Shaheed V. Omar

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study.

Summary Background Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. Methods Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. Findings We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. Interpretation A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early. Funding Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Transmission of Extensively Drug-Resistant Tuberculosis in South Africa.

BACKGROUND Drug‐resistant tuberculosis threatens recent gains in the treatment of tuberculosis and human immunodeficiency virus (HIV) infection worldwide. A widespread epidemic of extensively drug‐resistant (XDR) tuberculosis is occurring in South Africa, where cases have increased substantially since 2002. The factors driving this rapid increase have not been fully elucidated, but such knowledge is needed to guide public health interventions. METHODS We conducted a prospective study involving 404 participants in KwaZulu‐Natal Province, South Africa, with a diagnosis of XDR tuberculosis between 2011 and 2014. Interviews and medical‐record reviews were used to elicit information on the participants’ history of tuberculosis and HIV infection, hospitalizations, and social networks. Mycobacterium tuberculosis isolates underwent insertion sequence (IS)6110 restriction‐fragment–length polymorphism analysis, targeted gene sequencing, and whole‐genome sequencing. We used clinical and genotypic case definitions to calculate the proportion of cases of XDR tuberculosis that were due to inadequate treatment of multidrug‐resistant (MDR) tuberculosis (i.e., acquired resistance) versus those that were due to transmission (i.e., transmitted resistance). We used social‐network analysis to identify community and hospital locations of transmission. RESULTS Of the 404 participants, 311 (77%) had HIV infection; the median CD4+ count was 340 cells per cubic millimeter (interquartile range, 117 to 431). A total of 280 participants (69%) had never received treatment for MDR tuberculosis. Genotypic analysis in 386 participants revealed that 323 (84%) belonged to 1 of 31 clusters. Clusters ranged from 2 to 14 participants, except for 1 large cluster of 212 participants (55%) with a LAM4/KZN strain. Person‐to‐person or hospital‐based epidemiologic links were identified in 123 of 404 participants (30%). CONCLUSIONS The majority of cases of XDR tuberculosis in KwaZulu‐Natal, South Africa, an area with a high tuberculosis burden, were probably due to transmission rather than to inadequate treatment of MDR tuberculosis. These data suggest that control of the epidemic of drug‐resistant tuberculosis requires an increased focus on interrupting transmission. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project

Summary Background Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available. Methods In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system. Findings Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0–42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0–16·6% for ofloxacin, to 0·5–12·4% for levofloxacin, and 0·9–14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries. Interpretation Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19–63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness. Funding Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

ABSTRACT A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Molecular identification and genotyping of MRSA isolates

The aim of this study was to identify and characterize 97 methicillin-resistant Staphylococcus aureus (MRSA) isolates. Two conventional multiplex PCR assays, a real-time PCR assay and two PCR-based genotyping techniques including the spa- and hypervariable region (HVR)-typing methods were used to identify and characterize 97 MRSA strains isolated between April 2006 to September 2007 from the Steve Biko Academic Hospital. All MRSA isolates were positive for 16S rRNA gene, 99% were positive for the mecA gene and 4% positive for the Panton-Valentine leukocidin (PVL) gene. Staphylococcal cassette chromosome mec (SCCmec) typing showed 67% of isolates were SCCmec II [health-care-associated MRSA (HA-MRSA)], 14% were SCCmec III (HA-MRSA) and 4% were SCCmec IVd [community-associated MRSA (CA-MRSA)]. These CA-MRSA isolates showed a prevalence of 100% for the PVL gene. Using spa typing, three distinct clusters could be identified while HVR typing revealed six different clusters. CA-MRSA isolates were clustered together using spa and HVR typing. This study showed the prevalence of the CA-MRSA strains, PVL genes, the SCCmec types and the clonality of the MRSA strains. The high prevalence of the PVL gene in CA-MRSA isolates already residing in intensive care units was alarming and indicated the emergence of new MRSA lineages with a particular fitness for community and hospital transmission.

