Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

Provision of HIV services by community health workers should be strengthened to achieve full programme potential: a cross-sectional analysis in rural South Africa.

OBJECTIVE: South Africa's community health workers (CHWs) provide a bridge between the primary healthcare (PHC) facility and its community. We conducted a cross-sectional analysis to determine the contribution of the community-based HIV programme (CBHP) to the overall HIV programme. METHODS: We collected service provision data from the daily activity register of CHWs attached to 12 PHC facilities in rural Mopani District, South Africa. Personal identifiers of individuals referred to the facility for HIV services were recorded and verified against facility routine patient registers to determine the effectiveness of referral. RESULTS: HIV services were provided on 18 927 occasions; 30% of the total activities performed by CHWs during the study period. CHWs assessed 12 159 individuals for HIV risk (13% coverage of the study population); only 290 (2%) were referred for HIV testing services. Referral was effective in 213 (73%) individuals; evidence of an HIV-positive status was found for 38 (18%) individuals. However, 30 (79%) of these individuals were referred by CHWs despite being on ART. Adherence support was provided during 5657 visits; only one individual was referred for complications. Finally, of the 864 individuals lost to the ART programme, CHWs managed to find 452 (52%) for referral back to the facility; only 241 (53%) of these were (re)initiated on ART. CONCLUSIONS: Provision of HIV services by CHWs should be strengthened to fully deliver on the programme's potential. Human resource investment, home-based HIV testing and improved tracing models constitute potential strategies to enhance CHWs impact on the HIV programme.

OBJECTIF: Les agents de santé communautaires (ASC) d'Afrique du Sud constituent un pont entre les établissements de soins de santé primaires et leur communauté. Nous avons effectué une analyse transversale pour déterminer la contribution du programme VIH basé sur la communauté (PVBC) au programme global de lutte contre le VIH. MÉTHODES: Nous avons collecté des données sur la prestation de services à partir du registre des activités quotidiennes des ASC rattachés à 12 établissements de soins de santé primaires dans le district rural de Mopani, en Afrique du Sud. Les identifiants individuels des personnes référées vers l'établissement pour des services VIH ont été enregistrés et vérifiés par rapport aux registres de routine des patients de l'établissement afin de déterminer l'efficacité de l'aiguillage. RÉSULTATS: Les services VIH ont été fournis dans 18.927 occasions; 30% du total des activités réalisées par les ASC au cours de la période d'étude. Les ASC ont évalué 12.159 personnes pour les risques du VIH (couverture de 13% de la population étudiée); seuls 290 (2%) ont été référés pour des services de dépistage du VIH. L'aiguillage a été efficace chez 213 personnes (73%). L'évidence de séropositivité a été trouvée chez 38 personnes (18%). Cependant, 30 (79%) de ces personnes ont été référées par des ASC alors qu'elles étaient sous ART. Un soutien à la compliance a été fourni au cours de 5.657 visites; un seul individu a été référé pour des complications. Enfin, sur 864 personnes perdues du programme ART, les ASC ont réussi à retrouver 452 (52%) pour les référer de nouveau à l'établissement; seuls 241 (53%) de celles-ci ont été (ré) initiées sous ART. CONCLUSIONS: La fourniture de services VIH par les ASC devrait être renforcée pour exploiter pleinement le potentiel du programme. Les investissements dans les ressources humaines, le dépistage du VIH à domicile et les modèles de traçage améliorés constituent des stratégies potentielles pour améliorer l'impact des ASC sur le programme VIH.

The impact of implementing a Xpert MTB/RIF algorithm on drug-sensitive pulmonary tuberculosis: a retrospective analysis.

