N. C. Gey van Pittius

Bio: N. C. Gey van Pittius is an academic researcher from Stellenbosch University. The author has contributed to research in topics: Mycobacterium tuberculosis & Drug resistance. The author has an hindex of 18, co-authored 22 publications receiving 1606 citations. Previous affiliations of N. C. Gey van Pittius include DST Systems & University of Cape Town.

Differentiation of Mycobacterium tuberculosis complex by PCR amplification of genomic regions of difference.

Abstract: Differentiation of members of the Mycobacterium tuberculosis complex by conventional mycobacteriological methods is time consuming, making surveillance of species-specific disease difficult A two-step, multiplex polymerase chain reaction (PCR) method based on genomic regions of difference (RD1, RD1(mic), RD2(seal), RD4, RD9 and RD12) was developed for the differentiation of M canettii, M tuberculosis, M africanum, M microti, M pinnipedii, M caprae, M bovis and M bovis BCG The size of the respective multiplex PCR amplification products corresponded to the presence of the different M tuberculosis complex members This method allows for rapid differentiation, making it suitable for routine laboratories and surveillance purposes

A balancing act: efflux/influx in mycobacterial drug resistance.

Abstract: Since the discovery of the tubercle bacillus by Robert Koch in 1882 ([110][1]), a greater understanding of the dynamics and survival mechanisms of this pathogen has led to more questions than answers. Despite stringent control strategies and many advances in our knowledge of the epidemiology of

Mutation rate and the emergence of drug resistance in Mycobacterium tuberculosis

Abstract: The emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis remains a major concern of tuberculosis control programmes worldwide, as treatment depends on low-efficacy, toxic compounds that often lead to poor outcomes. M. tuberculosis develops drug resistance exclusively through chromosomal mutations, in particular single-nucleotide polymorphisms. Moreover, in laboratory assays the organism exhibits a spontaneous mutation rate that is at the lower end of the bacterial spectrum. Despite this, whole-genome sequencing technology has identified unexpected genetic diversity among clinical M. tuberculosis populations. This suggests that the mycobacterial mutation rate may be modulated within the host and, in turn, implies a potential role for constitutive and/or transient mutator strains in adaptive evolution. It also raises the possibility that environmental factors might act as key mutagens during M. tuberculosis infection. Here we consider the elements that might influence the mycobacterial mutation rate in vivo and evaluate the potential roles of constitutive and transient mutator states in the generation of drug resistance mutations. In addition, we identify key research questions that will influence future efforts to develop novel therapeutic strategies for a disease that continues to impose a significant global health burden.

The Diagnostic Performance of the GenoType MTBDRplus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay

Abstract: Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.

Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults.

