Geographic distribution and predictors of diagnostic delays among possible TB patients in Uganda.

BACKGROUND: Understanding the geographic distribution and factors associated with delayed TB diagnosis may help target interventions to reduce delays and improve patient outcomes. METHODS: We conducted a secondary analysis of adults undergoing TB evaluation within a public health demonstration project in Uganda. Using Global Moran's I (GMI) and Getis-Ord GI* statistics, we evaluated for residential clustering and hotspots associated with patient-related and health system-related delays. We performed multivariate logistic regression to identify individual predictors of both types of delays. RESULTS: Of 996 adults undergoing TB evaluation (median age: 37 years, IQR 28-49), 333 (33%) experienced patient delays, and 568 (57%) experienced health system delays. Participants were clustered (GMI 0.47-0.64, P ⩽ 0.001) at the sub-county level, but there were no statistically significant hotspots for patient or health system delays. Married individuals were less likely to experience patient delays (OR 0.6, 95% CI 0.48-0.75; P < 0.001). Those aged 38-57 years (OR 1.2, 95% CI 1.07-1.38; P = 0.002) were more likely than those aged ⩾58 years to experience patient delays. Knowledge about TB (OR 0.8, 95% CI 0.63-0.98; P = 0.03) protected against health system delays. CONCLUSIONS: We did not identify geographic hotspots for TB diagnostic delays. Instead, delays were associated with individual factors such as age, marital status and TB knowledge.

CONTEXTE: Comprendre la distribution géographique et les facteurs associés aux retards de diagnostic de la TB peut aider à cibler les interventions visant à réduire les retards et à améliorer les résultats pour les patients. MÉTHODES: Nous avons effectué une analyse secondaire des adultes soumis à une évaluation de la TB dans le cadre d'un projet de démonstration de santé publique en Ouganda. À l'aide des statistiques Global Moran's I (GMI) et Getis-Ord GI*, nous avons évalué les regroupements résidentiels et les points critiques associés aux retards liés aux patients et au système de santé. Nous avons effectué une régression logistique multivariée pour identifier les prédicteurs individuels des deux types de retards. RÉSULTATS: Sur les 996 adultes soumis à une évaluation de la TB (âge médian : 37 ans, IQR 28­49), 333 (33%) ont subi des retards liés aux patients et 568 (57%) ont subi des retards liés au système de santé. Les participants étaient regroupés (GMI 0,47­0,64 ; P ⩽ 0,001) au niveau du sous-comté, mais il n'y avait pas de points critiques statistiquement significatifs pour les retards des patients ou du système de santé. Les personnes mariées étaient moins susceptibles de subir des retards de la part des patients (OR 0,6 ; 95% CI 0,48­0,75 ; P < 0,001). Les personnes âgées de 38 à 57 ans (OR 1,2 ; 95% CI 1,07­1,38 ; P = 0,002) étaient plus susceptibles que celles âgées de ⩾58 ans de subir des retards. Les connaissances sur la TB (OR 0,8 ; 95% CI 0,63­0,98 ; P = 0,03) protégeaient contre les retards du système de santé. CONCLUSIONS: Nous n'avons pas identifié de points critiques géographiques pour les retards de diagnostic de la TB. Les retards étaient plutôt associés à des facteurs individuels tels que l'âge, la situation matrimoniale et les connaissances sur la TB.

Virulence genes and plasmid replicon profiles of selected ß-lactamase-producing Acinetobacter baumannii from orthopaedic patients and the environment in a tertiary referral hospital in Tanzania, East Africa.

