluation of the Molecular Line Probe Assay for Multidrug Resistant Mycobacterium Tuberculosis Detection in China.

In order to evaluate the performance of a molecular Hain line probe assay (Hain LPA) for rapid detection of rifampicin and isoniazid resistance of Mycobacterium tuberculosis in China, 1612 smear positive patients were consecutively enrolled in this study. Smear positive sputum specimens were collected for Hain LPA and conventional drug susceptibility testing (DST). The sensitivity and specificity of Hain LPA were analyzed by using conventional DST as golden reference. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for rifampicin resistance detection were 88.33%, 97.66%, 81.54%, and 98.62%, respectively. The sensitivity, specificity, PPV and NPV for isoniazid resistance detection were 80.25%, 98.07%, 87.25%, and 96.78%, respectively. These findings suggested that Hain LPA can be an effective method worthy of broader use in China.

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before. RESULTS: Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium. CONCLUSIONS: We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Ethambutol resistance as determined by broth dilution method correlates better than sequencing results with embB mutations in multidrug-resistant Mycobacterium tuberculosis isolates.

We evaluated the correlation of phenotypic ethambutol (EMB) susceptibility as determined by two drug susceptibility methods with embB mutations in multidrug-resistant (MDR) Mycobacterium tuberculosis strains. The concordance rate for EMB resistance between broth dilution method and sequencing results (83.6%) was significantly higher than between the proportion method and sequencing results (61.7%) (P = 0.004). Of the embB mutants, 75.4% (46/61) possessed a mutation at embB306. Our results demonstrated that ethambutol resistance determined by broth dilution method reveals better correlation with embB mutations than the proportion method in MDR isolates.

Association of polymorphisms in the Chr18q11.2 locus with tuberculosis in Chinese population.

A GWAS study has reported that two single nucleotide polymorphisms (SNPs) were associated with predisposition to tuberculosis (TB) in African populations. These two loci represented the long-waited GWAS hits for TB susceptibility. To determine whether these two SNPs are associated with TB in Chinese population, we attempted an replication in a cohort of over one thousand Chinese TB patients and 1,280 healthy controls using melting temperature shift allele-specific genotyping analysis. We found that only SNP rs4331426 was significantly associated with TB in Chinese population (p = 0.011). However, the effect was opposite. The G allele of the SNP in Chinese population is a protective allele (OR = 0.62, 95 % CI 0.44-0.87), while it was the risk allele for African population (OR = 1.19, 95 % CI 1.12-1.26). No significance was found for SNP rs2335704. The results provided an independent support for a role in susceptibility to TB for SNP rs4331426. However, it also indicated that direct predisposition element to TB and the association effects may vary across ethnic groups.

CISH and susceptibility to infectious diseases.

BACKGROUND: The interleukin-2-mediated immune response is critical for host defense against infectious pathogens. Cytokine-inducible SRC homology 2 (SH2) domain protein (CISH), a suppressor of cytokine signaling, controls interleukin-2 signaling. METHODS: Using a case-control design, we tested for an association between CISH polymorphisms and susceptibility to major infectious diseases (bacteremia, tuberculosis, and severe malaria) in blood samples from 8402 persons in Gambia, Hong Kong, Kenya, Malawi, and Vietnam. We had previously tested 20 other immune-related genes in one or more of these sample collections. RESULTS: We observed associations between variant alleles of multiple CISH polymorphisms and increased susceptibility to each infectious disease in each of the study populations. When all five single-nucleotide polymorphisms (SNPs) (at positions -639, -292, -163, +1320, and +3415 [all relative to CISH]) within the CISH-associated locus were considered together in a multiple-SNP score, we found an association between CISH genetic variants and susceptibility to bacteremia, malaria, and tuberculosis (P=3.8x10(-11) for all comparisons), with -292 accounting for most of the association signal (P=4.58x10(-7)). Peripheral-blood mononuclear cells obtained from adult subjects carrying the -292 variant, as compared with wild-type cells, showed a muted response to the stimulation of interleukin-2 production--that is, 25 to 40% less CISH expression. CONCLUSIONS: Variants of CISH are associated with susceptibility to diseases caused by diverse infectious pathogens, suggesting that negative regulators of cytokine signaling have a role in immunity against various infectious diseases. The overall risk of one of these infectious diseases was increased by at least 18% among persons carrying the variant CISH alleles.

Transmission of multidrug-resistant and extensively drug-resistant tuberculosis in a metropolitan city.

