Eligibility for Isoniazid Preventive Therapy in South African Gold Mines

Setting

The “Thibela TB” cluster randomised trial of community-wide isoniazid preventive therapy (IPT) to reduce tuberculosis incidence in the South African gold mines.

Objectives

To determine the proportion of participants eligible for IPT and the reasons and risk factors for ineligibility, to inform the scale-up of IPT.

Design

Cross-sectional survey of participants in intervention clusters (mine shafts) consenting to tuberculosis screening and assessment for eligibility to start IPT.

Results

Among 27,126 consenting participants, 94.7% were male, the median age was 41 years, 12.2% reported previous tuberculosis, 0.6% reported ever taking IPT and 2.5% reported currently taking antiretroviral therapy. There were 24,430 (90.1%) assessed as eligible to start IPT, of whom 23,659 started IPT. The most common reasons for ineligibility were having suspected tuberculosis that was subsequently confirmed by a positive smear and/or culture (n=705), excessive alcohol consumption (n=427) and being on tuberculosis treatment at time of initial screen (n=241). Ineligibility was associated with factors including older age, female gender, prior history of tuberculosis and being in “HIV care”. However, at least 78% were eligible for IPT in all of these sub-groups.

Conclusions

The vast majority of participants in this community-wide intervention were eligible for IPT.     

Autopsy Prevalence of Tuberculosis and Other Potentially Treatable Infections among Adults with Advanced HIV Enrolled in Out-Patient Care in South Africa

BackgroundEarly mortality among HIV-positive adults starting antiretroviral therapy (ART) remains high in resource-limited settings, with tuberculosis (TB) the leading cause of death. However, current methods to estimate TB-related deaths are inadequate and most autopsy studies do not adequately represent those attending primary health clinics (PHCs). This study aimed to determine the autopsy prevalence of TB and other infections in adults enrolled at South African PHCs in the context of a pragmatic trial of empiric TB treatment (“TB Fast Track”).ConclusionsTB, followed by bacterial infections, were the leading findings at autopsy among adults with advanced HIV enrolled from primary care clinics. To reduce mortality, strategies are needed to identify and direct those at highest risk into a structured pathway that includes expedited investigation and/or treatment of TB and other infections.     

Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes

AIMS: To analyse the frequencies of prion (PrP) gene haplotypes in UK sheep flocks and evaluate their relevance to transmissible spongiform encephalopathies (TSEs) and TSE resistance breeding programmes in sheep. METHODS AND RESULTS: Genomic DNA isolated from sheep blood was PCR amplified for the coding region of the PrP gene and then sequenced. This study has analysed the sequence of PrP between codons 110 and 245 in 6287 ARQ haplotypes revealing a total of eight variant sequences, which represent a higher than expected 41% of all ARQ haplotypes. The additional PrP gene dimorphisms were M112T, L141F, M137T, H143R, H151C, P168L, Q175E and P241S. CONCLUSION: The results do not suggest a correlation between the occurrence of a specific ARQ haplotype and the scrapie disease status of a flock. The ARQ haplotype variability appears to be different in the UK sheep flocks compared with sheep flocks from outside the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional PrP dimorphisms may impact on the methodologies used for standard PrP genotyping in sheep breeding programmes. Some of these polymorphisms were found with significant frequencies in the UK sheep flocks and should therefore be considered in breeding programmes.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Missed Opportunities for TB Investigation in Primary Care Clinics in South Africa: Experience from the XTEND Trial

Setting

40 primary health clinics (PHCs) in four provinces in South Africa, June 2012 –February 2013.

Objective

To determine whether health care worker (HCW) practice in investigating people with TB symptoms was altered when the initial test for TB was changed from smear microscopy to Xpert MTB/RIF.

Design

Cross-sectional substudy at clinics participating in a pragmatic cluster randomised trial, Xpert for TB: Evaluating a New Diagnostic "XTEND", which evaluated the effect of Xpert MTB/RIF implementation in South Africa.

Methods

Consecutive adults exiting PHCs reporting at least one TB symptom (defined as any of cough, weight loss, night sweats and fever) were enrolled. The main outcome was the proportion who self-reported having sputum requested by HCW during the clinic encounter just completed.

Results

3604 adults exiting PHCs (1676 in Xpert arm, 1928 in microscopy arm) were enrolled (median age 38 years, 71.4% female, 38.8% reported being HIV-positive, 70% reported cough). For 1267 participants (35.2%) the main reason for attending the clinic was TB symptom(s).Overall 2130/3604 (59.1%) said they reported their symptom(s) to HCW. 22.7% (818/3604) reported having been asked to give sputum for TB investigation. Though participants in the Xpert vs. microscopy arm were more likely to have sputum requested by HCW, this was not significantly different: overall (26.0% [436/1676] vs 19.8% [382/1928]; adjusted prevalence ratio [aPR] 1.31, [95% CI 0.78–2.20]) and when restricted to those presenting at clinics due to symptoms (49.1% [260/530] vs 29.9% [220/737]; aPR 1.38 [0.89–2.13]) and those reporting being HIV-positive (29.4% [190/647] vs 20.8% [156/749]; aPR 1.38[0.88–2.16]).Those attending clinic due to TB symptoms, were more likely to have sputum requested if they had increasing number of symptoms; longer duration of cough, unintentional weight loss and night sweats and if they reported symptoms to HCW.