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its high resistance to multi-drugs and disinfectants plus its ability to survive in hospital environments. Rectal swabs were collected for screening ß-lactamases-producing Acinetobacter baumannii among hospitalized orthopedic patients at a tertiary referral hospital in Tanzania. Swabs were also taken from patients' caretakers, healthcare workers, and the neighboring inanimate environment. A total of 26 confirmed ß-lactamases producing Acinetobacter baumannii were isolated, of which 4 representative isolates (two from patients and two from hospital environment) underwent whole-genome sequencing (WGS) to detect sequence types (ST), ß-lactamases genes, plasmid replicon types, and virulence genes. All four isolates harbored multiple ß-lactamases genes including blaADC-25(3), blaOXA(4), blaCTX-M-15(2) and blaNDM-1(2). Furthermore, isolates harbored virulence genes encoding outer membrane protein (ompA), curli protein (csg), siderophore biosynthesis systems (enterobactin [entABCDEFS, fepABCDG, fes]; yersiniabactin [ybtAEPQSTUX, irp1, irp2, fyuA] and aerobactin [iucABCD, iutA]), transport secretion system type II (T2SS) and type III (T3SS), E. coli common pilus (ecpRABCDE operon), type 1 fimbriae (fim), arylsulfatase (aslA) and adhesions (fedC). Only isolates from patients harbored 4 plasmid replicons each, with the most common plasmid replicons being IncFIA_1; IncY_1 and IncFIB(AP001918)_1. Admitted orthopedic patients and the hospital environment act as a reservoir of multiple ß-lactamases producing Acinetobacter baumannii (including those against carbapenems like blaOXA and blaNDM-1) endowed with virulence genes, highlighting the necessity to routinely screening of orthopedic patients with open fractures on admission as well as reinforcing infection prevention and control measures to reduce the dissemination of nosocomial infection within the hospital environment.

Quantification of multidrug-resistant M. tuberculosis bacilli in sputum during the first 8 weeks of treatment.

SETTING: Mulago Hospital, Kampala, Uganda.OBJECTIVE: To quantify Mycobacterium tuberculosis in sputum during the first 8 weeks of pulmonary multidrug-resistant TB (MDR-TB) treatment.DESIGN: We enrolled consecutive adults with pulmonary MDR-TB treated according to national guidelines. We collected overnight sputum samples before treatment and weekly. Sputum samples were cultured on Middlebrook 7H11S agar to measure colony-forming units per mL (cfu/mL) and in MGIT™ 960™ media to measure time to detection (TTD). Linear mixed-effects regression was used to estimate the relational change in log10 cfu/mL and TTD.RESULTS: Twelve adults (median age: 27 years) were enrolled. Half were women, and two-thirds were HIV-positive. At baseline, median log10 cfu/mL was 5.1, decreasing by 0.29 log10 cfu/mL/week. The median TTD was 116.5 h, increasing in TTD by 36.97 h/week. The weekly change was greater in the first 2 weeks (-1.04 log10 cfu/mL/week and 120.02 h/week) than in the remaining 6 weeks (-0.17 log10 cfu/mL/week and 26.11 h/week).CONCLUSION: Serial quantitative culture measures indicate a slow, uneven rate of decline in sputum M. tuberculosis over 8 weeks of standardized pulmonary MDR-TB treatment.

The transition to Xpert MTB/RIF ultra: diagnostic accuracy for pulmonary tuberculosis in Kampala, Uganda.

BACKGROUND: The World Health Organization (WHO) has endorsed the next-generation Xpert MTB/RIF Ultra (Ultra) cartridge, and Uganda is currently transitioning from the older generation Xpert MTB/RIF (Xpert) cartridge to Ultra as the initial diagnostic test for pulmonary tuberculosis (TB). We assessed the diagnostic accuracy of Ultra for pulmonary TB among adults in Kampala, Uganda. METHODS: We sampled adults referred for Xpert testing at two hospitals and a health center over a 12-month period. We enrolled adults with positive Xpert and a random 1:1 sample with negative Xpert results. Expectorated sputum was collected for Ultra, and for solid and liquid culture testing for Xpert-negative patients. We measured sensitivity and specificity of Ultra overall and by HIV status, prior history of TB, and hospitalization, in reference to Xpert and culture results. We also assessed how classification of results in the new "trace" category affects Ultra accuracy. RESULTS: Among 698 participants included, 211 (30%) were HIV-positive and 336 (48%) had TB. The sensitivity of Ultra was 90.5% (95% CI 86.8-93.4) and specificity was 98.1% (95% CI 96.1-99.2). There were no significant differences in sensitivity and specificity by HIV status, prior history of TB or hospitalization. Xpert and Ultra results were concordant in 670 (96%) participants, with Ultra having a small reduction in specificity (difference 1.9, 95% CI 0.2 to 3.6, p=0.01). When "trace" results were considered positive for all patients, sensitivity increased by 2.1% (95% CI 0.3 to 3.9, p=0.01) without a significant reduction in specificity (- 0.8, 95% CI - 0.3 to 2.0, p=0.08). CONCLUSIONS: After 1 year of implementation, Ultra had similar performance to Xpert. Considering "trace" results to be positive in all patients increased case detection without significant loss of specificity. Longitudinal studies are needed to compare the benefit of greater diagnoses to the cost of overtreatment.

