Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures

<p>Abstract</p> <p>Background</p> <p>Capilia TB is a simple immunochromatographic assay based on the detection of MPB64 antigen specifically secreted by the <it>Mycobacterium tuberculosis </it>complex (MTC). Capilia TB was evaluated for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 systems in Kampala, Uganda. Since most studies have mainly dealt with respiratory samples, the performance of Capilia TB on blood culture samples was also evaluated.</p> <p>Methods</p> <p>One thousand samples from pulmonary and disseminated tuberculosis (TB) suspects admitted to the JCRC clinic and the TB wards at Old Mulago hospital in Kampala, Uganda, were cultured in automated BACTEC MGIT 960 and BACTEC 9120 blood culture systems. BACTEC-positive samples were screened for purity by sub-culturing on blood agar plates. Two hundred and fifty three (253) samples with Acid fast bacilli (AFB, 174 BACTEC MGIT 960 and 79 BACTEC 9120 blood cultures) were analyzed for presence of MTC using Capilia TB and in-house PCR assays.</p> <p>Results</p> <p>The overall Sensitivity, Specificity, Positive and Negative Predictive values, and Kappa statistic for Capilia TB assay for identification of MTC were 98.4%, 97.6%, 97.7%, 98.4% and 0.96, respectively. Initially, the performance of in-house PCR on BACTEC 9120 blood cultures was poor (Sensitivity, Specificity, PPV, NPV and Kappa statistic of 100%, 29.3%,7%, 100% and 0.04, respectively) but improved upon sub-culturing on solid medium (Middlebrook 7H10) to 100%, 95.6%, 98.2%, 100% and 0.98, respectively. In contrast, the Sensitivity and Specificity of Capilia TB assay was 98.4% and 97.9%, respectively, both with BACTEC blood cultures and Middlebrook 7H10 cultured samples, revealing that Capilia was better than in-house PCR for identification of MTC in blood cultures. Additionally, Capilia TB was cheaper than in-house PCR for individual samples ($2.03 vs.$12.59, respectively), and was easier to perform with a shorter turnaround time (20 min vs. 480 min, respectively).</p> <p>Conclusion</p> <p>Capilia TB assay is faster and cheaper than in-house PCR for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 culture systems in real-time testing of AFB positive cultures.</p

Evaluation of the SD Bioline TB Ag MPT64 test for identification of <i>Mycobacterium tuberculosis</i> complex from liquid cultures in Southwestern Uganda

Background: To confirm presence of Mycobacterium tuberculosis complex, some tuberculosis culture laboratories still rely on para-nitrobenzoic acid (PNB), a traditional technique that requires sub-culturing of clinical isolates and two to three weeks to give results. Rapid identification tests have improved turnaround times for mycobacterial culture results. Considering the challenges of the PNB method, we assessed the performance of the SD Bioline TB Ag MPT64 assay by using PNB as gold standard to detect M. tuberculosis complex from acid-fast bacilli (AFB) positive cultures. Objectives: The aim of this study was to determine the sensitivity, specificity and turnaround time of the SD MPT64 assay for identification of M. tuberculosis complex, in a setting with high prevalence of tuberculosis and HIV. Methods: A convenience sample of 690 patients, with tuberculosis symptoms, was enrolled at Epicentre Mbarara Research Centre between April 2010 and June 2011. The samples were decontaminated using NALC-NaOH and re-suspended sediments inoculated in Mycobacterium Growth Indicator Tubes (MGIT) media, then incubated at 37 °C for a maximum of eight weeks. A random sample of 50 known negative cultures and 50 non-tuberculous mycobacteria isolates were tested for specificity, while sensitivity was based on AFB positivity. The time required from positive culture to reporting of results was also assessed with PNB used as the gold standard. Results: Of the 138 cultures that were AFB-positive, the sensitivity of the SD MPT64 assay was 100.0% [95% CI: 97.3 – 100] and specificity was 100.0% (95% CI, 96.4 – 100). The median time from a specimen receipt to confirmation of strain was 10 days [IQR: 8–12] with SD MPT64 and 24 days [IQR: 22–26] with PNB. Conclusion: The SD MPT64 assay is comparable to PNB for identification of M. tuberculosis complex and reduces the time to detection

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

Background: There are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy. Methods: Archived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes. Results: We tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencing Conclusion: We found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods

Predictors of delayed culture conversion among Ugandan patients.

