Molecular and phenotypic characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to kanamycin, amikacin, and capreomycin in China.

Although second-line anti-tuberculosis (TB) injectable drugs have been widely used to improve treatment outcomes of multidrug-resistant TB (MDR-TB), little is known about the prevalence and mechanism of second-line injectable drug resistance among MDR Mycobacterium tuberculosis isolates in China. Here, we found that 12.7 % (20/158) of isolates showed resistance to at least one second-line injectable drug among 158 MDR isolates. At the same time, there were 16 (10.1 %) strains resistant to kanamycin (KAN), 9 (5.7 %) to amikacin (AMK), and 12 (7.6 %) to capreomycin (CAP). In addition, our data revealed no significant difference in the drug resistance patterns for Beijing versus non-Beijing genotype strains (p > 0.05). The most frequently observed mutation was A-to-G substitution at position 1401 of the rrs gene, conferring high-level resistance to KAN and AMK, but had varying minimum inhibitory concentrations (MICs) for CAP. The mutations in the eis promoter and tlyA gene were responsible for low-level resistance to CAP. 83.3 % of A1401G substitutions in the rrs gene was observed in Beijing genotype strains, while the difference was not significant (p = 0.157). Our data demonstrated that the hot-spot regions localized in the rrs gene serve as excellent markers for AMK, but is not a sensitive marker for KAN and CAP. In addition, the cross-resistance patterns and MICs differed among different genetic mutation types, which challenge the practice in China of generalizing resistance to AMK and CAP based on the resistance to KAN alone. Our findings suggested that the individualized drug susceptibility to three major second-line injectable drugs is essential in order to generate more effective treatment regimens for MDR patients.

[Role of ocular ischaemic syndrome in establishing indications for revasculization of the carotid basin in stenotic lesions of extracranial portions of carotid arteries].

The article is dedicated to the problem concerning indications for revascularization of the carotid basin. The aim was to determine significance of ocular ischaemic syndrome in establishing indications for revascularization of the carotid basin in stenotic lesions of extracranial portions of carotid arteries.

Usefulness of resistant gene markers for predicting treatment outcome on second-line anti-tuberculosis drugs.

AIM: Mutations in rrs [nucleotide (nt) 1401], gyrA gene (codons 90, 91 or 94), tlyA, ethA and thyA genes of Mycobacterium tuberculosis (MTB) were evaluated for their usefulness in predicting treatment outcome of kanamycin (KM), capreomycin (CPM), ofloxacin (OFX), ethionamide (ETH) and para-aminosalicylic acid (PAS). METHODS AND RESULTS: DNA sequence analyses of these genes were performed against 188 MTB isolates obtained from patients put on second-line anti-TB drugs (SLDs) with well-documented clinical history and treatment outcome. Mutations in rrs and gyrA have 100% positive predictive value (PPV) in predicting treatment failure for KM and OFX, while 88·9 and 80% were obtained, respectively, when tlyA and rrs mutations were considered in CPM. For ETH and PAS, the PPV of using ethA and thyA mutations to predict treatment failure was 82·5 and 89·3%, respectively. CONCLUSIONS: Our study demonstrated high specificities of gene mutations in predicting poor treatment outcome; however, further technical advancement is required to make the molecular detection of resistances to other SLDs feasible in clinical laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to correlate different polymorphisms of major SLD resistance gene markers with predicted treatment outcome, using an international set of well-documented clinical MTB strains.

Comparative evaluation of the INNO-LIA syphilis score and the MarDx Treponema pallidum immunoglobulin G Marblot test assays for the serological diagnosis of syphilis.

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.

Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results.

The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 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 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. 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.

Serogroup, virulence, and genetic traits of Vibrio parahaemolyticus in the estuarine ecosystem of Bangladesh.

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., "clonal cluster," as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.

Variations in quality of carbol fuchsin stains collected from routine tuberculosis laboratories.