Accuracy and incremental yield of urine Xpert MTB/RIF Ultra versus Determine TB-LAM for diagnosis of pulmonary tuberculosis.

The performance of urine Xpert MTB/RIF Ultra (Xpert Ultra) for pulmonary TB diagnosis is unknown. HIV-positive and HIV-negative adults were enrolled at two health facilities in Kampala, Uganda. We compared the accuracy of urine Xpert Ultra and Determine TB-LAM in reference to sputum-based testing (positive Xpert MTB/RIF or culture), and assessed incremental yield. Urine Xpert Ultra had low sensitivity (17.2%, 95% CI 12.3-23.2) but high specificity (98.1%, 95% CI 94.4-99.6). Sensitivity reached 50.0% (95% CI 28.2-71.8) among HIV-positive patients with CD4 <100 cells/µL. Compared to Determine TB-LAM, urine Xpert Ultra was 9.4% (95% CI 3.8-14.9, P = 0.01) more sensitive, and 17.2% (95% CI 4.5-29.8, P = 0.01) more sensitive among HIV-positive patients. However, the incremental sensitivity of urine Xpert Ultra relative to sputum Xpert MTB/RIF was only 1% (95% CI -0.9 to 2.8). Urine Xpert Ultra could be an alternative for patients with advanced HIV infection unable to produce sputum.

Sputum smear microscopy in the Xpert® MTB/RIF era.

A balanced perspective is advocated for the assessment and application of the most recent and the oldest diagnostic methods for pulmonary tuberculosis (TB)-the molecular Xpert® MTB/RIF assay and microscopy for acid-fast bacilli. We discuss their respective merits and shortcomings and identify threats that may hamper their use in TB control. Neither test on its own provides all the information needed for diagnosis and treatment monitoring. Considering all aspects important for both individual patient care and disease control, neither seems 'better' than the other. The required advancement of microscopy had already been hampered before the introduction of the GeneXpert technology by unsuccessful and probably misguided attempts to decentralise culture-based diagnosis and drug susceptibility testing. It seems evident that systematic replacement of microscopy by Xpert is not a viable option for the foreseeable future. Instead, the two methods should complement each other to arrive at a comprehensive, accessible and continuous service for a maximum number of patients. This will intrinsically prioritise targeting the most potent transmitters with the worst prognosis, simultaneously offering optimised prospects for efficient TB control. New microscopy and Xpert applications are expected to ultimately make control programmes independent of culture-based methods in diagnosis, treatment monitoring and outcome assessment.

Sputum smear-positive, culture-negative state during anti-tuberculosis treatment in the MGIT liquid culture era.

The sputum smear-positive, culture-negative state poses a challenge for clinicians. Previous studies have shown that most samples with positive smears during the later stages of treatment are culture-negative. Earlier studies generally used solid culture media, which tend to be less sensitive than current liquid culture systems. We examined the smear-positive, culture-negative state in the era of MGIT™ 960™ liquid cultures. We found that the smear-positive, culture-negative state occurred less frequently with MGIT culture, and that the majority of the samples with late positive smears were culture-negative, regardless of media type.

Feasibility of a streamlined tuberculosis diagnosis and treatment initiation strategy.

OBJECTIVE: To assess the feasibility of a streamlined strategy for improving tuberculosis (TB) diagnostic evaluation and treatment initiation among patients with presumed TB. DESIGN: Single-arm interventional pilot study at five primary care health centers of a streamlined, SIngle-saMPLE (SIMPLE) TB diagnostic evaluation strategy: 1) examination of two smear results from a single spot sputum specimen using light-emitting diode fluorescence microscopy, and 2) daily transportation of smear-negative sputum samples to Xpert® MTB/RIF testing sites. RESULTS: Of 1212 adults who underwent sputum testing for TB, 99.6% had two smears examined from the spot sputum specimen. Sputum was transported for Xpert testing within 1 clinic day for 83% (907/1091) of the smear-negative patients. Of 157 (13%) patients with bacteriologically positive TB, 116 (74%) were identified using sputum smear microscopy and 41 (26%) using Xpert testing of smear-negative samples. Anti-tuberculosis treatment was initiated in 142 (90%) patients with bacteriologically positive TB, with a median time to treatment of 1 day for smear-positive patients and 6 days for smear-negative, Xpert-positive patients. CONCLUSION: The SIMPLE TB strategy led to successful incorporation of Xpert testing and rapid treatment initiation in the majority of patients with bacteriologically confirmed TB in a resource-limited setting.