Multidrug-resistant (MDR)- tuberculosis (TB) and extensively drug resistant (XDR)-TB reportedly lead to increased household transmission. This is a retrospective cohort study of active TB occurring among household contacts exposed to MDR-TB. Of 704 contacts in 246 households, initial screening identified 12 (1.7%) TB cases (prevalent cases) and 17 (2.4%) contacts that subsequently developed active TB (secondary cases) after a median (range) duration of 17 (5-62.5) months. Eight prevalent cases and three secondary cases had MDR-TB. TB incidence rates per 100,000 person-years were 254.9 overall and 45.0 for MDR-TB. XDR-TB in the index MDR-TB patient significantly increased the odds of identifying a prevalent TB case to 4.8 (95% CI 1.02-22.5), and the hazard of finding a secondary TB case to 4.7 (95% CI 1.7-13.5). Molecular fingerprinting confirmed household transmission of MDR-TB. Of 20 retrievable isolates from 27 XDR-TB index cases, restriction fragment length polymorphism analysis showed clustering among 13 (65%), with 11 (55%) due to recent transmission by n-1 method and an identifiable household source in only three (27.2%) of the 11 cases. XDR-TB relative to MDR-TB significantly increases household transmission of TB, probably reflecting prolonged/higher infectivity, and indicating a need for prolonged household surveillance. XDR-TB may largely transmit outside of the household settings.

Multicenter evaluation of genechip for detection of multidrug-resistant Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB), especially multidrug-resistant TB (MDR-TB), is still one of the most serious threats to TB control worldwide. Early diagnosis of MDR-TB is important for effectively blocking transmission and establishing an effective protocol for chemotherapy. Genechip is a rapid diagnostic method based on molecular biology that overcomes the poor biosafety, time consumption, and other drawbacks of traditional drug sensitivity testing (DST) that can detect MDR-TB. However, the Genechip approach has not been effectively evaluated, especially in limited-resource laboratories. In this study, we evaluated the performance of Genechip for MDR-TB in 1,814 patients in four prefectural or municipal laboratories and compared its performance with that of traditional DST. The results showed that the sensitivity and specificity of Genechip were 87.56% and 97.95% for rifampin resistance and 80.34% and 95.82% for isoniazid resistance, respectively. In addition, we found that the positive grade of the sputum smears influenced the judgment of results by Genechip. The test judged only 75% of the specimens of "scanty" positive grade. However, the positive grade of the specimens showed no influence on the accuracy of Genechip. Overall, the study suggests that, in limited-resource laboratories, Genechip showed high sensitivity and specificity for rifampin and isoniazid resistance, making it a more effective, rapid, safe, and cost-beneficial method worthy of broader use in limited-resource laboratories in China.

Development of an ICR mouse bioassay for toxicity evaluation in neurotoxic poisoning toxins-contaminated shellfish.

OBJECTIVE: To develop an ICR (female) mouse bioassay (MBA) for toxicity confirmation and evaluation of neurotoxins (brevetoxins)-contaminated shellfish. METHODS: Brevetoxins (BTX-B) as a causative agent of neurotoxic shellfish poisoning (NSP) under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. RESULTS: BTX-B could be detected in shellfish tissues at concentration of 50-400 µg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit (80 µg/100 g shellfish tissues). The LD50 identified was 455 mg/kg for BTX-B under general shellfish matrices (excluding oyster matrices) dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. CONCLUSION: The two ELISA analyses agree favorably (correlation coefficient, r³â‹0.96; Student's t-tests, P>0.05) with the developed bioassay.

Draft genome sequence of Salmonella enterica serovar Typhimurium ST1660/06, a multidrug-resistant clinical strain isolated from a diarrheic patient.

Salmonella enterica serovar Typhimurium is one of the most prevalent serovars of Salmonella that causes human gastroenteritis. Here, we report the draft genome sequence of the S. Typhimurium multidrug-resistant strain ST1660/06. Comparative genomic analysis unveiled three strain-specific genomic islands that potentially confer the multidrug resistance and virulence of the strain.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

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.

Emergence of antibiotic-resistant Mycoplasma genitalium as the cause of non-gonococcal urethritis in male patients at a sexually transmitted infection clinic.

