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.

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.

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.

Utility of GeneXpert in analysis of bronchoalveolar lavage samples from patients with suspected tuberculosis in an intermediate-burden setting.

OBJECTIVES: To explore the clinical role of GeneXpert in managing pulmonary tuberculosis (TB) in an intermediate burden city. METHODS: Sputum acid-fast-bacilli (AFB) smear negative patients underwent bronchoscopy for bronchial alveolar lavage (BAL). Fluids collected were examined for AFB smear, TB culture, TB polymerase chain reaction (PCR) (Cobas Taqman) and for GeneXpert. RESULTS: From October 2015 to February 2017, 227 BAL samples were collected. Cough and haemoptysis were the presenting symptoms in 70.0% and 37.4%, respectively. Apical shadows on chest X-rays (CXR) and apical cavitations on computed tomography (CT) were commoner in GeneXpert positive cases (p = 0.01 and 0.02, respectively). Sensitivity and specificity of GeneXpert for TB diagnosis was 80% and 98% respectively. Positive and negative predictive value of the test was 92.3 and 95.1%, respectively. There were 9 false negative GeneXpert samples (8 were Cobas Taqman TB PCR negative): 6 were diagnosed by BAL culture, 2 by biopsy and one by Cobas Taqman TB PCR. There were 3 false positive cases with negative culture; 2 were put on empirical treatment with favourable clinical responses, while one defaulted follow-p. CONCLUSION: GeneXpert in BAL samples has high sensitivity and specificity. It enabled timely initiation of anti-TB treatment in clinical suspicious cases.

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.

Low cost automated whole smear microscopy screening system for detection of acid fast bacilli.

BACKGROUND: In countries with high tuberculosis (TB) burden, there is urgent need for rapid, large-scale screening to detect smear-positive patients. We developed a computer-aided whole smear screening system that focuses in real-time, captures images and provides diagnostic grading, for both bright-field and fluorescence microscopy for detection of acid-fast-bacilli (AFB) from respiratory specimens. OBJECTIVES: To evaluate the performance of dual-mode screening system in AFB diagnostic algorithms on concentrated smears with auramine O (AO) staining, as well as direct smears with AO and Ziehl-Neelsen (ZN) staining, using mycobacterial culture results as gold standard. METHODS: Adult patient sputum samples requesting for M. tuberculosis cultures were divided into three batches for staining: direct AO-stained, direct ZN-stained and concentrated smears AO-stained. All slides were graded by an experienced microscopist, in parallel with the automated whole smear screening system. Sensitivity and specificity of a TB diagnostic algorithm in using the screening system alone, and in combination with a microscopist, were evaluated. RESULTS: Of 488 direct AO-stained smears, 228 were culture positive. These yielded a sensitivity of 81.6% and specificity of 74.2%. Of 334 direct smears with ZN staining, 142 were culture positive, which gave a sensitivity of 70.4% and specificity of 76.6%. Of 505 concentrated smears with AO staining, 250 were culture positive, giving a sensitivity of 86.4% and specificity of 71.0%. To further improve performance, machine grading was confirmed by manual smear grading when the number of AFBs detected fell within an uncertainty range. These combined results gave significant improvement in specificity (AO-direct:85.4%; ZN-direct:85.4%; AO-concentrated:92.5%) and slight improvement in sensitivity while requiring only limited manual workload. CONCLUSION: Our system achieved high sensitivity without substantially compromising specificity when compared to culture results. Significant improvement in specificity was obtained when uncertain results were confirmed by manual smear grading. This approach had potential to substantially reduce workload of microscopists in high burden countries.

Insights into intercontinental spread of Zika virus.

The epidemic of Zika virus (ZIKV) infection in South America has led to World Health Organization's declaration of a Public Health Emergency of International Concern. To further inform effective public health policy, an understanding of ZIKV's transmission mechanisms is crucial. To characterize the intercontinental transmission of ZIKV, we compiled and analyzed more than 250 gene sequences together with their sequence-related geographic and temporal information, sampled across 27 countries spanning from 1947 to 2016. After filtering and selecting appropriate sequences, extensive phylogenetic analyses were performed. Although phylogeographic reconstruction supported the transmission route of the virus in Africa, South-eastern Asia, Oceania and Latin America, we discovered that the Eastern Africa origin of ZIKV was disputable. On a molecular level, purifying selection was found to be largely responsible for the evolution of non-structural protein 5 and envelope protein E. Our dataset and ancestral sequences reconstruction analysis captured previously unidentified amino acid changes during evolution. Finally, based on the estimation of the time to the most recent common ancestors for the non-structural protein 5 gene, we hypothesized potential specific historic events that occurred in the 1940s and might have facilitated the spread of Zika virus from Africa to South-eastern Asia. Our findings provide new insights into the transmission characteristics of ZIKV, while further genetic and serologic studies are warranted to support the design of tailored prevention strategies.

Assessing the risk of dengue virus transmission in a non-endemic city surrounded by endemic and hyperendemic areas.

OBJECTIVE: To assess the potential risk of dengue transmission in a non-endemic city using a spatial epidemiological approach. METHODS: Past dengue exposure of the general population was examined by dengue virus (DENV) IgG testing of archived samples from voluntary blood donors. Vector intensities were determined by local ovitrap index (OI). Analyses were made in the context of population statistics at both the district and sub-district level. RESULTS: The overall prevalence of DENV IgG was low at 2.25%. Positive donors were more likely to be older, non-Chinese, and female. Neither the OI nor the location of residence was associated with DENV serology. The sub-district level OI was clustered, but no correlation could be confirmed with the location of residence of positive blood donors. CONCLUSIONS: The cumulative exposure of Hong Kong residents to dengue has so far been low. Coupled with the lack of a spatial relationship between exposed cases and vector intensities, a high risk of local transmission of DENV is not supported. The apparently higher exposure likelihood of females could be explained by past infection in workers from dengue endemic countries, while frequent travel could have exposed older adults to DENV. Continued surveillance, risk assessment, and intensive vector control remain essential to prevent the transformation of a non-endemic to an endemic city.