Tuberculosis resistance-conferring mutations with fitness cost among HIV-positive individuals in Uganda.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is considered to be less transmissible due to the fitness cost associated with drug resistance-conferring mutations in essential genes. OBJECTIVE: To test the hypothesis that TB drug resistance-conferring mutations with fitness cost are more frequent among human immunodeficiency virus (HIV) positive than among HIV-negative patients. DESIGN: We analysed all strains from the two TB drug resistance surveys conducted in Uganda between 2008 and 2011. Strains phenotypically susceptible to rifampicin and/or isoniazid were assumed to be wild-type; in all other cases, we performed whole-genome sequencing. Mutations at the rpoB531 and katG315 codons were considered without fitness loss, whereas other rpoB codons and non-katG were considered with fitness loss. RESULTS: Of the 897 TB patients, 286 (32.1%) were HIV-positive. Mutations with fitness loss in HIV-positive and HIV-negative patients were respectively as follows: non-531 rpoB: 1.03% (n = 3), 0.71% (n = 4) (OR 1.46, 95%CI 0.58-3.68); non-katG: 0.40% (n = 1), 1.0% (n = 6) (OR 0.40, 95%CI 0.07-2.20); rpoB531: 1.49% (n = 4), 0.69% (n = 4) (OR 2.29, 95%CI 0.83-5.77); katG315: 3.86% (n = 11), 2.55% (n = 15) (OR 1.54, 95%CI 0.81-2.90). The odds of mutations with and without fitness cost were higher for patients with a history of previous anti-tuberculosis treatment. CONCLUSIONS: Our data do not support the hypothesis that resistance-conferring mutations with fitness cost are likely to be often present in HIV-positive individuals.

Impact of mycobacterial culture among HIV-infected adults with presumed TB in Uganda: a prospective cohort study.

BACKGROUND: Implementation of new tuberculosis (TB) diagnostic strategies in resource-constrained settings is challenging. We measured the impact of solid and liquid mycobacterial cultures on treatment practices for patients undergoing TB evaluation in Kampala, Uganda. METHODS: We enrolled consecutive smear-negative, human immunodeficiency virus positive adults with cough of ⩾2 weeks from September 2009 to April 2010. Laboratory technicians performed mycobacterial cultures on solid and liquid media. We compared empiric treatment decisions with solid and liquid culture in terms of diagnostic yield and time to results, and assessed impact on patient management. RESULTS: Of 200 patients enrolled, 26 (13%) had culture-confirmed TB: 22 (85%) on solid culture alone, 2 (8%) on liquid culture alone, and 2 (8%) on both solid and liquid culture. Thirty-four patients received empiric anti-tuberculosis treatment, but only 10 (29%) were culture-positive. Median time to a positive result on solid culture was 92 days (interquartile range [IQR] 69-148) compared to 106 days (IQR 66-157) for liquid culture. No patients initiated treatment following a positive result on liquid culture. CONCLUSION: The introduction of mycobacterial culture did not influence care for patients undergoing evaluation for TB in Kampala, Uganda. Attention to contextual factors surrounding implementation is needed to ensure the effective introduction of new testing strategies in low-income countries.