Xpert MTB/RIF (Xpert) is the preferred first-line test for all persons with tuberculosis (TB) symptoms in South Africa in line with a diagnostic algorithm. This study evaluates pre- and post-implementation trends in diagnostic practices for drug-sensitive, pulmonary TB in adults in an operational setting, following the introduction of the Xpert-based algorithm. We retrospectively analysed data from the national TB database for Greater Tzaneen sub-district, Limpopo Province. Trends in a number of cases, diagnosis and outcome and characteristics associated with death are reported. A total of 8407 cases were treated from 2008 until 2015, with annual cases registered decreasing by 31·7% over that time period (from 1251 to 855 per year). After implementation of Xpert, 69·9% of cases were diagnosed by Xpert, 29·4% clinically, 0·6% by smear microscopy and 0·1% by culture. Cases with a recorded microbiological test increased from 76·2% to 96·4%. Cases started on treatment without confirmation, but with a negative microbiological test increased from 7·1% to 25·7%. Case fatality decreased from 15·0% to 9·8%, remaining consistently higher in empirically treated groups, regardless of HIV status. Implementation of the algorithm coincided with a reduced number of TB cases treated and improved coverage of microbiological testing; however, a substantial proportion of cases continued to start treatment empirically.

Acceptability and Feasibility of Integrating Point-of-Care Diagnostic Testing of Sexually Transmitted Infections into a South African Antenatal Care Program for HIV-Infected Pregnant Women.

Background: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) infections may increase the risk of vertical transmission of the human immunodeficiency virus (HIV). In resource-limited settings, symptomatic screening, and syndromic management of sexually transmitted infections (STIs) during pregnancy continue to be the standard of care. In the absence of diagnostic testing, asymptomatic infections in pregnant women go untreated. Objective: To describe the acceptability and feasibility of integrating diagnostic STI screening into first antenatal care visits for HIV-infected pregnant women. Methods: HIV-infected pregnant women were recruited during their first antenatal care visit from three antenatal care clinics in Tshwane District, South Africa, between June 2016 and October 2017. Self-collected vaginal swabs were used to screen for CT, NG, and TV with a diagnostic point-of-care (POC) nucleic acid amplification test. Those with STIs were provided treatment per South African national guidelines. Results: Of 442 eligible women, 430 (97.3%) agreed to participate and were tested. Of those with a positive STI test result (n = 173; 40.2%), 159 (91.9%) received same-day results and treatment; 100% of STI-infected women were treated within seven days. Conclusions: Integration of POC diagnostic STI screening into first-visit antenatal care services was feasible and highly acceptable for HIV-infected pregnant women.

A 10-year cohort analysis of routine paediatric ART data in a rural South African setting.

South Africa's paediatric antiretroviral therapy (ART) programme is managed using a monitoring and evaluation tool known as TIER.Net. This electronic system has several advantages over paper-based systems, allowing profiling of the paediatric ART programme over time. We analysed anonymized TIER.Net data for HIV-infected children aged <15 years who had initiated ART in a rural district of South Africa between 2005 and 2014. We performed Kaplan-Meier survival analysis to assess outcomes over time. Records of 5461 children were available for analysis; 3593 (66%) children were retained in care. Losses from the programme were higher in children initiated on treatment in more recent years (P < 0·0001) and in children aged ≤1 year at treatment initiation (P < 0·0001). For children aged <3 years, abacavir was associated with a significantly higher rate of loss from the programme compared to stavudine (hazard ratio 1·9, P < 0·001). Viral load was suppressed in 48-52% of the cohort, with no significant change over the years (P = 0·398). Analysis of TIER.Net data over time provides enhanced insights into the performance of the paediatric ART programme and highlights interventions to improve programme performance.

HIV-infected individuals on long-term antiretroviral therapy are at higher risk for ocular disease.