SETTING: In-use carbol fuchsin stains were collected from 10 different routine acid-fast bacilli smear microscopy laboratories. OBJECTIVE: To examine the variations in the composition of carbol fuchsin stains. METHOD: Carbol fuchsin concentrations were first determined spectrophotometrically by measuring absorbance at 547 nm. High-performance liquid chromatography (HPLC) separated and quantified the four basic fuchsin homologues: para-rosaniline, rosaniline, magenta II and new fuchsin, and identity was confirmed by mass spectrometry (MS). RESULTS: Absorbance measurement showed that three of 10 (30%) samples contained insufficient carbol fuchsin (<70%). Wide variations in relative proportions of fuchsin homologues were found. CONCLUSION: The relative abundance of rosaniline + new fuchsin was quite stable among the different laboratories. Spectrophotometry and HPLC/MS are necessary and sensitive tools for monitoring fuchsin quality.

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory.

AIMS: To facilitate efficient identification of commonly encountered mycobacteria species (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum complex, Mycobacterium chelonae/abscessus, Mycobacterium kansasii, Mycobacterium gordonae) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated. METHODS AND RESULTS: Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40 M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88.9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1.1 with an average turn around time of less than 3 days. CONCLUSIONS: A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting. SIGNIFICANCE AND IMPACT OF THE STUDY: With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.

TB surveillance in correctional institutions in Hong Kong, 1999-2005.

OBJECTIVE: To understand the epidemiology of tuberculosis (TB) inside the prison system of Hong Kong. METHOD: Prospective territory-wide TB surveillance was conducted among prisoners in 24 correctional institutions. RESULTS: From 1999 to 2005, 622 prevalent TB cases diagnosed before or within 3 months of incarceration and 214 incident cases diagnosed after 3 months were reported by prison staff to a paper-based central prison TB registry. Both crude prevalence and incidence were falling (chi(2) for trend, both P < 0.001), despite a higher sex- and age-adjusted prison TB incidence as compared to the general population (indirectly standardised rate [ISR] 280.6 vs. 108.0/100000, P < 0.001). Illegal immigrants (odds ratio [OR] 3.6, 95% confidence interval [CI] 1.8-7.4) and drug addicts (OR 2.04, 95%CI 1.13-3.7) were two major risk groups. The TB incident risk disappeared after their exclusion (ISR 117.1 vs. 108.0/100000, P = 0.52). No significant difference in the multidrug-resistant rate was found when comparing the group with the general population (3.5% vs. 1.0%, OR 3.6, 95%CI 0.5-28.4). No extensively drug-resistant (XDR) cases were identified. CONCLUSION: TB remains a significant disease in local prisons. Further strengthening of TB control programmes in prisons, especially targeting the higher risk groups, is recommended.

Risk factors for multidrug-resistant tuberculosis in Hong Kong.

SETTING: Previous anti-tuberculosis treatment is a widely reported risk factor for multidrug-resistant tuberculosis (MDR-TB), whereas other risk factors are less well described. In Hong Kong, the clinical characteristics of MDR-TB have not been systematically evaluated. OBJECTIVE: To explore the risk factors for MDR-TB in Hong Kong. DESIGN: MDR-TB patients treated between 1999 and 2003 were compared with non-MDR-TB patients by stratification of previous anti-tuberculosis treatment. RESULTS: One hundred and fifty-six MDR-TB patients, including 93 with and 63 without a previous history of anti-tuberculosis treatment, were identified. Among the 322 non-MDR-TB controls, respectively 192 and 130 patients did and did not have a previous history of anti-tuberculosis treatment. Using logistic regression analysis, non-permanent residents (OR 6.85, 95%CI 1.38-34.09), frequent travel (OR 2.48, 95%CI 1.07-5.74) and younger age were found to be independent predictors of MDR-TB in previously treated patients, whereas living on financial assistance just failed to reach statistical significance (OR 2.75, 95%CI 0.98-7.68, P=0.05). In the treatment-naïve group, despite significant differences in baseline characteristics among MDR-TB and non-MDR-TB patients, no independent predictor variables could be identified. CONCLUSION: In Hong Kong, non-permanent residents, frequent travel and young age were independent predictors of MDR-TB among previously treated patients.