Mycoplasma genitalium is the cause of an emerging sexually transmitted infection (STI) with high propensity for development of antimicrobial resistance. In a prevalence study conducted at the public STI service in Hong Kong, the first void urine samples of 38 (8%) of 493 male patients with non-gonococcal urethritis (NGU) tested positive for M. genitalium using reverse transcription polymerase chain reaction. Patients with M. genitalium infection were younger [31 vs 33 years, odds ratio (OR) 0.96, 95% confidence interval (CI) 0.93-0.996; P=0.03], more likely to present with urethral discharge (12% vs 6%, OR 2.16, 95% CI 1.10-4.23; P=0.02) and had symptom duration >2 weeks (14% vs 6%, OR 2.34, 95% CI 1.10-4.97; P=0.03) compared with patients without M. genitalium infection. The prevalence of M. genitalium infection was lower in patients co-infected with Chlamydia trachomatis compared with patients with isolated infection (4% vs 10%, OR 0.38, 95% CI 0.17-0.84; P=0.02). The prevalence of M. genitalium infection was not higher in men who have sex with men. Antimicrobial-resistance-conferring mutations were present in 24 (63%) patients with M. genitalium - 23S rRNA 18 (47%) and parC 19 (53%). Similar to neighbouring countries in the Asia Pacific region, concurrent resistance mutations against both macrolides and fluoroquinolones were demonstrated in 14 (37%) patients. Histories of azithromycin and moxifloxacin use were significantly associated with a diagnosis of M. genitalium infection. Characteristically, NGU in Hong Kong featured the co-existence of mono-resistance against macrolides or fluoroquinolones, and the presence of dual class resistance. The geographic variability of antimicrobial resistance against M. genitalium is attributed not just to the different transmission networks formed in separate population groups, but the antimicrobial prescriptions for the treatment of urethritis in the community.

Cluster analysis of toxins profile pattern as a tool for tracing shellfish contaminated with PSP-toxins.

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.

Epidemiology of antituberculosis drug resistance 2002-07: an updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance.

BACKGROUND: The Global Project on Anti-Tuberculosis Drug Resistance has been gathering data since 1994. This study provides the latest data on the extent of drug resistance worldwide. METHODS: Data for drug susceptibility were gathered from 90 726 patients in 83 countries and territories between 2002 and 2007. Standardised collection of results enabled comparison both between and within countries. Where possible, data for HIV status and resistance to second-line drugs were also obtained. Laboratory data were quality assured by the Supranational Tuberculosis Reference Laboratory Network. FINDINGS: The median prevalence of resistance to any drug in new cases of tuberculosis was 11.1% (IQR 7.0-22.3). The prevalence of multidrug resistance in new tuberculosis cases ranged from 0% in eight countries to 7% in two provinces in China, 11.1% in Northern Mariana Islands (although reporting only two cases), and between 6.8% and 22.3% in nine countries of the former Soviet Union, including 19.4% in Moldova and 22.3% in Baku, Azerbaijan (median for countries surveyed 1.6%, IQR 0.6-3.9). Trend analysis showed that between 1994 and 2007, the prevalence of multidrug-resistant (MDR) tuberculosis in new cases increased substantially in South Korea and in Tomsk Oblast and Orel Oblast, Russia, but was stable in Estonia and Latvia. The prevalence of MDR tuberculosis in all tuberculosis cases decreased in Hong Kong and the USA. 37 countries and territories reported representative data on extensively drug-resistant (XDR) tuberculosis. Five countries, all from the former Soviet Union, reported 25 cases or more of XDR tuberculosis each, with prevalence among MDR-tuberculosis cases ranging between 6.6% and 23.7%. INTERPRETATION: MDR tuberculosis remains a threat to tuberculosis control in provinces in China and countries of the former Soviet Union. Data on drug resistance are unavailable in many countries, especially in Africa, emphasising the need to develop easier methods for surveillance of resistance in tuberculosis. FUNDING: Global Project: United States Agency for International Development and Eli Lilly and Company. Drug resistance surveys: national tuberculosis programmes, the Government of the Netherlands, the Global Fund to Fight AIDS, Tuberculosis and Malaria, Japan International Cooperation Agency, and Kreditanstalt für Wiederaufbau.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Streptococcus suis in retail markets: how prevalent is it in raw pork?

Streptococcus suis present in raw pork meats sold in local retail markets was enumerated by Most Probable Number (MPN)-PCR method. This method combined the conventional MPN technique with a specifically designed PCR assay based on the amplification of a 294-bp S. suis species-specific 16S rRNA gene sequence. A total of 78 raw pork lean meat samples purchased at two different supermarkets (Site A and B) and a wet market (Site C) were tested. Results indicated that S. suis could be detected from the enriched MPN tubes of all, except one, sample homogenates. The concentration of S. suis ranged from <3 to 4600 MPN/g of pork meat, with a total bacterial count (TBC) varying from 3.6 log to 7.4 log CFU/g. Statistical analyses indicated that pork meats purchased from the supermarket at Site B in summer contained significantly higher concentration of S. suis organisms than those from other retailers in any season. A significant correlation existed between log S. suis concentration and log TBC of the samples. This study revealed that raw pork meats available in local supermarkets or wet markets could contain S. suis at concentrations that were usually difficult to detect with traditional culture method. Field application of this method may contribute to a measurable evaluation, and thus the effective control, of human S. suis infection due to raw pork or pig carcass handling.