Phylogenetic and Structural Significance of Dihydrofolate Synthase (folC) Mutations in Drug-Resistant Mycobacterium tuberculosis.

The aim of this study is to investigate the mutation pattern of the folC gene in drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates of global and Hong Kong cohorts. The public sequence read archives of 1,124 MTB genomes from three independent studies were retrieved and folC mutations existing solely in drug-resistant MTB strains were identified. A phylogenetic tree was constructed to analyze the segregation of mutation-related amino acid residues in the FolC structure. These mutation sites were further supported by direct Sanger sequencing of the folC gene among 254 clinical MTB isolates in a Hong Kong cohort. Homology modeling of wild-type and mutated FolC was performed, and the predicted structures were docked with hydroxydihydropteroate, the metabolic derivative of para-aminosalicylic acid (PAS), to evaluate the resultant binding affinity changes. Combining the results of three previous cohorts and our cohort, E40, I43, S150, and E153 are the most frequently affected amino acid residues in resistant isolates. Based on the distribution of mutations in the genome-based phylogenetic tree, lineage-specific mutation patterns were observed. Regarding the segregation of affected amino acid residues, the four most frequently affected residues are all in close proximity of the binding pocket for the PAS derivative. Molecular modeling results showed that mutations at E40, I43, and S150 can alter the structure of FolC putative binding pocket, causing the PAS derivative to bind outside of the now deformed pocket. This might ablate the interaction between the protein and the PAS derivative. To conclude, this study is the first comprehensive mutation pattern and bioinformatics analysis of the folC gene in MTB drug-resistant isolates. The distribution of mutations in phylogenetic lineages and protein structure is reported, analyzed, and discussed.

Concurrent Outbreaks of Tuberculosis in a School and the Wider Community in Macau.

Between 2009 and 2012, 22 adolescents of age 15-20 from a day school in Macau were diagnosed with tuberculosis. Detection of multiple molecular clusters may suggest the presence of concurrent outbreaks, and could reflect also ongoing transmissions in the community. Careful interpretation of molecular epidemiology data is crucial in contact investigations.

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.

Cost-effectiveness analysis of the Xpert MTB/RIF assay for rapid diagnosis of suspected tuberculosis in an intermediate burden area.

OBJECTIVES: We examined, from a Hong Kong healthcare providers' perspective, the cost-effectiveness of rapid diagnosis with Xpert in patients hospitalized for suspected active pulmonary tuberculosis (PTB). METHODS: A decision tree was designed to simulate outcomes of three diagnostic assessment strategies in adult patients hospitalized for suspected active PTB: conventional approach, sputum smear plus Xpert for acid-fast bacilli (AFB) smear-negative, and a single sputum Xpert test. Model inputs were derived from the literature. Outcome measures were direct medical cost, one-year mortality rate, quality-adjusted life-years (QALYs) and incremental cost per QALY (ICER). RESULTS: In the base-case analysis, Xpert was more effective with higher QALYs gained and a lower mortality rate when compared with smear plus Xpert by an ICER of USD99. A conventional diagnostic approach was the least preferred option with the highest cost, lowest QALYs gained and highest mortality rate. Sensitivity analysis showed that Xpert would be the most cost-effective option if the sensitivity of sputum AFB smear microscopy was ≤74%. The probabilities of Xpert, smear plus Xpert and a conventional approach to be cost-effective were 94.5%, 5.5% and 0%, respectively, in 10,000 Monte Carlo simulations. CONCLUSIONS: The Xpert sputum test appears to be a highly cost-effective diagnostic strategy for patients with suspected active PTB in an intermediate burden area like Hong Kong.

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.

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.

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.

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.

Use of cerebrospinal fluid enzyme immunoassay for diagnosis of neurosyphilis.

Neurosyphilis is a difficult clinical stage of syphilis as there is no ideal method for diagnosis and workup requires lumbar puncture which may sometimes provide ambiguous results especially in HIV co-infected patients. Enzyme immunoassay is a widely used screening test for syphilis in serum, but its test performance was not well studied in cerebrospinal fluid. To examine the diagnostic performance of cerebrospinal fluid-enzyme immunoassay (CSF-EIA) in neurosyphilis, we conducted a prospective study for two years. All consecutive patients admitted for workup of neurosyphilis under the Social Hygiene Service, in Hong Kong, were included. Laboratory tests on CSF included several serological tests, CSF cell count, and protein. Forty-five patients were prospectively recruited, of which 29 people were living with HIV / AIDS. Using diagnostic case definition standard stipulated in the IUSTI 2008 guidelines, 17 patients satisfied the diagnosis of neurosyphilis. The CSF-EIA test provided 100% in both sensitivity and negative predictive value; its specificity was 46.4% (13/28, 95% CI 31.8-61%). Specificity improved to 80.8% (95% CI: 68.4-93.2%) with optical density cut-off value at 1.4 for cases with CSF red cell counts <600/mm(3) This is the first study on use of CSF-EIA in neurosyphilis. CSF-EIA showed high sensitivity and high negative predictive value in the study population and the presence of CSF red cells < 600/mm(3)might not affect its accuracy.