Introduction of antiretroviral therapy (ART) has dramatically reduced the incidence of infectious ocular diseases in human immunodeficiency virus (HIV)-infected individuals. However, the effects of long-term ART and chronic HIV infection on the eye are ill-defined. This study determined the occurrence and severity of ocular diseases among 342 participants in a rural South African setting: HIV-naïve (n = 105), HIV-infected ART-naïve (n = 16), HIV-infected on ART for 36 months (long-term ART; n = 165). More HIV-infected participants presented with an external eye condition, in particular blepharitis, than HIV-naïve individuals (18% vs. 7%; age-adjusted odds ratio (aOR) = 2·8, P < 0·05). Anterior segment conditions (particularly keratoconjunctivitis sicca and pterygium) were also more common (50% vs. 27%; aOR = 2·4; P < 0·01). Compared with individuals on short-term ART, participants receiving long-term ART were more likely to have clinically detectable cataract (57% vs. 38%; aOR = 2·2, P = 0·01) and posterior segment diseases, especially HIV retinopathy (30% vs. 11%; aOR = 3·4, P < 0·05). Finally, long-term ART was significantly associated with presence of HIV retinopathy (P < 0·01). These data implicate that ocular disease is more common and of more diverse etiology among HIV-infected individuals, especially those on long-term ART and suggest that regular ophthalmological monitoring of HIV-infected individuals on ART is warranted.

Reduction in extrapulmonary tuberculosis in context of antiretroviral therapy scale-up in rural South Africa.

Scale-up of antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection has reduced the incidence of pulmonary tuberculosis (PTB) in South Africa. Despite the strong association of HIV infection with extrapulmonary tuberculosis (EPTB), the effect of ART on the epidemiology of EPTB remains undocumented. We conducted a retrospective record review of patients initiated on treatment for EPTB in 2009 (ART coverage <5%) and 2013 (ART coverage 41%) at four public hospitals in rural Mopani District, South Africa. Data were obtained from TB registers and patients' clinical records. There was a 13% decrease in overall number of TB cases, which was similar for cases registered as EPTB (n = 399 in 2009 vs. 336 in 2013; P < 0·01) and for PTB (1031 vs. 896; P < 0·01). Among EPTB cases, the proportion of miliary TB and disseminated TB decreased significantly (both P < 0·01), TB meningitis and TB of bones increased significantly (P < 0·01 and P = 0·02, respectively) and TB pleural effusion and lymphadenopathy remained the same. This study shows a reduction of EPTB cases that is similar to that of PTB in the context of the ART scale-up. The changing profile of EPTB warrants attention of healthcare workers.

Clinical and corneal microbial profile of infectious keratitis in a high HIV prevalence setting in rural South Africa.

The purpose of this investigation was to determine the clinical and corneal microbial profile of infectious keratitis in a high human immunodeficiency virus (HIV) prevalence setting in rural South Africa. Data in this cross-sectional study were collected from patients presenting with symptoms of infectious keratitis (n = 46) at the ophthalmology outpatient department of three hospitals in rural South Africa. Corneal swabs were tested for herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV) and adenovirus DNA by real-time polymerase chain reaction (PCR) and for bacteria and fungi by culture. Based on clinical history, disease characteristics and laboratory results, 29 (63 %) patients were diagnosed as viral keratitis, including 14 (48 %) viral keratitis cases complicated by bacterial superinfection, and 17 (37 %) as bacterial keratitis. VZV and HSV-1 DNA was detected in 11 (24 %) and 5 (11 %) corneal swabs, respectively. Among clinically defined viral keratitis cases, a negative viral swab was predominantly (93 %) observed in cases with subepithelial inflammation and was significantly associated with an increased duration of symptoms (p = 0.003). The majority of bacteria cultured were Gram-positive (24/35), including Staphylococcus epidermidis and S. aureus. Viral aetiology was significantly associated with a history of herpes zoster ophthalmicus (p < 0.001) and a trend was observed between viral aetiology and HIV infection (p = 0.06). Twenty-one (47 %) keratitis cases were complicated by anterior uveitis, of which 18 (86 %) were HIV-infected cases with viral keratitis. The data implicate a high prevalence of herpetic keratitis, in part complicated by bacterial superinfection and/or uveitis, in HIV-infected individuals presenting with infectious keratitis in rural South Africa.

Serogroup distribution of urogenital Chlamydia trachomatis in urban ethnic groups in The Netherlands.