Early and late tuberculosis risks among close contacts in Hong Kong.

SETTING: Tuberculosis (TB) notification is a statutory requirement in Hong Kong, where contact investigations are performed by the Tuberculosis and Chest Service. OBJECTIVES: 1) To evaluate the risk of active TB in close contacts within 5 years, and 2) to identify risk factors associated with early and late development of active TB disease. DESIGN: The characteristics of consecutive TB cases notified from 18 January to 17 April 2000 were collected together with those of their contacts. Contacts were prospectively followed up through the territory-wide TB notification registry for 5 years for the development of disease. RESULTS: A total of 1537 index cases and 4661 close contacts were analysed. Screening found 31 (0.67%) active TB cases within a 3-month period, and another 58 (1.24%) cases presented subsequently. Index cases with cough or pulmonary cavities and diabetic contacts were independent risk factors of early cases (all P<0.05). Adjusted at risk index characteristics for late TB development included positive sputum smear (2.79, 95%CI 1.31-5.95) and family history of TB (4.26, 95%CI 2.01-9.03). Contact risk factors included diabetes mellitus (3.44, 95%CI 1.04-11.33) and institutionalisation (3.61, 95%CI 1.70-7.65). CONCLUSION: Considerable TB risk remains after initial contact screening. A number of possible risk factors were identified.

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler.

AIMS: To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus. METHODS AND RESULTS: Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates (n = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10(7)-10(1) CFU ml(-1) and that for tdh2 was 10(7)-10(4) CFU ml(-1). CONCLUSIONS: The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed allows accurate detection of pathogenic V. parahaemolyticus, which is valuable for rapid tracing of infection source during outbreaks.

Prevalence and determinants of positive tuberculin reactions of residents in old age homes in Hong Kong.

OBJECTIVE: To determine the prevalence of tuberculous infection and the predictors of positive tuberculin reactivity in old age home residents in Hong Kong. DESIGN: Cross-sectional study. METHODS: A questionnaire-interview and review of medical records were carried out, together with measurement of weight and height/arm span and assessment of nutritional status. A one-stage tuberculin skin test (TST) was performed using two units of PPD RT23. An induration > or = 10 mm was considered as positive. RESULTS: Of 3682 residents (71.7% participation, mean age 82 years) who agreed to undergo a TST, 46.3% had a positive reaction. Factors associated with a significantly higher risk of a positive TST included being male, an ex- or current smoker and having a past history of tuberculosis (TB). Factors associated with reduced positive tuberculin reactivity included older age groups (> 70 years), a history of cancer and chronic obstructive pulmonary disease and low body mass index quartiles. CONCLUSION: In old age homes, the high prevalence of latent tuberculous infection is responsible for the high rate of active TB due to reactivation. Early diagnosis and treatment are necessary to prevent transmission of disease in these crowded environments with susceptible individuals.

Denaturing HPLC for high-throughput screening of rifampicin-resistant Mycobacterium tuberculosis isolates.

OBJECTIVE: To evaluate the use of denaturation high-performance liquid chromatography (dHPLC) as a rapid method to detect rifampicin (RMP) resistance based on mutations in the rpoB gene in a high-volume laboratory setting. METHODS: A total of 132 RMP-resistant Mycobacterium tuberculosis strains with different rpoB mutation were used to optimise the running condition of dHPLC as a pilot study. A blind correlation study was subsequently done between dHPLC and in vitro RMP susceptibility tests on 3167 M. tuberculosis strains in a high-throughput clinical setting. RESULTS: In the pilot study, rpoB mutation could be detected on 116/132 (87.9%) RMP-resistant strains by dHPLC. In the second phase of the study, 84/3107 (2.7%) clinical M. tuberculosis isolates were RMP-resistant. The sensitivity and specificity of dHPLC in the prediction of RMP resistance were 70/84 (83.3%) and 70/77 (91.0%), respectively. The specificity became 100% when 511 Leu to Pro mutation was excluded from the RMP resistance-related genetic changes. CONCLUSION: In the detection of RMP resistance in a high-throughput laboratory setting, dHPLC has been demonstrated to be rapid, simple, workable, automatable and inexpensive in terms of running costs and the labour involved.