Contexte : La mise en œuvre de nouvelles stratégies de diagnostic de la tuberculose (TB) dans les contextes de ressources limitées constitue un défi. Nous avons mesuré l'impact des cultures mycobactériennes en milieu solide et liquide sur les pratiques de traitement des patients ayant une évaluation de la TB à Kampala, Ouganda.Méthodes : Nous avons enrôlé des patients adultes consécutifs à frottis négatif, positifs pour le virus de l'immunodéficience humaine avec toux de ⩾2 semaines, de septembre 2009 à avril 2010. Les techniciens de laboratoire ont réalisé des cultures mycobactériennes en milieu solide et liquide. Nous avons comparé les décisions de traitement empirique aux cultures en milieu solide et liquide en termes de rendement diagnostique et de délai d'obtention des résultats et nous avons évalué l'impact sur la gestion des patients.Résultats : Des 200 patients enrôlés, 26 (13%) avaient une TB confirmée par culture, 22 (85%) par culture en milieu solide seule, 2 (8%) par culture en milieu liquide seul et 2 (8%) par culture à la fois en milieu solide et liquide. Trente-quatre patients ont reçu un traitement de TB empirique, mais seulement 10 (29%) ont eu une TB à culture positive. Le délai médian d'obtention d'un résultat de culture positive en milieu solide a été de 92 jours (IQR 69­148). Le délai médian d'obtention d'un résultat de culture positive en milieu liquide a été de 106 jours (IQR 66­157). Aucun patient n'a commencé un traitement à la suite d'un résultat de culture positive en milieu liquide.Conclusion : L'introduction de la culture mycobactérienne n'a pas influencé les soins aux patients bénéficiant d'une évaluation de TB à Kampala, Ouganda. Il est nécessaire d'être attentif aux facteurs contextuels entourant la mise en œuvre afin d'assurer une introduction effective de nouvelles stratégies de tests dans les pays à faible revenu.

Marco de referencia: La ejecución de nuevas estrategias de diagnóstico de la tuberculosis (TB) en los entornos con limitación de recursos es problemática. En el presente estudio se midió la repercusión del uso del cultivo de micobacterias en medio sólido o liquido sobre las prácticas de tratamiento de los pacientes en curso de investigación diagnóstica de la TB en Kampala, Uganda.Métodos: Se incluyeron de manera consecutiva en el estudio los pacientes adultos, seronegativos frente al virus de la inmunodeficiencia humana, que consultaban por tos de ⩾2 semanas de duración y presentaban baciloscopia negativa, de septiembre del 2009 a abril del 2010. Los auxiliares de laboratorio practicaron el cultivo de micobacterias en medio sólido y medio líquido. Se compararon las decisiones empíricas de tratamiento y los tipos de cultivo, con respecto al rendimiento diagnóstico y al lapso hasta obtener los resultados y se evaluó su repercusión en el manejo de los pacientes.Resultados: De los 200 pacientes que participaron en el estudio, 26 obtuvieron confirmación del diagnóstico de TB mediante el cultivo (13%), 22 de ellos con el cultivo en medio sólido únicamente (85%), dos con el cultivo en medio líquido exclusivamente y dos con ambos tipos de cultivo (8%). Treinta y cuatro pacientes recibieron tratamiento antituberculoso empírico, pero solo 10 de ellos obtuvieron un cultivo positivo (29%). La mediana del lapso hasta obtener el resultado del cultivo en medio sólido fue 92 días (IQR 69­148). La mediana de este lapso con los cultivos en medio líquido fue 106 días (IQR 66­157). Ningún paciente inició el tratamiento antituberculoso después de haber obtenido el resultado positivo del cultivo en medio líquido.Conclusión: La introducción del cultivo para micobacterias no tiene ninguna influencia en la atención que reciben los pacientes en quienes se investiga la TB en Kampala, Uganda. Es importante prestar atención a los factores contextuales que rodean la ejecución, a fin de lograr una introducción eficaz de las nuevas estrategias diagnósticas en los países con recursos limitados.

Chest X-ray vs. Xpert® MTB/RIF assay for the diagnosis of sputum smear-negative tuberculosis in Uganda.

SETTING: An out-patient clinic in a country with high rates of tuberculosis-human immunodeficiency virus (TB-HIV) co-infection. DESIGN: Cross-sectional analytical study of 123 adults with chronic cough and no previous anti-tuberculosis treatment. Demographic, clinical, chest X-ray (CXR) and GeneXpert® MTB/RIF data were collected. Proportions of TB diagnoses using both tests were calculated and compared using an unpaired t-test. RESULTS: Sixty-six patients (53.7%) were female and 35 (28.5%) tested positive for HIV; 21 (17.1%) were Xpert-positive, while 51 (42.5%) had CXR suggestive of TB (P = 0.0018), of whom only 15 (29.4%) were Xpert-positive. CXR was suggestive of pulmonary TB in 15 (71.4%) of the 21 patients with a positive Xpert test. CONCLUSIONS: The majority of the sputum smear-negative patients did not have TB on single Xpert testing. CXR gave an overestimate of sputum smear-negative TB cases.

