Rise in rifampicin-monoresistant tuberculosis in Western Cape, South Africa.

SETTING: Brewelskloof Hospital, Western Cape, South Africa. OBJECTIVES: To verify the perceived increase in rifampicin monoresistant tuberculosis (RMR-TB) in the Cape Winelands-Overberg region and to identify potential risk factors. DESIGN: A retrospective descriptive study of trends in RMR-TB over a 5-year period (2004-2008), followed by a case-control study of RMR and isoniazid (INH) monoresistant TB cases, diagnosed from April 2007 to March 2009, to assess for risk factors. RESULTS: The total number of RMR-TB cases more than tripled, from 31 in 2004 to 98 in 2008. The calculated doubling time was 1.63 years (95%CI 1.18-2.66). For the assessment of risk factors, 95 RMR-TB cases were objectively verified on genotypic and phenotypic analysis. Of 108 specimens genotypically identified as RMR cases, 13 (12%) were misidentified, multidrug-resistant TB. On multivariate analysis, previous use of antiretroviral therapy (OR 6.4, 95%CI 1.3-31.8), alcohol use (OR 4.8, 95%CI 2.0-11.3) and age ≥ 40 years (OR 5.8, 95%CI 2.4-13.6) were significantly associated with RMR-TB. CONCLUSION: RMR-TB is rapidly increasing in the study setting, particularly among patients with advanced human immunodeficiency virus (HIV) disease. Routine drug susceptibility testing should be considered in all TB-HIV co-infected patients, and absence of INH resistance should be confirmed phenotypically if genotypic RMR-TB is detected.

Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa.

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C(T)) variability of three DCS batches was ≤ 3.47. The study of longer-term DCS stability is ongoing.

inhA promoter mutations: a gateway to extensively drug-resistant tuberculosis in South Africa?

SETTING: Western Cape and Eastern Cape Provinces, South Africa. OBJECTIVE: To assess the potential association between the evolution of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis and mutations in the inhA promoter or the katG gene. DESIGN: Analysis of the frequency distribution of isoniazid (INH) resistance conferring mutations in a population sample of drug-resistant isolates of M. tuberculosis. RESULTS: In the Western Cape and Eastern Cape Provinces, the percentage of isolates exhibiting inhA promoter mutations increased significantly from respectively 48.4% and 62.4% in multidrug-resistant tuberculosis (MDR-TB) isolates to 85.5% and 91.9% in XDR isolates. Data from the Western Cape revealed that significantly more XDR-TB isolates showed mutations in the inhA promoter than in katG (85.5% vs. 60.9%, P < 0.01), while the respective proportions were equal for INH-resistant non-MDR-TB isolates (∼30%). CONCLUSIONS: inhA promoter mutations are strongly associated with XDR-TB in South Africa. We suggest that this is due to the dual resistance to ethionamide and (low-dose) INH conferred by inhA promoter mutations. The use of molecular probe assays such as the GenoType® MTBDRplus assay, which allows the detection of inhA promoter mutations, could enable treatment regimens to be adjusted depending on the pharmacogenetic properties of the mutations detected.

Drug-resistant tuberculosis epidemic in the Western Cape driven by a virulent Beijing genotype strain.

Temporal analysis of drug-resistant tuberculosis (TB) cases in the Western Cape, South Africa, showed a 1.5-fold increase over a 2-year period, suggesting a doubling time of 8.2 years. This increase was strongly associated with multidrug resistance and the Beijing genotype. Forty-two per cent of the overall increase was due to the Beijing genotype strain R220, suggesting that this strain had evolved unique properties that allowed for both acquisition and transmission of drug resistance. To curb the drug-resistant TB epidemic in this setting, it will be essential to implement rapid diagnostics and efficient infection control measures, improve contact screening and ensure treatment adherence.

Validation of a Rapid Tuberculosis PCR Assay for Detection of MDR-TB Patients in Gauteng; South Africa

Nucleic acid amplification tests offer shorter turnaround times for diagnosis of tuberculosis (TB) and drug resistance of isolates compared to conventional culture methods. The rapid molecular-based multidrug-resistant (MDR)-TB assay; GenoTyper MTBDRplus (Hain Lifescience) was evaluated in Gauteng; South Africa; as a pilot investigation to assess its performance for detection of MDR-TB in patients who were at high risk of drug-resistant TB. A total of 945 sputum specimens sequentially received within a period of six weeks from seven hospitals were assessed by MTBDRplus and compared to liquid culture drug susceptibility tests (DST) using the MGIT 960 system (BD Diagnostic Systems) as the `gold standard'. Of the 945 specimens processed; 731 had interpretable results from both tests and therefore were included in the analysis. The overall sensitivities of the MTBDRplus in detecting individual resistance to rifampicin (RMP) and isoniazid (INH); as well as MDR were 95.0; 93.4and 100; respectively. The specificities were 99.7for RMP; and 100for INH and MDR. The Genotyper MTBDRplus assay showed excellent concordance with the conventional `gold standard' MGIT DST; and it detected all the MDR-TB cases analysed

Eliminating protein from reusable laryngeal mask airways. A study comparing routinely cleaned masks with three alternative cleaning methods.

