Epidemic Levels of Drug Resistant Tuberculosis (MDR and XDR-TB) in a High HIV Prevalence Setting in Khayelitsha, South Africa

BACKGROUND: Although multidrug-resistant tuberculosis (MDR-TB) is emerging as a significant threat to tuberculosis control in high HIV prevalence countries such as South Africa, limited data is available on the burden of drug resistant tuberculosis and any association with HIV in such settings. We conducted a community-based representative survey to assess the MDR-TB burden in Khayelitsha, an urban township in South Africa with high HIV and TB prevalence. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted among adult clinic attendees suspected for pulmonary tuberculosis in two large primary care clinics, together constituting 50% of the tuberculosis burden in Khayelitsha. Drug susceptibility testing (DST) for isoniazid and rifampicin was conducted using a line probe assay on positive sputum cultures, and with culture-based DST for first and second-line drugs. Between May and November 2008, culture positive pulmonary tuberculosis was diagnosed in 271 new and 264 previously treated tuberculosis suspects (sample enriched with previously treated cases). Among those with known HIV status, 55% and 71% were HIV infected respectively. MDR-TB was diagnosed in 3.3% and 7.7% of new and previously treated cases. These figures equate to an estimated case notification rate for MDR-TB of 51/100,000/year, with new cases constituting 55% of the estimated MDR-TB burden. HIV infection was not significantly associated with rifampicin resistance in multivariate analyses. CONCLUSIONS/SIGNIFICANCE: There is an extremely high burden of MDR-TB in this setting, most likely representing ongoing transmission. These data highlight the need to diagnose drug resistance among all TB cases, and for innovative models of case detection and treatment for MDR-TB, in order to interrupt transmission and control this emerging epidemic

Differential Genetic Susceptibility to Child Risk at Birth in Predicting Observed Maternal Behavior

This study examined parenting as a function of child medical risks at birth and parental genotype (dopamine D4 receptor; DRD4). Our hypothesis was that the relation between child risks and later maternal sensitivity would depend on the presence/absence of a genetic variant in the mothers, thus revealing a gene by environment interaction (GXE). Risk at birth was defined by combining risk indices of children's gestational age at birth, birth weight, and admission to the neonatal intensive care unit. The DRD4-III 7-repeat allele was chosen as a relevant genotype as it was recently shown to moderate the effect of environmental stress on parental sensitivity. Mothers of 104 twin pairs provided DNA samples and were observed with their children in a laboratory play session when the children were 3.5 years old. Results indicate that higher levels of risk at birth were associated with less sensitive parenting only among mothers carrying the 7-repeat allele, but not among mothers carrying shorter alleles. Moreover, mothers who are carriers of the 7-repeat allele and whose children scored low on the risk index were observed to have the highest levels of sensitivity. These findings provide evidence for the interactive effects of genes and environment (in this study, children born at higher risk) on parenting, and are consistent with a genetic differential susceptibility model of parenting by demonstrating that some parents are inherently more susceptible to environmental influences, both good and bad, than are others

Atypical Balance between Occipital and Fronto-Parietal Activation for Visual Shape Extraction in Dyslexia

Reading requires the extraction of letter shapes from a complex background of text, and an impairment in visual shape extraction would cause difficulty in reading. To investigate the neural mechanisms of visual shape extraction in dyslexia, we used functional magnetic resonance imaging (fMRI) to examine brain activation while adults with or without dyslexia responded to the change of an arrow’s direction in a complex, relative to a simple, visual background. In comparison to adults with typical reading ability, adults with dyslexia exhibited opposite patterns of atypical activation: decreased activation in occipital visual areas associated with visual perception, and increased activation in frontal and parietal regions associated with visual attention. These findings indicate that dyslexia involves atypical brain organization for fundamental processes of visual shape extraction even when reading is not involved. Overengagement in higher-order association cortices, required to compensate for underengagment in lower-order visual cortices, may result in competition for top-down attentional resources helpful for fluent reading.Ellison Medical FoundationMartin Richmond Memorial FundNational Institutes of Health (U.S.). (Grant UL1RR025758)National Institutes of Health (U.S.). (Grant F32EY014750-01)MIT Class of 1976 (Funds for Dyslexia Research

ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers

Risk factors associated with isoniazid resistance in tuberculosis

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2005.Tuberculosis (TB) is one of the most serious infectious diseases known to mankind, with devastating outcomes in the poorest countries in the world. Isoniazid is the cornerstone of all first-line anti-TB regimens. Forty-eight percent of all drug resistant TB isolates in the Western Cape are Isoniazid mono-resistant, and the majority of these isolates belong to the Beijing/W strain family. Currently, the known molecular mechanisms which confer Isoniazid resistance in these isolates are attributed to mutations within the katG gene and account for up to 70% of all drug resistant TB isolates. Risk factors for the development of Isoniazid resistance can be attributed to either pathogen or host related factors and may partially account for the other 30% of Isoniazid resistant isolates. In this study, three aspects which may contribute to Isoniazid resistance were investigated: DNA repair in the bacterium, host response to anti-TB treatment and socioeconomic factors. A PCR based dot-blot strategy was used to screen for previously reported missense mutations in the mutT2, Rv3908 and ogt DNA repair genes of different strains of M. tuberculosis. All the Beijing isolates (drug resistant and susceptible), in contrast to the Atypical Beijing strains and other dominant strain families, exhibited missense mutations in all three base excision repair genes. It is therefore speculated that defects in the DNA repair genes (mutator phenotypes) of the Beijing isolates may contribute to the development of drug resistance and hence, may account for the large proportion of isolates that are Isoniazid mono-resistant. A novel method, based on primer extension, was initially developed to screen the NAT2 gene and then used to type individuals into fast, intermediate and slow acetylators of Isoniazid. The newly develop method, which is sensitive and accurate, improves the detection of Single Nucleotide Polymorphisms within the NAT2 gene, in contrast to the traditionally used methods. Utilising this method, it was found that the combination of fast and intermediate acetylators was significantly associated with Isoniazid resistance in the study community. This finding may have an important impact on TB control programmes, since it may allow for the administration of higher dosages of Isoniazid to fast/intermediate acetylators and a lower dose for slow acetylators. Clinical factors (compliance and retreatment after cure) and socio-economic factors (education, employment and income) were found to be significantly associated with the development of INH resistance. Diagnostic delay was also found to be a risk factor, since it may allow for transmission of TB during this period. The HIV prevalence in the study population is low and subsequently HIV status was not associated with the development of INH resistance. This study indicates that a combination of risk factors, both pathogen and host related, are involved in the development of Isoniazid resistance

Diagnostic utility of the line probe assay for the detection of drug resistance in Mycobacterium tuberculosis