The prevalence of Chlamydia trachomatis varies between ethnic groups in The Netherlands. It is, however, unknown whether this is associated with specific serogroups. The objective of this study was to determine whether serogroup distribution is associated with ethnic origin in the region of The Hague, The Netherlands. Serogroups of 370 microbiologically confirmed C. trachomatis-positive samples were analysed. The samples were obtained from 247 women and 123 men between January and October 2008, of self-reported Dutch Caucasian, Dutch Antillean, Surinamese, N. African/Turkish or other descent. We observed a difference in serogroup distribution comparing Dutch Caucasian women to Dutch Antillean women (χ2 for distribution P = 0·035). Serogroup C was more common in Dutch Antillean women, whereas serogroup B was less common (P = 0·03). This difference was not observed for Dutch Antillean men. The observed difference in distribution of C. trachomatis serogroups between ethnic groups is relevant for further transmission studies.

Analyses of multiple-site and concurrent Chlamydia trachomatis serovar infections, and serovar tissue tropism for urogenital versus rectal specimens in male and female patients.

OBJECTIVES: The aims of this study were: to determine the incidence of concurrent infections on a serovar level; to determine the incidence of multiple anatomical infected sites on a detection and genotyping level and analyse site-specific serovar distribution; to identify tissue tropism in urogenital versus rectal specimens. METHODS: Chlamydia trachomatis-infected patients in two populations were analysed: 75 visiting the outpatient department of obstetrics and gynaecology of the MC Haaglanden, and 358 visiting the outpatient sexually transmitted disease clinic, The Hague, The Netherlands. The PACE 2 assay (Gen-Probe) was used to detect C trachomatis from urethral, cervical, vaginal, oropharyngeal and anorectal swabs. C trachomatis genotyping was performed on all C trachomatis positive samples, using the CT-DT genotyping assay. RESULTS: Samples from 433 patients (256 female and 177 male) with confirmed C trachomatis infection were analysed. In 11 patients (2.6%), concurrent serovars in one anatomical sample site were present. In 62 (34.1%) female and four (9.3%) male patients, multiple sample site infections were found. A substantial percentage of women tested at the cervical/vaginal and rectal site were found to be positive at both sites (36.1%, 22/61). In men, D/Da and G/Ga serovars were more prevalent in rectal than urogenital specimens (p=0.0081 and p=0.0033, respectively), while serovar E was more prevalent in urogenital specimens (p=0.0012). CONCLUSIONS: The prevalence of multiple serovar infections is relatively low. Significant differences in serovar distribution are found in rectal specimens from men, with serovar G/Ga being the most prominent, suggesting tissue tropism.

Antimicrobial resistance in Neisseria gonorrhoeae and Mycoplasma genitalium isolates from the private healthcare sector in South Africa: A pilot study.

BACKGROUND: Reports have emerged globally of antimicrobial resistance (AMR) in Neisseria gonorrhoeae and Mycoplasma genitalium infections. In South Africa (SA), there are substantial differences between private and public healthcare with regard to antimicrobial drug prescribing practice, which could affect AMR patterns of private and public healthcare patients. OBJECTIVES: To perform a pilot study to determine the frequency of AMR of N. gonorrhoeae and M. genitalium in patients accessing SA's private healthcare sector. METHODS: In this cross-sectional study, N. gonorrhoeae-positive cultures and M. genitalium DNA samples were collected from a private healthcare reference laboratory from August 2018 to August 2019. In N. gonorrhoeae-positive cultures, antimicrobial susceptibility testing was performed, followed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) to determine genetic relatedness of the isolates. To determine macrolide and fluoroquinolone resistance rates, M. genitalium-positive samples were analysed by sequencing the 23S rRNA, gyrA and parC genes. RESULTS: Twenty-one N. gonorrhoeae- and 27 M. genitalium-positive specimens were included in this analysis. High rates of resistance were detected among gonococcal isolates, with 90% resistance to tetracycline, 86% to penicillin and 62% to ciprofloxacin, but no resistance to azithromycin, cefixime and ceftriaxone. NG-MAST revealed genetically diverse isolates with 83% novel NG-MAST sequence types. Macrolide and fluoroquinolone resistance-associated mutations were detected in 18.5% (n=5/27) and 7.4% (n=2/27) of M. genitalium strains, respectively. CONCLUSIONS: We observed high frequencies of ciprofloxacin, penicillin and tetracycline resistance in N. gonorrhoeae and macrolide resistance-associated mutations in M. genitalium in private healthcare sector patients in SA. This finding highlights the need to use diagnostics for sexually transmitted infections and to include the private healthcare sector in antimicrobial surveillance and stewardship programmes.