Multicenter Noninferiority Evaluation of Hain GenoType MTBDRplus Version 2 and Nipro NTM+MDRTB Line Probe Assays for Detection of Rifampin and Isoniazid Resistance

ABSTRACT Less than 30% of multidrug-resistant tuberculosis (MDR-TB) patients are currently diagnosed, due to laboratory constraints. Molecular diagnostics enable rapid and simplified diagnosis. Newer-version line probe assays have not been evaluated against the WHO-endorsed Hain GenoType MTBDRplus (referred to as Hain version 1 [V1]) for the rapid detection of rifampin (RIF) and isoniazid (INH) resistance. A two-phase noninferiority study was conducted in two supranational reference laboratories to allow head-to-head comparisons of two new tests, Hain Genotype MTBDRplus version 2 (referred to as Hain version 2 [V2]) and Nipro NTM+MDRTB detection kit 2 (referred to as Nipro), to Hain V1. In phase 1, the results for 379 test strains were compared to a composite reference standard that used phenotypic drug susceptibility testing (DST) and targeted sequencing. In phase 2, the results for 644 sputum samples were compared to a phenotypic DST reference standard alone. Using a challenging set of strains in phase 1, the values for sensitivity and specificity for Hain V1, Hain V2, and Nipro, respectively, were 90.3%/98.5%, 90.3%/98.5%, and 92.0%/98.5% for RIF resistance detection and 89.1%/99.4%, 89.1%/99.4%, and 89.6%/100.0% for INH resistance detection. Testing of sputa in phase 2 yielded values for sensitivity and specificity of 97.1%/97.1%, 98.2%/97.8%, and 96.5%/97.5% for RIF and 94.4%/96.4%, 95.4%/98.8%, and 94.9%/97.6% for INH. Overall, the rates of indeterminate results were low, but there was a higher rate of indeterminate results with Nipro than with Hain V1 and V2 in samples with low smear grades. Noninferiority of Hain V2 and Nipro to Hain V1 was demonstrated for RIF and INH resistance detection in isolates and sputum specimens. These results serve as evidence for WHO policy recommendations on the use of line probe assays, including the Hain V2 and Nipro assays, for MDR-TB detection.

Rapid antibiotic resistance predictions from genome sequence data for S. aureus and M. tuberculosis.

Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment. Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S. aureus and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.

Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant mycobacterium tuberculosis isolates from the Northern Region of South Africa

ABSTRACT Despite South Africa being one of the high-burden multidrug-resistant tuberculosis (MDR-TB) countries, information regarding the population structure of drug-resistant Mycobacterium tuberculosis strains is limited from many regions of South Africa. This study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates in a high-burden setting of South Africa as well as the possible association of genotypes with drug resistance and demographic characteristics. A total of 336 consecutive MDR-TB isolates from four provinces of South Africa were genotyped using spoligotyping and mycobacterial interspersed repetitive-unit–variable number tandem repeat (MIRU-VNTR) typing. Drug susceptibility testing for ofloxacin, kanamycin, and capreomycin was performed using the agar proportion method. The results showed that 4.8% of MDR-TB isolates were resistant to ofloxacin, 2.7% were resistant to kanamycin, and 4.5% were resistant to capreomycin, while 7.1% were extensively drug resistant (XDR), and the remaining 83.6% were susceptible to all of the second-line drugs tested. Spoligotyping grouped 90.8% of the isolates into 25 clusters, while 9.2% isolates were unclustered. Ninety-one percent of the 336 isolates were assigned to 21 previously described shared types, with the Beijing family being the predominant genotype in the North-West and Limpopo Provinces, while the EAI1_SOM family was the predominant genotype in the Gauteng and Mpumalanga Provinces. No association was found between genotypes and specific drug resistance patterns or demographic information. The high level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggest that the transmission of TB in the study settings is not caused by the clonal spread of a specific M. tuberculosis strain.

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa

BackgroundThe increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs.MethodConsecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method.ResultThe agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively.ConclusionsThe BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+IL2+TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.