Abstract: Background Accurate, rapid detection of tuberculosis (TB) and TB drug resistance is critical for improving patient care and decreasing TB transmission. Xpert® MTB/RIF assay is an automated test that can detect both TB and rifampicin resistance, generally within two hours after starting the test, with minimal hands-on technical time. The World Health Organization (WHO) issued initial recommendations on Xpert® MTB/RIF in early 2011. A Cochrane Review on the diagnostic accuracy of Xpert® MTB/RIF for pulmonary TB and rifampicin resistance was published January 2013. We performed this updated Cochrane Review as part of a WHO process to develop updated guidelines on the use of the test. Objectives To assess the diagnostic accuracy of Xpert® MTB/RIF for pulmonary TB (TB detection), where Xpert® MTB/RIF was used as both an initial test replacing microscopy and an add-on test following a negative smear microscopy result. To assess the diagnostic accuracy of Xpert® MTB/RIF for rifampicin resistance detection, where Xpert® MTB/RIF was used as the initial test replacing culture-based drug susceptibility testing (DST). The populations of interest were adults presumed to have pulmonary, rifampicin-resistant or multidrug-resistant TB (MDR-TB), with or without HIV infection. The settings of interest were intermediate- and peripheral-level laboratories. The latter may be associated with primary health care facilities. Search methods We searched for publications in any language up to 7 February 2013 in the following databases: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; ISI Web of Knowledge; MEDION; LILACS; BIOSIS; and SCOPUS. We also searched the metaRegister of Controlled Trials (mRCT) and the search portal of the WHO International Clinical Trials Registry Platform to identify ongoing trials. Selection criteria We included randomized controlled trials, cross-sectional studies, and cohort studies using respiratory specimens that allowed for extraction of data evaluating Xpert® MTB/RIF against the reference standard. We excluded gastric fluid specimens. The reference standard for TB was culture and for rifampicin resistance was phenotypic culture-based DST. Data collection and analysis For each study, two review authors independently extracted data using a standardized form. When possible, we extracted data for subgroups by smear and HIV status. We assessed the quality of studies using QUADAS-2 and carried out meta-analyses to estimate pooled sensitivity and specificity of Xpert® MTB/RIF separately for TB detection and rifampicin resistance detection. For TB detection, we performed the majority of analyses using a bivariate random-effects model and compared the sensitivity of Xpert® MTB/RIF and smear microscopy against culture as reference standard. For rifampicin resistance detection, we undertook univariate meta-analyses for sensitivity and specificity separately to include studies in which no rifampicin resistance was detected. Main results We included 27 unique studies (integrating nine new studies) involving 9557 participants. Sixteen studies (59%) were performed in low- or middle-income countries. For all QUADAS-2 domains, most studies were at low risk of bias and low concern regarding applicability. As an initial test replacing smear microscopy, Xpert® MTB/RIF pooled sensitivity was 89% [95% Credible Interval (CrI) 85% to 92%] and pooled specificity 99% (95% CrI 98% to 99%), (22 studies, 8998 participants: 2953 confirmed TB, 6045 non-TB). As an add-on test following a negative smear microscopy result, Xpert®MTB/RIF pooled sensitivity was 67% (95% CrI 60% to 74%) and pooled specificity 99% (95% CrI 98% to 99%; 21 studies, 6950 participants). For smear-positive, culture-positive TB, Xpert® MTB/RIF pooled sensitivity was 98% (95% CrI 97% to 99%; 21 studies, 1936 participants). For people with HIV infection, Xpert® MTB/RIF pooled sensitivity was 79% (95% CrI 70% to 86%; seven studies, 1789 participants), and for people without HIV infection, it was 86% (95% CrI 76% to 92%; seven studies, 1470 participants). Among 180 specimens with nontuberculous mycobacteria (NTM), Xpert® MTB/RIF was positive in only one specimen that grew NTM (14 studies, 2626 participants). Comparison with smear microscopy In comparison with smear microscopy, Xpert® MTB/RIF increased TB detection among culture-confirmed cases by 23% (95% CrI 15% to 32%; 21 studies, 8880 participants). For TB detection, if pooled sensitivity estimates for Xpert® MTB/RIF and smear microscopy are applied to a hypothetical cohort of 1000 patients where 10% of those with symptoms have TB, Xpert® MTB/RIF will diagnose 88 cases and miss 12 cases, whereas sputum microscopy will diagnose 65 cases and miss 35 cases. Rifampicin resistance For rifampicin resistance detection, Xpert® MTB/RIF pooled sensitivity was 95% (95% CrI 90% to 97%; 17 studies, 555 rifampicin resistance positives) and pooled specificity was 98% (95% CrI 97% to 99%; 24 studies, 2411 rifampicin resistance negatives). For rifampicin resistance detection, if the pooled accuracy estimates for Xpert® MTB/RIF are applied to a hypothetical cohort of 1000 individuals where 15% of those with symptoms are rifampicin resistant, Xpert® MTB/RIF would correctly identify 143 individuals as rifampicin resistant and miss eight cases, and correctly identify 833 individuals as rifampicin susceptible and misclassify 17 individuals as resistant. Where 5% of those with symptoms are rifampicin resistant, Xpert® MTB/RIF would correctly identify 48 individuals as rifampicin resistant and miss three cases and correctly identify 931 individuals as rifampicin susceptible and misclassify 19 individuals as resistant. Authors' conclusions In adults thought to have TB, with or without HIV infection, Xpert® MTB/RIF is sensitive and specific. Compared with smear microscopy, Xpert® MTB/RIF substantially increases TB detection among culture-confirmed cases. Xpert® MTB/RIF has higher sensitivity for TB detection in smear-positive than smear-negative patients. Nonetheless, this test may be valuable as an add-on test following smear microscopy in patients previously found to be smear-negative. For rifampicin resistance detection, Xpert® MTB/RIF provides accurate results and can allow rapid initiation of MDR-TB treatment, pending results from conventional culture and DST. The tests are expensive, so current research evaluating the use of Xpert® MTB/RIF in TB programmes in high TB burden settings will help evaluate how this investment may help start treatment promptly and improve outcomes.