Lung microbiome of stable and exacerbated COPD patients in Tshwane, South Africa.

Chronic obstructive pulmonary disease (COPD) is characterised by the occurrence of exacerbations triggered by infections. The aim of this study was to determine the composition of the lung microbiome and lung virome in patients with COPD in an African setting and to compare their composition between the stable and exacerbated states. Twenty-four adult COPD patients were recruited from three hospitals. Sputum was collected and bacterial DNA was extracted. Targeted metagenomics was performed to determine the microbiome composition. Viral DNA and RNA were extracted from selected samples followed by cDNA conversion. Shotgun metagenomics sequencing was performed on pooled DNA and RNA. The most abundant phyla across all samples were Firmicutes and Proteobacteria. The following genera were most prevalent: Haemophilus and Streptococcus. There were no considerable differences for alpha and beta diversity measures between the disease states. However, a difference in the abundances between disease states was observed for: (i) Serratia (3% lower abundance in exacerbated state), (ii) Granulicatella (2.2% higher abundance in exacerbated state), (iii) Haemophilus (5.7% higher abundance in exacerbated state) and (iv) Veillonella (2.5% higher abundance in exacerbated state). Virome analysis showed a high abundance of the BeAn 58058 virus, a member of the Poxviridae family, in all six samples (90% to 94%). This study is among the first to report lung microbiome composition in COPD patients from Africa. In this small sample set, no differences in alpha or beta diversity between stable and exacerbated disease state was observed, but an unexpectedly high frequency of BeAn 58058 virus was observed. These observations highlight the need for further research of the lung microbiome of COPD patients in African settings.

In vitro approaches for generation of Mycobacterium tuberculosis mutants resistant to bedaquiline, clofazimine or linezolid and identification of associated genetic variants.

Bedaquiline, clofazimine and linezolid are pertinent drugs for drug-resistant tuberculosis. Drug-resistant mutants provide insight into important resistance acquisition mechanisms. Methods for in vitro Mycobacterium tuberculosis mutant generation are poorly described. Induction (serial passaging) and spontaneous (adapted Luria-Delbrück assay) approaches using M. tuberculosis ATCC reference strains (one fully-susceptible, four unique mono-resistant) were performed. Mutant MIC values were confirmed (MGIT960) and resultant RAVs compared between approaches and to a catalog of previously published RAVs. Mutant MIC values showed a 3-4-fold (induced) and a 1-4-fold (spontaneous) increase compared to baseline. The pyrazinamide-resistant strain had higher baseline MIC values and acquired resistance (≥4-fold) in fewer passages than other strains (induction approach) for bedaquiline. Previously described and novel RAVs in atpE (8 vs. 1) and rv0678 (4 vs. 12) genes were identified in bedaquiline- and clofazimine-resistant mutants. No rv1979c and rv2535c RAVs were identified. Previously described RAVs were identified in rplC and rrl genes for linezolid-resistant mutants. Both approaches successfully led to in vitro mutants with novel RAVs being described in atpE and rv0678 genes. It was observed that pre-existing resistance may influence mutant phenotypic and genotypic characteristics and warrants further attention.

Collated data of mutation frequencies and associated genetic variants of bedaquiline, clofazimine and linezolid resistance in Mycobacterium tuberculosis.

