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.

Use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening enzyme immunoassay results for the serological diagnosis of syphilis.

We studied the use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening results of the Murex ICE Syphilis enzyme immunoassay (EIA) with the confirmatory results of both the Serodia Treponema pallidum particle agglutination (TPPA) and the fluorescent treponemal antibody-absorption (FTA-Abs) assays, for the serological diagnosis of syphilis. This was an observational study on the serum samples received by the Syphilis Laboratory, Hong Kong, during the period from January 2006 to December 2012. A total of 801 serum samples with discordant positive screening EIA results were used. Consensus results of such serum samples were derived from results of the EIA, TPPA and FTA-abs assays. The age range of the individuals was 14 to 104 years (median of 52). There were 369 males and 432 females. Of 378 serum samples, 139 showed agreement among positive results, 23 of 310 showed agreement among indeterminate results and 277 of 465 showed agreement among negative results. The proportions of agreement among positive, indeterminate and negative results were 0.37 (95% CI 0.32-0.42), 0.07 (95% CI 0.05-0.11) and 0.60 (95% CI 0.55-0.64), respectively; kappa 0.55 (95% CI 0.49-0.60). There were 60 serum samples with positive consensus results but negative INNO-LIA syphilis score results and 10 with negative consensus results but positive INNO-LIA syphilis score results. Although the INNO-LIA syphilis score assay can be considered a valid alternative confirmatory test for the serological diagnosis of syphilis, the present study showed that its use in the resolution of discordant positive screening EIA results was moderate. A more extensive characterization of serum samples with discordant reactive screening treponemal test results is necessary.

First proficiency testing of second-line anti-tuberculosis drug susceptibility testing in 12 provinces of China.

OBJECTIVE: To evaluate the performance of drug susceptibility testing (DST) against the main second-line (SL) anti-tuberculosis drugs in tuberculosis (TB) laboratories in China. METHOD: The supranational TB reference laboratory issued 30 Mycobacterium tuberculosis isolates to the participating laboratories. Each participating laboratory performed DST against kanamycin (KM), amikacin (AMK), capreomycin (CPM) and ofloxacin (OFX) using the proportion method in Löwenstein-Jensen medium per World Health Organization recommendations. Reported results were checked and compared with the judicial results. RESULT: The main performance indicators for the four anti-tuberculosis drugs evaluated (KM, AMK, CPM, OFX) were as follows: accordance rates: 91.62%, 99.16%, 96.93% and 96.37%; reproducibility: 99.16%, 99.16%, 94.96% and 94.12%; specificity: 99.12%, 99.64%, 98.00% and 98.41%; sensitivity: 78.03%, 97.62%, 94.44% and 91.51%. The accordance rates and sensitivity values of the four drugs showed statistically significant differences, while specificities showed no significant differences. CONCLUSION: Eight (66.7%) participating laboratories met the set requirement criteria; however, DST in four (33.3%) laboratories requires greater attention. Of the four drugs tested, the results for KM were lower than those for the other drugs. External quality assessment can lead to effective evaluation of laboratory performance in SL DST.

Multicentre evaluation of Ziehl-Neelsen and light-emitting diode fluorescence microscopy in China.

OBJECTIVE: To assess the feasibility of using light-emitting diode fluorescence microscopy (LED-FM) in peripheral laboratories in China. DESIGN: The performance of LED-FM and Ziehl-Neelsen (ZN) microscopy was compared on slides directly prepared from the sputum of tuberculosis (TB) suspects and follow-up patients on treatment. The examination time, fading of fluorescence-stained slides, average unit cost and qualitative user appraisal of LED-FM were also analysed. RESULTS: Among 11 276 slides, the smear-positive rate for LED-FM was 11.2% (1263/11 276), 2.6% (294/11 276) higher than that of ZN (8.6%, 969/11 276; χ(2) 263.5, P < 0.05). The examination time for LED-FM (120.0 ± 38.9 seconds) was shorter than that for ZN (206.3 ± 75.9 s; t = 28.12, P < 0.05). For smear fading, quantitative and qualitative errors occurred within respectively 7.8 and 7.7 weeks. The average unit costs for ZN and LED-FM were respectively US$2.20 ± 0.58 and US$1.97 ± 0.71 (t = 5.08, P < 0.05). LED-FM was accepted by most laboratory technicians. CONCLUSION: LED-FM compared favourably with ZN, with a higher smear-positive detection rate, a shorter examination time and lower unit examination cost. LED-FM may be an alternative to ZN as a cost-effective method for detecting bacilli in peripheral laboratories in China.

A pilot external quality assurance programme for line-probe assay detection of anti-tuberculosis drug resistance.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB; resistance to isoniazid and rifampicin) is difficult to detect and control. Line-probe assays (LiPA) are widely used for the rapid detection of MDR-TB. OBJECTIVE: To ensure the quality of the test, a pilot external quality assurance (EQA) programme was initiated to assess the feasibility of running such a programme and the possibility of improving the proficiency of TB laboratories in performing the test. DESIGN: Prepared filter-paper-based Mycobacterium tuberculosis DNA samples were shipped to participant laboratories for LiPA EQA. The tests were performed blind, and the results were returned to the organising laboratory for comparison and analysis. RESULTS: A total of four rounds of EQA samples were dispatched to five laboratories in four countries. Overall inter- and intra-laboratory reproducibility was respectively 97% and 96%. The strengths and weaknesses of the participant laboratories in performing the test were discussed. CONCLUSION: A LiPA EQA programme can ensure quality and improve the performance of TB laboratories. This is a critical step during the initial stages at the time of setting up this method of testing.