Thesis(PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: The epidemic levels of drug-resistant tuberculosis (DR-TB) in high-burden countries such as South Africa, which is currently ranked as third highest in the world, is the result of a synergistic relationship between the increased transmission of DR strains, poor patient adherence as well as Human-Immunodeficiency Virus (HIV)-coinfection. The impact of these combined factors on the rise of DR-TB led to an urgent need for the development of new diagnostic tools to rapidly detect TB and its associated drug susceptibility profile. The Foundation for Innovative New Diagnostics (FIND) has taken the onus upon them to ensure that laboratory strengthening becomes a reality by having developed, and still developing, new diagnostic assays in order to improve the laboratory turn-around time (TAT), whereby the transmission of DR-TB strains can be stopped. Laboratory strengthening does not solely rely on new diagnostic assays alone, and thus a Quality Management System, discussed in the dissertation, must be in place to ensure that the rapid result is accurate and reliable. The series of studies encompassed in this dissertation includes methodological validations (both technical and operational) of rapid TB diagnostic assays in order to rapidly and accurately diagnose the disease, and thus reducing the diagnostic delay associated with conventional diagnostic platforms. The studies were conducted “in-house” at the National Health Laboratory Service (NHLS) Reference TB laboratory in Green Point, Cape Town, which is a high-volume public health laboratory. The need to rapidly detect resistance to the first line anti-tubercular drugs Isoniazid and Rifampicin was a priority and thus the performance of a commercial line probe assay (LPA), the GenoType®MTBDRplus Ver1.0 LPA, was assessed for use on smear positive direct patient material. The performance characteristics was superior to that of conventional drug susceptibility testing, where the sensitivity and specificity for the detection of multi-drug resistant TB (MDR-TB) was 98.8 and 100%, respectively, with results in 1-2 days. Based on this study, the World Health Organization (WHO) endorsed the use of molecular LPA for the rapid detection of DR-TB. Furthermore, the need for quality assurance associated with the GenoType®MTBDRplus LPA in the diagnostic laboratory is essential and thus a user manual for the molecular detection of Drug Resistant Tuberculosis in resource-limited settings has also been developed (http://www.finddiagnostics.org/export/sites/default/resource-center/reports brochures/docs/LPA LaboratoryManual22Mar2012.pdf) for which Global Laboratory Initiative (GLI) status is pending. With the outbreak of extensively drug resistant TB (XDR-TB) in Tugela Ferry area in KwaZulu-Natal and the rest of the world, the need to rapidly detect resistance to the second line drugs arose, and thus the performance characteristics of the GenoType®MTBDRsl LPA was assessed for use on smear positive direct patient material. The performance characteristics proved to be excellent once again, with a 93.3% reduction in TAT. The data was scrutinized by the WHO, where it may be used as a triage test to guide treatment, but to date, no final policies on the use thereof has been finalized. The need for rapid point-of-care (POC) testing led to the implementation of the Xpert®MTB/RIF assay in the referral laboratories, for use on both smear positive and smear negative direct patient material. In order to accommodate for laboratories where the LPA has been implemented already, the GenoType®MTBDRplus Ver2.0 LPA was developed, which is aimed for use on all smear types as well. A head-to-head assessment was done between these assays to determine their performance characteristics and it was shown to be equally good. In this study we have shown the utility of molecular diagnostic assays to rapidly diagnose TB and its associated drug susceptibility patterns. This will have a significant impact on diagnostic delay and clinical decision making as well as patient outcome.AFRIKAANSE OPSOMMING: Die epidemiese vlakke van middel-weerstandige tuberkulose (MW-TB) in hoë-lading lande, soos Suid Afrika wat tans derde hoogste op die wêreld ranglys is, is die nagevolge van 'n sinergistiese verband tussen die verhoogde voorkoms van transmissie van MW stamme, swak pasiënt deelname aan die voorgeskrewe behandelings programme, asook Menslike Immuniteitsgebreksvirus (MIV) ko-infeksie. Die impak van hierdie drie faktore saam, gee aanleiding tot 'n verhoging in MW-TB en dus was daar 'n daadwerklike behoefte vir die ontwikkeling van nuwe diagnostiese toetse wat nie net TB kan identifiseer nie, maar wat ook die gepaardgaande middel-weerstandigheids profiel aandui. Die “Foundation for Innovative New Diagnostics” (FIND) het die onus van laboratorium versterking op hulself geneem, deur te verseker dat die