Efflux-mediated drug resistance in bacteria: an update.

Abstract: Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome, although they can also be plasmid-encoded. A previous article in this journal provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past 5 years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.

Mycobacterium abscessus: a new antibiotic nightmare

Abstract: The intrinsic and acquired resistance of Mycobacterium abscessus to commonly used antibiotics limits the chemotherapeutic options for infections caused by these mycobacteria. Intrinsic resistance is attributed to a combination of the permeability barrier of the complex multilayer cell envelope, drug export systems, antibiotic targets with low affinity and enzymes that neutralize antibiotics in the cytoplasm. To date, acquired resistance has only been observed for aminoglycosides and macrolides, which is conferred by mutations affecting the genes encoding the antibiotic targets (rrs and rrl, respectively). Here we summarize previous and recent findings on the resistance of M. abscessus to antibiotics in light of what has been discovered for other mycobacteria. Since we can now distinguish three groups of strains belonging to M. abscessus (M. abscessus sensu stricto, Mycobacterium massiliense and Mycobacterium bolletii), studies on antibiotic susceptibility and resistance should be considered according to this new classification. This review raises the profile of this important pathogen and highlights the work needed to decipher the molecular events responsible for its extensive chemotherapeutic resistance.

Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis

Abstract: Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are generally thought to have high mortality rates. However, many cases can be treated with the right combination and rational use of available antituberculosis drugs. This Review describes the evidence available for each drug and discusses the basis for recommendations for the treatment of patients with MDR and XDR tuberculosis. The recommended regimen is the combination of at least four drugs to which the Mycobacterium tuberculosis isolate is likely to be susceptible. Drugs are chosen with a stepwise selection process through five groups on the basis of efficacy, safety, and cost. Among the first group (the oral first-line drugs) high-dose isoniazid, pyrazinamide, and ethambutol are thought of as an adjunct for the treatment of MDR and XDR tuberculosis. The second group is the fluoroquinolones, of which the first choice is high-dose levofloxacin. The third group are the injectable drugs, which should be used in the following order: capreomycin, kanamycin, then amikacin. The fourth group are called the second-line drugs and should be used in the following order: thioamides, cycloserine, then aminosalicylic acid. The fifth group includes drugs that are not very effective or for which there are sparse clinical data. Drugs in group five should be used in the following order: clofazimine, amoxicillin with clavulanate, linezolid, carbapenems, thioacetazone, then clarithromycin.

Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology

Abstract: Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in diagnostic and reference laboratories in the near future. The objective of this article is to consider at a very practical level where, in the context of a modern diagnostic microbiology laboratory, WGS might be cost-effective compared to current alternatives. We propose that molecular epidemiology performed for surveillance and outbreak investigation and genotypic antimicrobial susceptibility testing for microbes that are difficult to grow represent the most immediate areas for application of WGS, and discuss the technical and infrastructure requirements for this to be implemented.