Andries W. Dreyer

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.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women

BackgroundGenital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women.MethodsSelf-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum.ResultsSeventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance.ConclusionsTreatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined.

Comparison of the VersaTREK blood culture system against the Bactec9240 system in patients with suspected bloodstream infections

BackgroundTo evaluate the VersaTREK (TREK Diagnostic Systems, Cleveland, Ohio) blood culture system against the Bactec9240 (BD Microbiology, Cockeysville, MD), for the recovery of bloodstream pathogens.MethodsVenous blood from patients with suspected bacterial sepsis was evenly distributed into bottles of each system. Positive signals were recorded and bottles processed onto standard media for organism recovery. False positive signals were regarded if no organisms were seen on Gram stain and no growth was observed.Results177 bottles were available for analysis; the Bactec9240 system yielded 43 positive, 134 negative results and no false positive signals. The VersaTREK system had 58 positive signals with 14 being false positives.ConclusionsIn our setting with high background burden of immuno-compromised patients, the VersaTREK system compared favourably with the Bactec9240 in recovering blood stream aerobic and facultative anaerobic pathogens from patients with suspected bacterial sepsis. A concern is the high false positivity rate. Due to its versatility to accommodate small and large workloads as well as using smaller volumes of blood, this system may establish itself as a useful alternative for the recovery of bloodstream pathogens.

Normal flora and bacterial vaginosis in pregnancy: an overview

Abstract The female genital tract is an intricate, yet balanced ecosystem that hosts a variety of different residential microflora. The physiological changes that occur during pregnancy may disrupt this balanced ecosystem and predispose women to a potentially pathogenic microbiota. Bacteria that are associated with bacterial vaginosis (BV) are opportunistic pathogens that frequently form part of this microbiota. The overgrowth of and infections with these bacteria are linked to poor obstetric outcomes and increased transmission of other reproductive tract infections (RTIs). These infections increase womens susceptibility of acquiring HIV, the rates of HIV shedding and the development of acquired immune deficiency syndrome (AIDS) in HIV-infected patients. It is unknown how the plethora of bacterial species associated with BV contributes to the dynamics of this condition. The use of high-throughput methods have led to the in-depth investigation of different BV-related bacterial species and the functional capabilities of these species. However, the pathogenesis of BV is still poorly defined and the role of individual BV-related bacterial species in specific pregnancy complications is unclear and controversial. The majority of BV infections are asymptomatic and successful diagnosis is complicated by the lack of reliable and standardized diagnostic tests.

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study

Summary Background In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. Methods Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. Findings Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87–94) for rpoB (rifampicin resistance), 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39–68) for pncA (pyrazinamide resistance), 85% (77–91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81–92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. Interpretation Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. Funding Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

Prevalence of drug-resistant tuberculosis and imputed burden in South Africa: a national and sub-national cross-sectional survey

BACKGROUND Globally, per-capita, South Africa reports a disproportionately high number of cases of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis. We sought to estimate the prevalence of resistance to tuberculosis drugs in newly diagnosed and retreated patients with tuberculosis provincially and nationally, and compared these with the 2001-02 estimates. METHODS A cross-sectional survey was done between June 15, 2012-June 14, 2014, using population proportionate randomised cluster sampling in the nine provinces in South Africa. 343 clusters were included, ranging between 31 and 48 per province. A patient was eligible for inclusion in the survey if he or she presented as a presumptive case during the intake period at a drug resistance survey enrolling facility. Consenting participants (≥18 years old) completed a questionnaire and had a sputum sample tested for resistance to first-line and second-line drugs. Analysis was by logistic regression with robust SEs, inverse probability weighted against routine data, and estimates were derived using a random effects model. FINDINGS 101 422 participants were tested in 2012-14. Nationally, the prevalence of MDR tuberculosis was 2·1% (95% CI 1·5-2·7) among new tuberculosis cases and 4·6% (3·2-6·0) among retreatment cases. The provincial point prevalence of MDR tuberculosis ranged between 1·6% (95% CI 0·9-2·9) and 5·1% (3·7-7·0). Overall, the prevalence of rifampicin-resistant tuberculosis (4·6%, 95% CI 3·5-5·7) was higher than the prevalence of MDR tuberculosis (2·8%, 2·0-3·6; p=0·01). Comparing the current survey with the previous (2001-02) survey, the overall MDR tuberculosis prevalence was 2·8% versus 2·9% and prevalance of rifampicin-resistant tuberculosis was 3·4% versus 1·8%, respectively. The prevalence of isoniazid mono-resistant tuberculosis was above 5% in all provinces. The prevalence of ethionamide and pyrazinamide resistance among MDR tuberculosis cases was 44·7% (95% CI 25·9-63·6) and 59·1% (49·0-69·1), respectively. The prevalence of XDR tuberculosis was 4·9% (95% CI 1·0-8·8). Nationally, the estimated numbers of cases of rifampicin-resistant tuberculosis, MDR tuberculosis, and isoniazid mono-resistant tuberculosis for 2014 were 13 551, 8249, and 17 970, respectively. INTERPRETATION The overall prevalence of MDR tuberculosis in South Africa in 2012-14 was similar to that in 2001-02; however, prevalence of rifampicin-resistant tuberculosis almost doubled among new cases. Furthermore, the high prevalence of isoniazid mono-resistant tuberculosis, not routinely screened for, and resistance to second-line drugs has implications for empirical management. FUNDING Presidents Emergency Plan for AIDS Relief through the Centers for Disease Control and Prevention under the terms of 1U19GH000571.

