Prevalence of Extensively Drug Resistant Tuberculosis among Archived Multidrug Resistant Tuberculosis Isolates in Zimbabwe

We conducted a cross-sectional study of second line drug resistance patterns and genetic diversity of MDR-TB isolates archived at the BRTI-TB Laboratory, Harare, between January 2007 and December 2011. DSTs were performed for second line antituberculosis drugs. XDR-TB strains were defined as MDR-TB strains with resistance to either kanamycin and ofloxacin or capreomycin and ofloxacin. Strain types were identified by spoligotyping. No resistance to any second line drugs was shown in 73% of the isolates, with 23% resistant to one or two drugs but not meeting the definition of XDR-TB. A total of 26 shared types were identified, and 18 (69%) matched preexisting shared types in the current published spoligotype databases. Of the 11 out of 18 clustered SITs, 4 predominant (>6 isolates per shared type) were identified. The most and least abundant types were SIT 1468 (LAM 11-ZWE) with 12 (18%) isolates and SIT 53 (T1) with 6 (9%) isolates, respectively. XDR-TB strains are rare in Zimbabwe, but the high proportion of "pre-XDR-TB" strains and treatment failure cases is of concern. The genetic diversity of the MDR-TB strains showed no significant association between SITs and drug resistance

Clinic-based SAMBA-II vs centralized laboratory viral load assays among HIV-1 infected children, adolescents and young adults in rural Zimbabwe: A randomized controlled trial.

BACKGROUND In Zimbabwe, children, adolescents and young adults living with HIV (CALWH) who are on public health antiretroviral therapy (ART) have inadequate viral load (VL) suppression. We assessed whether a clinic-based VL monitoring could decrease 12-month virologic failure rates among these CALWH. METHODS The study was registered on ClinicalTrials.gov: NCT03986099. CALWH in care at Chidamoyo Christian Hospital (CCH) and 8 rural outreach sites (ROS) on long-term community-based ART were randomized (1:1) to 6 monthly VL monitoring by COBAS®Ampliprep®/Taqman48® HIV-1 at the provincial referral laboratory (PRL) as per standard of care (SOC) or by the clinic-based SAMBA II assay, Diagnostics for the Real World, at CCH. VL suppression, turn-around-time (TAT) for VL results, drug switching and drug resistance in second-line failure were assessed at 12 months. RESULTS Of 390 CALWH enrolled 347 (89%) completed 12 months follow-up. Median (IQR) age and ART duration were 14.1 (9.7-18.2) and 6.4 (3.7-7.9) years, respectively. Over half (57%) of the participants were female. At enrolment, 78 (20%) had VL ≥1,000 copies/ml and VL suppression of 80% was unchanged after 12 months, with no significant difference between the SOC (81%) and the clinic-based (80%) arms (p = 0.528). Median (IQR) months to confirmatory VL result at CCH vs PRL was 4.0 (2.1-4.4) vs 4.5 (3.5-6.3) respectively; p = 0.027 at 12 months. Drug switching was documented among 26/347 (7%) participants with no difference between the median (IQR) time to switch in SOC vs clinic-based arms (5.1 (3.9-10.0) months vs 4.4 (2.5-8.4) respectively; p = 0.569). Out of 24 confirmed second-line failures, only 4/19 (21%) had protease inhibitor resistance. CONCLUSION In rural Zimbabwe, the clinic-based SAMBA II assay was able to provide confirmatory VL results faster than the SOC VL assay at the PRL. However, this rapid TAT did not allow for a more efficient drug switch among these CALWH

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry

A proof of principle gas chromatography–mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36–68% for PDIMs, allowing some superior detection of the target analytes

Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis

Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test

Study of methylation and ethylation by thermochemolysis of tuberculostearic acid from Mycobacterium tuberculosis culture and sputum

The direct analysis of TBSA was performed by thermally assisted hydrolysis and alkylation in M.tb culture dilutions and in sputum deposits, using tetramethyl ammonium hydroxide (TMAH) and tetraethylammonium hydroxide (TEAH) as derivatizing agents. Thermochemolysis of fatty acids was done in a programmable temperature PTV inlet Optic3, combined with an automatic LINEX liner exchanger installed on an Agilent GC-MS system. The GC-MS response is discussed and the results are compared for the two alkylating reagents used

A study of online and offline derivatization methods for tuberculostearic acid - a biomarker for M. Tuberculosis

Mycobacteria such as M. tuberculosis have the ability of producing lipid components specific to their cell wall. The detection of these lipids can serve to diagnose tuberculosis, a contagious disease that still affects millions of people worldwide. Microscopic examination of sputum can give a quick diagnosis of pulmonary M. tuberculosis but it lacks sensitivity, while culturing may need up to 8 weeks for a definitive result. 10-R-Methyloctadecanoic acid or TBSA, a saturated methyl branched fatty acid, is one of the lipids found in the cell wall of mycobacteria and of certain related bacterial species in the order of Actinomycetales. In M. tuberculosis, TBSA may represent up to 25% of the total fatty acid content and has been identified in the sputum of TB infected patients, by using gas chromatography-mass spectrometry GC-MS. Complex sample extraction and preparation procedures are usually necessary prior to separating the derivatized analytes on the GC column. In the present work, aliquots of pooled positive and pooled negative sputum samples were analyzed by: (i) direct thermochemolysis of sputum deposit aliquots; (ii) thermochemolysis of TBSA extracted from sputum deposits, and (iii) the injection in the GC inlet of the boron trifluoride alkylated derivatives obtained from the sputum extracts. The derivatized compounds were separated on a DB-5MS capillary column and detected by a quadrupole mass spectrometer with electron impact ionisation source. The study discusses the implications of the various derivatization methods on the GC-MS response in selected ion monitoring, SIM mode, for the molecular ion of the methylated TBSA. The yield of TBSA extracted from sputum and submitted to TMAH derivatisation is only a fraction of that obtained from the direct thermochemolytic analysis of the vortexed deposit, but the extraction of TBSA also reduces the risk of overloading the GC capillary column, drops the baseline and improves the peaks shape

Study Flow Diagram.

<p><i>Definition of abbreviation</i>: MDR TB = multidrug resistant tuberculosis; MODS = microscopic-observation drug-susceptibility. ^†Six additional previously cultured isolates of known MDR status were included for analysis of drug susceptibility testing only and are not included here.</p

Drug-Susceptibility Test Results from the MODS Assay.

*<p>Analysis limited to samples with positive microscopic-observation drug-susceptibility and reference standard culture.</p>†<p>Among directly inoculated patient specimens.</p