nuut ontwikkelde diagnostiese toetse, asooks steeds ontwikkelende diagnostiese toetse, gebruik kan word om die konsep van laboratorium versterking 'n realiteit te maak. Die doel is dus om sodoende die tyd-tot-resultaat tussen geneesheer en laboratorium te verbeter, terwyl die transmissie van MW-TB ook die hok geslaan kan word. Nietemin, laboratorium versterking berus nie net op nuwe diagnostiese toetse nie, en dus is dit noodsaaklik dat 'n Kwaliteitbestuursisteem, soos bespreek in hierdie verhandeling, in plek is om te verseker dat die resultaat spoedig, akkuraat en betroubaar is. Die samevattende reeks studies in hierdie verhandeling behels metodologiese validasies (beide tegnies en operasioneel van aard) van spoedige TB diagnostiese toetse met die doel om die siekte so vinnig en akkuraat as moontlik te diagnoseer en dus die diagnostiese vertraging, wat histories met konvensionele metodes geassosiëerd is, te verminder. Al die studies is uitgevoer in die “National Health Laboratory Service (NHLS)” TB verwysingslaboratorium in Groenpunt, Kaapstad, wat 'n hoë-volume publieke gesondheidslaboratorium is. Die noodsaaklikheid om weerstandigheid teenoor die eerste-linie antituberkulose middels isoniasied en rifampisien so spoedig moontlik te diagnoseer het 'n groot bekommernis geword, en dus is die laboratorium daartoe genoop om die prestasie eienskappe van 'n kommersiëel beskikbare “line probe assay” (LPA), die “Genotype®MTBDRplus Ver1.0 LPA”, te asseseer vir die gebruik daarvan op direkte pasientmateriaal wat smeer positief is. Die prestasie eienskappe was beter as die van konvensionele middelvatbaarheidstoetse, waar die sensitiwiteit en spesifisiteit vir die diagnosering van MW-TB 98.8 en 100%, respektiewelik, was. Verder was die resultate ook binne 1-2 dae beskikbaar. Op grond van dié bevindinge het die Wêreldgesondheidsorganisasie (WGO) die gebruik van hierdie molekulêre “LPA” vir die spoedige diagnose van MW-TB onderskryf. Nietemin, die belangrikheid van gehalteversekering wat met die “GenoType®MTBDRplus LPA” in die diagnostiese laboratorium geassosieerd is, is essentiëel en dus is 'n gebruikershandleiding vir die molekulêre diagnose van MW-TB in beperkte hulpbron-instellings ontwikkel (http://www.finddiagnostics.org/export/sites/default/resource-center/reports brochures/docs/LPA LaboratoryManual22Mar2012.pdf) waarvoor daar op„n “Global Laboratory Initiative (GLI)” status in afwagting is. Met die uitbraak van ekstensiewemiddelweerstandige TB (EMW-TB) in die Tugela Ferry distrik in KwaZulu-Natal asook in die res van die wêreld, het die noodsaaklikheid onstaan om weerstandigheid teenoor die tweede-linie middels ook so spoedig moontlik te diagnoseer, en die laboratorium is dus weereens daartoe genoop om die prestasie eienskappe van die “GenoType®MTBDRsl LPA” (ook vir die gebruik op direkte pasient materiaal wat smeer positief is) te asseseer. Die prestasie eienskappe was weereens verbysterend, en het „n 93.3% afname in tyd-tot-resultaat getoon. Die data is deur die WGO aangevra, en daar is besluit dat die toets gebruik kan word om behandeling in werking te stel, maar geen finale onderskrywings is tot op hede nog gemaak nie. Die behoefte aan 'n punt-van-sorg toets het gelei tot die implementering van die “Xpert®MTB/RIF” toets in die verwysingslaboratorium, en is geoogmerk vir die gebruik op beide smeer positiewe en -negatiewe direkte pasient materiaal. Omrede die “LPA” al in verskeie laborotoriums geimplementeer was, is die “GenoType®MTBDRplus Ver2.0 LPA” ontwikkel, waarvan die gebruik onafhanklik is van die smeerresultaat. 'n Direkte assesering tussen die twee toetse was gedoen en daar is bevind dat beide se prestasie eienskappe vergelykend was. In hierdie studies het ons bewys dat die gebruik van molekulêre diagnostiese toetse in staat is om TB en die gepaargaande middel-weerstandigheids profiel spoedig te diagnoseer. Hierdie bevindinge sal 'n groot impak hê op die vetraging van tyd-tot-resultaat, op die mediese besluitneming asook op die uitkoms van die pasiënt.FIND (Foundation for Innovative New Diagnostics)Hain LifescienceNational Health Laboratory Service (NHLS

Rapid sequencing of the mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility

CITATION: Streicher, E. M., et al. 2014. Rapid sequencing of the mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility. Journal of Clinical Microbiology, 52(11): 4056-4057. doi: 10.1128/JCM.02438-14.The original publication is available at http://jcm.asm.orgWe developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.http://jcm.asm.org/content/52/11/4056.fullPublisher's versio

Univariate and multivariate analyses to assess associations with rifampicin resistance among all, previously treated, and HIV positive culture positive TB cases.

<p>Univariate and multivariate analyses to assess associations with rifampicin resistance among all, previously treated, and HIV positive culture positive TB cases.</p