Socio-demographic and geographic indicators and distribution of tuberculosis in Hong Kong: a spatial analysis.

OBJECTIVE: To determine the socio-demographic and geographic indicators responsible for the distribution and transmission of tuberculosis (TB) in Hong Kong using geographical information system (GIS) technology. MATERIALS AND METHODS: All patients with bacteriologically proven TB over a period of 3 years (May 1999-April 2002) residing within Hong Kong Island were studied. Molecular characterisation of their sputum isolates by IS6110-based restriction fragment length polymorphism (RFLP) technique was performed. Socio-demographic data were derived from the 2001 Hong Kong population census. Geographic coordinates of patients' addresses were linked to the GIS; large street block groups (LSBGs) were the units of analysis. RESULTS: Of 2387 patients with bacteriologically confirmed TB, 2332 had valid addresses distributed in 430 LSBGs in Hong Kong Island. Of the five socio-demographic indicators studied, significant correlations were found between the rate of TB in an LSBG and low educational attainment, elderly population and low-income household, but not population density or unemployment. The five socio-demographic indicators were not different between LSBG with clustered cases and those with unique cases. CONCLUSION: Low educational attainment, old age and poverty were significant determinants of the rate of TB in different parts of Hong Kong, while none of the socio-demographic indicators was related to disease transmission.

Chest radiograph screening for tuberculosis in a Hong Kong prison.

SETTING: Long-stay prisoners are not regularly screened for TB in Hong Kong. OBJECTIVE: To evaluate tuberculosis (TB) screening in prison. METHOD: All prisoners in a maximum security prison as of 31 October 2001 were screened by chest radiograph (CXR), except for those being followed up for TB or examined by CXR in the last 6 months. RESULTS: A total of 814 male prisoners aged 34.6 +/- 9.6 (mean +/- SD) years were successfully screened. Of 53 cases (6.51%) with radiographic abnormalities, 10 active TB cases (8 culture-negative, 2 culture-positive) were diagnosed, giving an overall yield of 1.23% (95%CI 0.59-2.26). There was no statistical difference in age, ethnicity, place of birth or residency status between those with and those without TB (all P > 0.05). Incarceration > or = 2 years, being in current prison > or = 2 years and not having CXR in last 2 years were associated with TB in univariate analysis (all P < 0.05), but only the last remained an independent predictor in multiple logistic regression (OR 16.8, 95%CI 2.1-132.9, P = 0.008). In that group, the yield was 3.1% (95%CI 1.42-5.89). No further cases were detected in the subsequent 2 years. CONCLUSION: CXR screening of long-stay prisoners gave a high yield in this study.

IS6110 dot blot hybridization for the identification of Mycobacterium tuberculosis complex.

To explore a simple, rapid, and inexpensive way to identify Mycobacterium tuberculosis complex culture, dot blot hybridization using IS6110 as the marker was performed against 2788 known clinical isolates of mycobacteria including M. tuberculosis (n = 721), M. kansasii (177), M. marinum (10), M. avium complex (700), M. terrae complex (203), M. fortuitum (476), M. chelonae (439), and other nonpigmented Runyon's Group IV mycobacteria (62). We found that the sensitivity and specificity of the test were 94.3% and 100%, respectively. When this method was evaluated in a laboratory blind study of 1253 initially unknown clinical isolates, its sensitivity and specificity were 91.2% and 100%, respectively. Because this identification test is technically simple, rapid, and can be done in batches, together with its high sensitivity and specificity, it is a cost-effective method for routine identification of M. tuberculosis complex in laboratories of areas with a high incidence of tuberculosis.