Distinct T-cell responses when BCG vaccination is delayed from birth to 6 weeks of age in Ugandan infants.

BACKGROUND: In Uganda, the tuberculosis vaccine BCG is administered on the first day of life. Infants delivered at home receive BCG vaccine at their first healthcare facility visit at 6 weeks of age. Our aim was to determine the effect of this delay in BCG vaccination on the induced immune response. METHODS: We assessed CD4(+) and CD8(+) T-cell responses with a 12-hour whole-blood intracellular cytokine/cytotoxic marker assay, and with a 6-day proliferation assay. RESULTS: We enrolled 92 infants: 50 had received BCG vaccine at birth and 42 at 6 weeks of age. Birth vaccination was associated with (1) greater induction of CD4(+) and CD8(+) T cells expressing either interferon γ (IFN-γ) alone or IFN-γ together with perforin and (2) induction of proliferating cells that had greater capacity to produce IFN-γ, tumor necrosis factor α (TNF-α), and interleukin 2 together, compared with delayed vaccination. CONCLUSIONS: Distinct patterns of T-cell induction occurred when BCG vaccine was given at birth and at 6 weeks of age. We propose that this diversity might impact protection against tuberculosis. Our results differ from those of studies of delayed BCG vaccination in South Africa and the Gambia, suggesting that geographical and population heterogeneity may affect the BCG vaccine-induced T-cell response.

Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment.

SETTING: Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. OBJECTIVE: To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN: Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS: Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. CONCLUSION: TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

Relapse more common than reinfection in recurrent tuberculosis 1-2 years post treatment in urban Uganda.

OBJECTIVE: To determine the proportion of recurrent tuberculosis (TB) due to relapse with the patient's initial strain or reinfection with a new strain of Mycobacterium tuberculosis 1-2 years after anti-tuberculosis treatment in Uganda, a sub-Saharan TB-endemic country. DESIGN: Records of patients with culture-confirmed TB who completed treatment at an urban Ugandan clinic were reviewed. Restriction fragment length polymorphism (RFLP) patterns were used to determine relapse or reinfection. Associations between human immunodeficiency virus (HIV) positivity and type of TB recurrence were determined. RESULTS: Of 1701 patients cured of their initial TB episode with a median follow-up of 1.24 years, 171 (10%) had TB recurrence (8.4 per 100 person-years). Rate and risk factors for recurrence were similar to other studies from sub-Saharan Africa. Insertion sequence (IS) 6110-based RFLP of paired isolates from 98 recurrences identified 80 relapses and 18 reinfections. Relapses among HIV-positive and -negative patients were respectively 79% and 85% of recurrences. CONCLUSIONS: Relapse was more common and presented earlier than reinfection in both HIV-positive and -negative TB patients 1-2 years after completing treatment. These findings impact both the choice of retreatment drug regimen, as relapsing patients are at higher risk for acquired drug resistance, and clinical trials of new TB regimens with relapse as clinical endpoint.

Characteristics and treatment outcomes of tuberculosis retreatment cases in three regional hospitals, Uganda.

SETTING: Three regional referral hospitals in Uganda with a high burden of tuberculosis (TB) and human immunodeficiency virus (HIV) cases. OBJECTIVE: To determine the treatment outcomes of TB retreatment cases and factors influencing these outcomes. DESIGN: A retrospective cohort study of routinely collected National Tuberculosis Programme data between 1 January 2009 and 31 December 2010. RESULTS: The study included 331 retreatment patients (68% males), with a median age of 36 years, 93 (28%) of whom were relapse smear-positive, 21 (6%) treatment after failure, 159 (48%) return after loss to follow-up, 26 (8%) relapse smear-negative and 32 (10%) relapse cases with no smear performed. Treatment success rates for all categories of retreatment cases ranged between 28% and 54%. Relapse smear-positive (P = 0.002) and treatment after failure (P = 0.038) cases were less likely to have a successful treatment outcome. Only 32% of the retreatment cases received a Category II treatment regimen; there was no difference in treatment success among patients who received Category II or Category I treatment regimens (P = 0.73). CONCLUSION: Management of TB retreatment cases and treatment success for all categories in three referral hospitals in Uganda was poor. Relapse smear-positive or treatment after failure cases were less likely to have a successful treatment outcome.