[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.

Cervical spinal osteosarcoma in an adolescent.

Primary cervical spine osteosarcoma is rare and is considered to have a poor prognosis. We describe an adolescent patient suffering from primary cervical spine osteosarcoma with delayed diagnosis. Nineteen months deficit and symptom-free survival was achieved after an aggressive multi-modality treatment comprising surgery and adjuvant chemoradiotherapy.

Random blinded rechecking of sputum acid-fast bacilli smear using fluorescence microscopy: 8 years' experience.

BACKGROUND: The Hong Kong TB Reference Laboratory is a high volume laboratory examining around 400 sputum acid-fast bacilli smears daily using fluorescence microscopy (FM). OBJECTIVE: To assess the effectiveness of blinded rechecking applied to FM in a high-throughput laboratory. METHOD: From 2003, 2.5% (5% in 2003 and 2004) of all smears were randomly selected, relabelled and assigned to each technician (rechecker) in turn. These smears were restained and re-examined. Discordance between initial screener and rechecker was resolved by a controller. RESULTS: From 2003 to 2010, low false-negative (LFN) errors (0.10-0.27%) were within the critical values, at 85% (1 year) and 90% (7 years) sensitivity. However, LFN error (0.28-0.62%) among recheckers was prominent. There were also low false-positive (LFP) cases (0.13-0.75%), but subsequent cultures showed these to be mycobacteria culture-positive. This relatively poor performance among the recheckers might be due to background fluorescence increase after restaining and/or inefficiency of the rechecking procedure. CONCLUSION: In a high-throughput laboratory, blind rechecking is a good means of quality assurance. To minimise false LFP, problems due to restaining should be resolved before blinded rechecking can be generally applied in the field for FM where mycobacterial cultures are not routinely performed.

Rapid molecular identification of mycobacterial species in positive culture isolates using the biochip test.

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

Methodological issues in quantifying the magnitude of the tuberculosis problem in a prison population.

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

Role of whole-blood interferon-gamma release assay in the diagnosis of smear-negative tuberculosis.

SETTING: Hong Kong Chest Clinics. OBJECTIVES AND METHODS: To conduct a prospective study investigating the role of a whole-blood interferon-gamma release assay, QuantiFERON®-TB Gold In-Tube (QFT-GIT), in the diagnosis of smear-negative tuberculosis (TB). The QFT-GIT result was compared with the final confirmed diagnosis after 12 months. RESULTS: Of 262 smear-negative subjects, 188 had active TB, 167 (88.8%) of whom were QFT-GIT-positive; 74 had inactive/non-TB, 30 (40.5%) of whom were QFT-GIT-negative. The positive (PPV) and negative predictive values for active TB were respectively 79.1% and 58.8%. For this target group with high TB prevalence (71.8%), a positive test increased the chance of active disease by only 7.3%. Despite a positive likelihood ratio (LR) of 1.49, the negative LR was 0.28, making the diagnosis of active TB much less likely after a negative test. Although sensitivity and specificity showed no difference across different age groups, the PPV decreased (P < 0.001) with increasing age, likely reflecting the increased prevalence of competing diagnoses. CONCLUSION: In an area with a high prevalence of latent TB infection, a positive QFT-GIT test does not add much to confirm the diagnosis of smear-negative TB, while a negative test indicates a need for further investigation.

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.

Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.

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

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.

Bulk acid-fast staining of sputum smears: time to end a taboo.

SETTING: A high-throughput laboratory routinely performing fluorescence microscopy for acid-fast bacilli (AFB) smear with automated bulk staining. OBJECTIVES: To determine the risk of false-positive AFB sputum smears from bulk staining showing as smear-positive, culture-negative specimens, or a decrease in smear- and culture-positives. DESIGN: Direct AFB smear and Löwenstein-Jensen culture were performed for a total of 39,350 routine sputum specimens. Of these, 6633 were randomly selected for individual AFB staining, while the remaining 32,717 were processed by bulk machine staining. Positives for smear and culture were compared. RESULTS: Overall, 111 specimens yielded a positive individually stained smear; of these, 100 (90.1%, 95%CI 83.0-95.0) were also culture-positive compared to 504/543 smear-positives after bulk staining (92.8%, 95%CI 90.6-95.0). The proportions of smear-positive, culture-negative and smear- and culture-positive specimens were respectively 1.8% vs. 2.2% and 90.1% vs. 92.8%, for individual and bulk staining (non-significant). CONCLUSIONS: The risk of transferring AFB from positive to negative smears during bulk AFB staining is negligible, if it occurs at all. Bulk staining should not be discouraged, as even in low-income countries this method will save significant resources, particularly manpower, and improve staining results in laboratories with a high workload.