A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in the rpoB, katG, inhA, ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 for rpoB, katG, inhA, ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Assessment of laboratory test utilization for HIV/AIDS care in urban ART clinics of Lilongwe, Malawi

Background The 2011 Malawi HIV guidelines promote CD4 monitoring for pre-ART assessment and considering HIVRNA monitoring for ART response assessment, while some clinics used CD4 for both. We assessed clinical ordering practices as compared to guidelines, and determined whether the samples were successfully and promptly processed. Methods We conducted a retrospective review of all patients seen in from August 2010 through July 2011,, in two urban HIV-care clinics that utilized 6-monthly CD4 monitoring regardless of ART status. We calculated the percentage of patients on whom clinicians ordered CD4 or HIVRNA analysis. For all samples sent, we determined rates of successful labprocessing, and mean time to returned results. Results Of 20581 patients seen, 8029 (39%) had at least one blood draw for CD4 count. Among pre-ART patients, 2668/2844 (93.8%) had CD4 counts performed for eligibility. Of all CD4 samples sent, 8082/9207 (89%) samples were successfully processed. Of those, mean time to processing was 1.6 days (s.d 1.5) but mean time to results being available to clinician was 9.3 days (s.d. 3.7). Regarding HIVRNA, 172 patients of 17737 on ART had a blood draw and only 118/213 (55%) samples were successfully processed. Mean processing time was 39.5 days (s.d. 21.7); mean time to results being available to clinician was 43.1 days (s.d. 25.1). During the one-year evaluated, there were multiple lapses in processing HIVRNA samples for up to 2 months. Conclusions Clinicians underutilize CD4 and HIVRNA as monitoring tools in HIV care. Laboratory processing failures and turnaround times are unacceptably high for viral load analysis. Alternative strategies need to be considered in order to meet laboratory monitoring needs.