A comprehensive literature search was conducted to obtain previously published resistance associated mutations for bedaquiline, clofazimine and linezolid for Mycobacterium tuberculosis. Where possible, mutation frequencies for these three drugs were also identified. This catalog of previously published mutations could serve as a reference for comparing mutations associated with either in vitro or clinical resistant mutants. The usage of these data was seen in our study relating to approaches for resistance mutant creation (in vitro approaches for generation of Mycobacterium tuberculosis mutants resistant to bedaquiline, clofazimine or linezolid and identification of associated genetic variants (Ismail et al., 2018 in press). Previously published mutations for clofazimine were described in the rv0678 and rv1979c genes, for bedaquiline in atpE, rv0678 and rv2535c (pepQ) genes and for linezolid in the rplC and rrl genes.

Application opportunities of geographic information systems analysis to support achievement of the UNAIDS 90-90-90 targets in South Africa.

In an effort to achieve control of the HIV epidemic, 90-90-90 targets have been proposed whereby 90% of the HIV-infected population should know their status, 90% of those diagnosed should be receiving antiretroviral therapy, and 90% of those on treatment should be virologically suppressed. In this article we present approaches for using relatively simple geographic information systems (GIS) analyses of routinely available data to support HIV programme management towards achieving the 90-90-90 targets, with a focus on South Africa (SA) and other high-prevalence settings in low- and middle-income countries. We present programme-level GIS applications to map aggregated health data and individual-level applications to track distinct patients. We illustrate these applications using data from City of Johannesburg Region D, demonstrating that GIS has great potential to guide HIV programme operations and assist in achieving the 90-90-90 targets in SA.

The role of procalcitonin in a decision tree for prediction of bloodstream infection in febrile patients.

Bloodstream infection (BSI) in febrile patients is associated with high mortality. Clinical and laboratory variables, such as procalcitonin (PCT), may predict BSI and help decision-making concerning empirical treatment. This study compared two models for prediction of BSI, and evaluated the role of PCT vs. clinical variables, collected daily in 300 consecutive febrile inpatients, for 48 h after onset of fever. Multiple logistic regression (MLR) and classification and regression tree (CART) models were compared for discriminatory power and diagnostic performance. BSI was present in 17% of cases. MLR identified the presence of intravascular devices, nadir albumin and thrombocyte counts, and peak temperature, respiratory rate and leukocyte counts, but not PCT, as independent predictors of BSI. In contrast, a peak PCT level of >2.45 ng/mL was the principal discriminator in the decision tree based on CART. The latter was more accurate (94%) than the model based on MLR (72%; p <0.01). Hence, the presence of BSI in febrile patients is predicted more accurately and by different variables, e.g., PCT, in CART analysis, as compared with MLR models. This underlines the value of PCT plus CART analysis in the diagnosis of a febrile patient.

Xpert(®) MTB/RIF detection of Mycobacterium tuberculosis from sputum collected in molecular transport medium.

BACKGROUND: The Xpert(®) MTB/RIF assay is widely used for Mycobacterium tuberculosis detection. However, specimen transport remains a challenge. PrimeStore Molecular Transport Medium(®) (PS-MTM) inactivates specimens and stabilizes DNA/RNA at ambient temperature for subsequent molecular detection. OBJECTIVE: To compare the detection of M. tuberculosis concentrations in PS-MTM using Xpert and real-time polymerase chain reaction (RT-PCR), and smear-positive sputum specimens collected using a flocked swab. METHODS: Dilutions of M. tuberculosis in PS-MTM and phosphate buffered saline (PBS) were analyzed using the Xpert assay and commercial RT-PCR. Smear-positive (1+ to 3+) sputum specimens (n = 17) were transferred by flocked swab into PS-MTM and PBS, and were compared to standard 1.0 ml sputum Xpert analysis. RESULTS: Using the Xpert assay, cycle threshold values from high M. tuberculosis concentrations in PS-MTM (>10(3) colony forming units [cfu]/ml) were increased compared to control. In contrast, M. tuberculosis samples containing <10(3) cfu/ml, i.e., low concentrations, suspended in PS-MTM resulted in detection down to 10 cfu/ml. Xpert detection efficiency in PS-MTM treated samples (63.2%) was improved compared to PBS controls (34.9%). Xpert detected M. tuberculosis in all sputum specimens collected by flocked swabs in PS-MTM, and correlated with routine Xpert detection. CONCLUSIONS: PS-MTM enhances M. tuberculosis detection at low concentrations of M. tuberculosis, and provides a simplified and efficient collection method for Xpert detection.