Laryngeal mask airways (LMAs) have the potential to act as a vector for the transmission of prion diseases. This study was undertaken to define the problem of protein contamination and to investigate three alternative cleaning methods. Forty-eight LMAs were allocated to one of four groups, stained with erythrosin and given a total stain score and a grid stain score in order to determine the degree of protein contamination. Eighteen randomly selected LMAs that had been routinely cleaned and sterilised (group 1) were compared with 12 LMAs that had been washed and scrubbed with the benefit of prior staining (group 2), 13 that had been washed and scrubbed without any visual guide (group 3) and 13 that had been cleaned using a Biosonic ultrasonic cleaning system (group 4). The results show that none of the cleaning methods achieved optimal results, as all methods left proteinaceous material on some masks. The study showed that: (i) staining as a guide to cleaning does not lead to more effective removal of proteinaceous material; (ii) systematic cleaning and scrubbing does lead to more effective removal of proteinaceous material from surfaces other than the grid area; and (iii) ultrasonic cleaning was more effective than other methods of cleaning for the removal of proteinaceous material from those areas of the mask most inaccessible, such as the grid.

The 5-year outcome of multidrug resistant tuberculosis patients in the Cape Province of South Africa.

Little is known about the outcome of multidrug resistant (MDR) tuberculosis (TB) in developing countries. In this study, 443 patients with MDR-TB, defined as resistance to two or more antituberculosis drugs, were identified over the 2-year period 1987 and 1988 in the Cape Province of South Africa. The 5-year outcome of the 343 (77%) patients that could be traced by questionnaire was evaluated retrospectively during 1992 and 1993. Of these, 240 (70%) were resistant to both isoniazid (H) and rifampicin (R) with or without resistance to other first-line antituberculosis drugs and 103 (30%) were resistant to H or R and/or other antituberculosis drugs. Mortality was 116 (48%) and 28 (27%) in these groups respectively with a significantly greater risk of death in the first group. Only 114 (33%) of all the MDR-TB patients were cured after 5 years, 50 (15%) were respiratory disabled and 44 (13%) were still bacteriology positive. Twenty-four (7%) patients were lost during follow-up. Taking into account the high costs involved in treating MDR-TB patients and the scarce resources available in developing countries, more emphasis should be placed on direct observed therapy to cure newly diagnosed infectious drug sensitive tuberculosis patients, thus preventing MDR-TB rather than treating it.

Strain-specific variation in the dnaJ gene of mycobacteria.

The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was evaluated for species identification among mycobacteria by analysis of the dnaJ gene. Nine clinical isolates of Mycobacterium tuberculosis with different fingerprint patterns all gave the same distinct SSCP banding pattern and could be distinguished from other mycobacteria, such as M. avium. In contrast, considerable strain-specific dnaJ gene variations were observed amongst 42 clinical isolates of M. avium and 13 other atypical mycobacterial strains. Only 62% of the M. avium isolates hybridised to an M. avium-specific probe and only 14% could be identified correctly as M. avium by both probe and restriction fragment length polymorphism analysis. This finding was supported by direct sequence analysis. Variations were also observed in M. gordonae and M. scrofulaceum isolates. Computerised analysis of M. avium samples broadly identified three clusters. Results suggest that although the SSCP procedure may be useful for distinguishing M. tuberculosis from other mycobacteria, this technique applied to the dnaJ gene may not be suitable for strain identification. The results stress the importance of testing a large collection of clinical isolates before new molecular procedures are introduced into routine laboratories.

Characterization of strains of Neisseria meningitidis recovered from complement-sufficient and complement-deficient patients in the Western Cape Province, South Africa.

Complement deficiency has been associated with increased susceptibility to meningococcal disease. In order to determine whether special meningococcal strains caused disease in complement-deficient (CD) patients, 17 Neisseria meningitidis strains recovered from patients in the Western Cape Province, South Africa, known to be CD were compared with 124 routine isolates obtained from patients living in the same area. Serogrouping of the strains from the CD subjects revealed that the common serogroups, particularly serogroup B, predominated. However, the prevalence of rare serogroups among isolates from CD subjects was significantly higher than that found among isolates from the control group. Sero- and subtyping of the class 1 and class 2 or 3 outer membrane proteins showed no significant difference between isolates from CD subjects and the routine clinical isolates. Multilocus enzyme electrophoresis of the 141 isolates revealed six clusters of electrophoretic types (ETs) and two unrelated ETs. The same degree of genetic diversity existed in ETs of isolates from CD subjects and the control group. However, the ET-5 complex, which is known to be associated with epidemic disease, was found in 22 (18%) of the routine clinical isolates but in none of the isolates from the CD subjects. This difference was marginally significant. What was highly significant was the finding that 8 of the 17 isolates from CD subjects were in one ET cluster, cluster F, which comprised a total of 20 isolates. Thus, our results show a difference in the clonal compositions of the strains that infect CD subjects in comparison with the clonal compositions of those that cause clinical infections in the population at large.