Kai Man Kam

PulseNet International: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.

Evaluation and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping Vibrio parahaemolyticus: an International Multicenter Collaborative Study

ABSTRACT 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.

Molecular and Conventional Epidemiology of Tuberculosis in Hong Kong: a Population-Based Prospective Study

ABSTRACT A prospective population-based molecular and conventional epidemiological study of 65.4% of bacteriologically confirmed cases of tuberculosis was carried out on the island of Hong Kong from May 1999 to Oct 2000 by the IS6110-based restriction fragment length polymorphism technique. Eleven of the isolates had five or fewer bands; 24.5% of the remaining 691 isolates belonged to clusters. The estimated proportion of recently transmitted disease was 15 to 20%.

Diagnostic accuracy of the PURE-LAMP test for pulmonary tuberculosis at the county-level laboratory in China.

Background Early and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China. Methodology/Principal Findings From April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture. Conclusions/Significance The study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.

Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis

ABSTRACT A prospective cross-sectional blinded study of 28 patients (21 male and 7 female patients; mean age, 44 years) with suspected active tuberculosis (TB) attending a TB and chest clinic is described. Blood was taken for immune cell enumeration, a whole-blood enzyme-linked immunosorbent assay (ELISA) for the detection of gamma interferon (IFN-γ) by the QuantiFERON-TB Gold (QFT-G) assay, and intracellular cytokine flow cytometry (ICC) analysis; and sputum was simultaneously taken for bacteriological culture for Mycobacterium tuberculosis. Twelve healthy subjects were included as controls. The performance characteristics of the QFT-G and ICC assays for the detection of active TB were compared. Among the patients with active TB, we found (i) normal to slightly elevated peripheral CD4+ and CD8+ T-cell counts but a significant reduction in the number of NK cells; (ii) CD4+ T cells were the major cell type producing IFN-γ, a type 1 cytokine; (iii) small percentages of CD8+ T cells were also primed for IFN-γ production; (iv) the production of interleukin-4 (IL-4), a type 2 cytokine, was not prominent; and (v) the sensitivity and the specificity of the QFT-G assay were 88.2% and 18%, respectively, and those of the ICC assay were 94.1% and 36.4%, respectively. The specificities of the blood tests were likely underestimated due to cross-reaction to a non-M. tuberculosis mycobacterial infection and the lack of a confirmatory test that could be used to diagnose latent M. tuberculosis infection. Flow cytometry accurately locates the pool of immunological effector cells responsible for cytokine production during active TB. The ICC assay is an additional useful tool for the diagnosis of active TB.

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.

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.

A feasibility study of the Xpert MTB/RIF test at the peripheral level laboratory in China

OBJECTIVE To evaluate the performance of Xpert MTB/RIF (MTB/RIF) in the county-level tuberculosis (TB) laboratory in China. METHODS From April 2011 to January 2012, patients with suspected multidrug-resistant tuberculosis (MDR-TB) and non-MDR-TB were enrolled consecutively from four county-level TB laboratories. The detection of Mycobacterium tuberculosis (MTB) by MTB/RIF was compared to detection by Löwenstein-Jensen culture. The detection of rifampin resistance was compared to detection by conventional drug-susceptibility testing. The impact of multiple specimens on the performance of MTB/RIF was also evaluated. RESULTS A total of 2142 suspected non-MDR-TB cases and 312 suspected MDR-TB cases were enrolled. For MTB detection in suspected non-MDR-TB cases, the sensitivity and specificity of MTB/RIF were 94.4% and 90.2%, respectively. The sensitivity in smear-negative patients was 88.8%. For the detection of rifampin resistance in suspected non-MDR-TB cases, the sensitivity and specificity of MTB/RIF were 87.1% and 97.9%, respectively. For the detection of rifampin resistance in suspected MDR-TB cases, the sensitivity and specificity of MTB/RIF were 87.1% and 91.0%, respectively. Using multiple sputum specimens had no significant influence on the performance of MTB/RIF for MTB detection. CONCLUSIONS The introduction of MTB/RIF could increase the accuracy of detection of MTB and rifampin resistance in peripheral-level TB laboratories in China. One single specimen is adequate for TB diagnosis by MTB/RIF.

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

BackgroundVibrio 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.ResultsHere 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.ConclusionsWe 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.

Cost-effectiveness comparison of Genechip and conventional drug susceptibility test for detecting multidrug-resistant tuberculosis in China.

Background Genechip (CapitalBio, Beijing, China) is a system for diagnosing resistance to rifampin and isoniazid, which shows high efficiency in detecting drug-resistant tuberculosis. Here, we firstly evaluated the costs of Genechip for detecting the drug susceptibility of Mycobacterium tuberculosis, compared to conventional drug susceptibility test (DST) in laboratories in China. Methodology/Principal Findings Data on the costs of the two tests were collected at four hospitals. Costs were calculated using the essential factor cost calculation method. The costs of diagnosing a single case of multidrug-resistant tuberculosis (MDR-TB) using Genechip and DST were US