Time to detection of Mycobacterium tuberculosis as an alternative to quantitative cultures.

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.

Sensitivity and specificity of herpes simplex virus-2 serological assays among HIV-infected and uninfected urban Ugandans.

Herpes simplex virus type 2 (HSV-2) is a risk factor for HIV-1 infection. We characterized HSV-2 serology assay performance in HIV-positive and HIV-negative Africans. Serostatus for HSV-2 and HIV-1 was determined in 493 serum specimens stored from a community HSV-2 prevalence survey in Kampala, Uganda. HSV-2 serology by Focus HerpeSelect ELISA, Biokit HSV-2 rapid assay and Kalon HSV-2 was compared with HSV-2 Western blot (WB) according to HIV-1 serostatus. Sensitivity/specificity was: 99.5%/70.2% for Focus, 97.0%/86.4% for Biokit and 97.5%/96.2% for Kalon. Focus with Biokit confirmation improved sensitivity/specificity (99.4%/96.8%, respectively). Use of a higher Focus index value cut-off of 2.2 instead of 1.1 increased specificity from 70.2% to 92.4%. Kalon had higher specificity than Focus (P < 0.001). Of commercially available HSV-2 serological assays, Kalon alone, or Focus ELISA followed by Biokit confirmation perform best. Improved HSV-2 assays are needed for HSV-2 and HIV-1 public health activities in Africa.

Role of microscopic examination of stool specimens in the diagnosis of campylobacter infection from children with acute diarrhoea in Kampala, Uganda.

Campylobacter species are a frequent cause of enteritis and less often of extraintestinal infections in humans. The diagnosis of campylobacter infection depends mainly on culture which is difficult and expensive to be done as routine in most clinical microbiology laboratories in the developing countries. This study was conducted to determine the sensitivity and specificity of Gram-stain of the stool in diagnosis of campylobacter infection, using culture as the gold standard. A total of 226 stool specimens were obtained from children with acute diarrhoea, attending Mulago Hospital in Kampala, Uganda. Stool smears were made and conventional Gram stain done using 0.3% carbol-fuschin as counter stain for 5 minutes. Mucous part of the stool was cultured in Charcoal Ceferaperazone Deoxycholate Agar and blood contained selective media. A total of 21 stool samples (9.3%) were positive by culture and 17 (7.5%) by Gram stain. Sensitivity and specificity of Gram stain in the diagnosis of campylobacter infection was 76% and 99.5%, respectively with positive predictive value of 94.1%. A total of 127 (56.2%) had white blood cells (WBC) in stool and there was strong association between WBC in stool and the presence of campylobacter infection (P=0.001). Gram stain is a good alternative in diagnosis of campylobacter infection in place where facilities for culture are limited.

Evaluation of seven tests for the rapid detection of multidrug-resistant tuberculosis in Uganda.

SETTINGS: National Tuberculosis (TB) Reference Laboratory and Department of Medical Microbiology, College of Health Sciences, Makerere University, Kampala, Uganda. OBJECTIVE: To evaluate head-to-head rapid tests for drug susceptibility testing (DST) of Mycobacterium tuberculosis against rifampicin (RMP) and isoniazid (INH) in a resource-limited setting. METHODS: Thirty-one well-characterised strains of M. tuberculosis were tested with the nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS), MGIT 960 (Mycobacterium Growth Indicator Tube 960), Genotype MTBDRplus, Alamar blue, MTT and resazurin assays. The proportion method on Löwenstein-Jensen medium was used as the reference test. RESULTS: NRA correctly identified the resistant strains, with 100% sensitivity and specificity. MGIT 960 detected all multidrug-resistant strains but missed one RMP-monoresistant strain. Genotype MTBDRplus detected all RMP-resistant strains, but the sensitivity for detection of INH resistance was lower (88%). Sensitivity and specificity ranged from 86% to 100% for MODS and from 57% to 100% for the Alamar blue, MTT and resazurin assays. Test results were obtained within 2-14 days. CONCLUSION: In the study setting, NRA, MGIT 960 and Genotype MTBDRplus gave excellent detection of multidrug-resistant tuberculosis, with significantly shorter time to results compared to conventional testing.

Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy.

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.