Evaluation of the GenoType MTBDRsl Version 2.0 Assay for second-line drug resistance detection of Mycobacterium tuberculosis isolates in South Africa

ABSTRACT Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDRsl version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (gyrA and gyrB genes) and second-line injectable drugs (SLID) (rrs and eis genes). The study evaluated the diagnostic performance of the GenoType MTBDRsl VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on Mycobacterium tuberculosis complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDRsl VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDRsl VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDRsl VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings.

Comparison of the new Mycofast Revolution assay with a molecular assay for the detection of genital mycoplasmas from clinical specimens

BackgroundGenital mycoplasmas are opportunistic bacteria that are associated with undesirable gynaecologic and reproductive events. Mycoplasmas are fastidious bacteria with increasing resistance to routine antimicrobials and often fail to grow on conventional culture methods. The commercial Mycofast Revolution assay permits the phenotypic detection and identification of genital mycoplasmas. Antimicrobial susceptibility testing against five antimicrobial agents with MICs corresponding to the CLSI guidelines can also be performed. This study aimed to compare the new commercially available Mycofast Revolution assay with a multiplex PCR assay.MethodsSelf-collected swabs were obtained from pregnant women attending the antenatal clinic of a tertiary academic hospital in Pretoria, South Africa from October 2012 to November 2012. These swabs were used to seed UMMt and modified Amies transport media. The seeded UMMt transported medium was used to inoculate the Mycofast Revolution assay for the identification, enumeration and antimicrobial susceptibility testing of genital mycoplasmas. Following DNA extraction from the modified Amies transport medium, specimens were subjected to a multiplex PCR assay for the detection of genital mycoplasmas.ResultsThe Mycofast Revolution kit had a sensitivity and specificity of 77.3% (95% CI: 62.15% to 88.51%) and 80% (95% CI: 28.81% to 96.70%), respectively, against the PCR assay. The positive and negative predictive values were 97.1% (95% CI: 85.03% to 99.52%) and 28.6% (95% CI: 8.57% to 58.08%). Genital mycoplasmas were detected in 71.4% (35/49) of samples with the Mycofast Revolution assay with 49% (24/49) being Ureaplasma spp. and 22.4% (11/49) mixed strains. The multiplex PCR assay had a positivity rate of 89.8% (44/49) for genital mycoplasmas; mixed strains were present in 51% (25/49) of samples, Ureaplasma spp. in 16.3% (8/49) and M. hominis in 22.4% (11/49) of samples.ConclusionsThere was a fair agreement (κ = 0.319) between the Mycofast Revolution assay and the mPCR assay. With the high prevalence rates of genital mycoplasmas, fast and efficient diagnostic methods are imperative to treat infections and minimise complications. The Mycofast Revolution assay is simple to use, has a short turn-around time and interpretation of results are straightforward. This assay circumvents common problems experienced with conventional culture and molecular methods in diagnostic laboratories where skilled personnel are limited and can be used as an alternative diagnostic assay.

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

Background In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. Methods A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods. Results Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases. Conclusion We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.