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings.

SETTING: Mopani District, South Africa. OBJECTIVE: To explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStore(®) Molecular Transport Medium (PS-MTM) compared to settings where microscopy or Xpert(®) MTB/RIF is used as the baseline test. DESIGN: Two sputum specimens were collected from patients with cough of ⩾ 2 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMix(®) polymerase chain reaction (PM-PCR). RESULTS: Of 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PS-MTM/PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P < 0.001), but similar to Xpert (P = 0.33). CONCLUSION: PCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.

Clinical indicators of mycobacteraemia in adults admitted to hospital in Blantyre, Malawi.

SETTING: Adult medical wards of a central hospital in Blantyre, Malawi. OBJECTIVE: To measure the prevalence and outcome of mycobacteraemia in febrile hospitalised adults, and to determine what proportion could be identified using routine methods; to assess clinical indicators of mycobacteraemia, and the usefulness of a diagnostic trial of anti-tuberculosis treatment. DESIGN: We prospectively examined adults admitted with fever or a history of fever. All had blood cultured for bacteria and mycobacteria, chest X-ray and sputum smears. FINDINGS: Mycobacterium tuberculosis was the commonest cause of blood stream infection (BSI), affecting 57 of 344 patients (17%). In 44 (77%) patients with mycobacteraemia, TB was identified using routine investigations; it was not suspected in six (11%). Strong clinical indicators of mycobacteraemia were anaemia, HIV seropositivity, cough, chronic fever and a clinical diagnosis of AIDS on the day of admission. Of nine patients selected for a therapeutic trial of tuberculosis (TB) treatment, six had mycobacteraemia, of whom five died during the trial. Mortality on short-course chemotherapy, on the TB ward after 1 month was similar whether patients had mycobacteremia (21%) or not (32%). CONCLUSION: TB can be identified with routine methods in most patients with mycobacteraemia. If treated, mycobacteraemia has as good an early outcome as TB without mycobacteraemia. Strengthening of basic facilities is likely to improve detection and treatment of mycobacterial disease.

Ocular disease in patients with tuberculosis and HIV presenting with fever in Africa.

AIMS: To investigate ocular disease in patients with tuberculosis (TB) and HIV in Africa presenting with fever, and to determine if indirect ophthalmoscopy is useful in the diagnosis of mycobacteraemia. METHODS: A prospective study of all adult patients admitted with fever to a large central hospital in Malawi, Africa. All recruited patients had an ophthalmic examination, HIV tests, chest x ray, sputum examinations, bacterial and mycobacterial blood cultures, and malaria slide to observe the presence of parasites. RESULTS: 307 patients were recruited; 109 (36%) had TB, including 53 (17%) with mycobacteraemia; 255 (83%) had HIV and 191 (62%) had AIDS. Of the patients with TB 102 (94%) had HIV. Choroidal granulomas were found in four patients, all of whom had AIDS; three (2.8% of those with TB) had disseminated TB with mycobacteraemia, and one had persistent fever but no other evidence of TB. Among the patients with AIDS, 32 (17%) had microangiopathy manifest by cotton wool spots; one (0.5%) had signs of active cytomegalovirus (CMV) retinitis. The presence of microangiopathy was not related to TB. CONCLUSIONS: In Malawian patients with TB presenting acutely with fever, choroidal granulomas were found in 2.8%, and were concurrent with mycobacteraemia and AIDS. Ophthalmoscopy was not a useful aid in the diagnosis of mycobacteraemia. Cytomegalovirus (CMV) retinitis is rarely seen in African AIDS patients. This may be the result of mortality early in the disease course, or differences in race, HIV subtype, or comorbidity.