Conclusions

A large proportion of people exiting PHCs reporting TB symptoms did not get tested. Implementation of Xpert MTB/RIF did not substantially change the probability of testing for TB. Better systems are needed to ensure that opportunities to identify active TB among PHC attendees are not missed.     

Performance Characteristics of the Cepheid Xpert MTB/RIF Test in a Tuberculosis Prevalence Survey

Background

Xpert MTB/RIF (“Xpert”) is a molecular test for detection of Mycobacterium tuberculosis (MTB) in sputum. Performance characteristics have been established for its use during passive tuberculosis (TB) case detection in symptomatic TB suspects, but Xpert performance has not been assessed in other settings. Objectives were to determine Xpert performance and costs in the context of a TB prevalence survey.

Methodology/Principal Findings

This was a diagnostic sub-study of a TB prevalence survey conducted in gold mining companies in South Africa. Sputa (one per participant) were tested using smear microscopy, liquid culture (reference comparator), and Xpert. Costs were collected using an ingredients approach and analyzed using a public health program perspective. 6893 participants provided a sputum specimen. 187/6893 (2.7%) were positive for MTB in culture, 144/6893 (2.1%) were positive for MTB by Xpert, and 91/6893 (1.3%) were positive for acid fast bacilli by microsocopy. Sensitivity, specificity, positive predictive value, and negative predictive value for detection of MTB by Xpert were 62.6% (95% confidence interval [CI] 55.2, 69.5), 99.6% (99.4, 99.7), 81.3% (73.9, 87.3), and 98.9 (98.6, 98.8); agreement between Xpert and culture was 98.5% (98.2, 98.8). Sensitivity of microscopy was 17.6% (12.5, 23.9). When individuals with a history of TB treatment were excluded from the analysis, Xpert specificity was 99.8 (99.7, 99.9) and PPV was 90.6 (83.3, 95.4) for detection of MTB. For the testing scenario of 7000 specimens with 2.7% of specimens culture positive for MTB, costs were $165,690 for Xpert and $115,360 for the package of microscopy plus culture.

Conclusion

In the context of a TB prevalence survey, the Xpert diagnostic yield was substantially higher than that of microscopy yet lower than that of liquid culture. Xpert may be useful as a sole test for TB case detection in prevalence surveys, particularly in settings lacking capacity for liquid culture.     

“Proof-Of-Concept” Evaluation of an Automated Sputum Smear Microscopy System for Tuberculosis Diagnosis

Background

“TBDx” is an innovative smear microscopy system that automatically loads slides onto a microscope, focuses and digitally captures images and then classifies smears as positive or negative using computerised algorithms.

Objectives

To determine the diagnostic accuracy of TBDx, using culture as the gold standard, and compare this to a microscopist''s diagnostic performance.

Methods

This study is nested within a cross-sectional study of tuberculosis suspects from South African gold mines. All tuberculosis suspects had one sputum sample collected, which was decontaminated prior to smear microscopy, liquid culture and organism identification. All slides were auramine-stained and then read by both a research microscopist and by TBDx using fluorescence microscopes, classifying slides based on the WHO classification standard of 100 fields of view (FoV) at 400× magnification.

Results

Of 981 specimens, 269 were culture positive for Mycobacterium tuberculosis (27.4%). TBDx had higher sensitivity than the microscopist (75.8% versus 52.8%, respectively), but markedly lower specificity (43.5% versus 98.6%, respectively). TBDx classified 520/981 smears (53.0%) as scanty positive. Hence, a proposed hybrid software/human approach that combined TBDx examination of all smears with microscopist re-examination of TBDx scanty smears was explored by replacing the “positive” result of slides with 1–9 AFB detected on TBDx with the microscopist''s original reading. Compared to using the microscopist''s original results for all 981 slides, this hybrid approach resulted in equivalent specificity, a slight reduction in sensitivity from 52.8% to 49.4% (difference of 3.3%; 95% confidence interval: 0.2%, 6.5%), and a reduction in the number of slides to be read by the microscopist by 47.0%.

Discussion

Compared to a research microscopist, the hybrid software/human approach had similar specificity and positive predictive value, but sensitivity requires further improvement. Automated microscopy has the potential to substantially reduce the number of slides read by microscopists.     

Programmatically Selected Multidrug-Resistant Strains Drive the Emergence of Extensively Drug-Resistant Tuberculosis in South Africa

Background

South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known.

Methods

We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations.

Results

Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin.

Conclusions

XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.