BACKGROUND: Estimates of month-2 culture conversion, a proxy indicator of tuberculosis (TB) treatment efficacy in phase-2 trials can vary by culture-type and geographically with lower rates reported among African sites. The sub-study aimed at comparing TB detection rates of different culture media, within and across rifampicin-based regimens (R10, 15 and 20 mg/Kg) over a 6-month treatment follow-up period, and to establish predictors of month-2 culture non-conversion among HIV-negative TB patients enrolled at RIFATOX trial site in Uganda. METHODS: Unlike in other Rifatox Trial sites, it is only in Uganda were Lowenstein-Jensen (LJ) and Mycobacteria growth indicator tube (MGIT) were used throughout 6-months for treatment monitoring. Conversion rates were compared at month-2, 4 and 6 across cultures and treatment-type. Binomial regression analysis performed for predictors of month-2 non-conversion. RESULTS: Of the 100 enrolled patients, 45% had converted based on combined LJ and MGIT by month-2, with no significant differences across treatment arms, p = 0.721. LJ exhibited higher conversion rates than MGIT at month-2 (58.4% vs 56.0%, p = 0.0707) and month-4 (98.9% vs 88.4%, p = 0.0391) respectively, more so within the high-dose rifampicin arms. All patients had converted by month-6. Time-to-TB detection (TTD) on MGIT and social service jobs independently predict month-2 non-conversion. CONCLUSION: The month-2 culture conversion used in phase 2 clinical trials as surrogate marker of treatment efficacy is influenced by the culture method used for monitoring mycobacterial response to TB treatment. Therefore, multi-centric TB therapeutic trials using early efficacy endpoint should use the same culture method across sites. The Time-to-detection of MTB on MGIT prior to treatment and working in Social service jobs bear an increased risk of culture non-conversion at month-2. TRIAL REGISTRATION: ISRCTN ISRCTN55670677 . Registered 09th November 2010. Retrospectively registered

Development of treatment-decision algorithms for children evaluated for pulmonary tuberculosis: an individual participant data meta-analysis.

Background: Many children with pulmonary tuberculosis remain undiagnosed and untreated with related high morbidity and mortality. Recent advances in childhood tuberculosis algorithm development have incorporated prediction modelling, but studies so far have been small and localised, with limited generalisability. We aimed to evaluate the performance of currently used diagnostic algorithms and to use prediction modelling to develop evidence-based algorithms to assist in tuberculosis treatment decision making for children presenting to primary health-care centres. Methods: For this meta-analysis, we identified individual participant data from a WHO public call for data on the management of tuberculosis in children and adolescents and referral from childhood tuberculosis experts. We included studies that prospectively recruited consecutive participants younger than 10 years attending health-care centres in countries with a high tuberculosis incidence for clinical evaluation of pulmonary tuberculosis. We collated individual participant data including clinical, bacteriological, and radiological information and a standardised reference classification of pulmonary tuberculosis. Using this dataset, we first retrospectively evaluated the performance of several existing treatment-decision algorithms. We then used the data to develop two multivariable prediction models that included features used in clinical evaluation of pulmonary tuberculosis-one with chest x-ray features and one without-and we investigated each model's generalisability using internal-external cross-validation. The parameter coefficient estimates of the two models were scaled into two scoring systems to classify tuberculosis with a prespecified sensitivity target. The two scoring systems were used to develop two pragmatic, treatment-decision algorithms for use in primary health-care settings. Findings: Of 4718 children from 13 studies from 12 countries, 1811 (38·4%) were classified as having pulmonary tuberculosis: 541 (29·9%) bacteriologically confirmed and 1270 (70·1%) unconfirmed. Existing treatment-decision algorithms had highly variable diagnostic performance. The scoring system derived from the prediction model that included clinical features and features from chest x-ray had a combined sensitivity of 0·86 [95% CI 0·68-0·94] and specificity of 0·37 [0·15-0·66] against a composite reference standard. The scoring system derived from the model that included only clinical features had a combined sensitivity of 0·84 [95% CI 0·66-0·93] and specificity of 0·30 [0·13-0·56] against a composite reference standard. The scoring system from each model was placed after triage steps, including assessment of illness acuity and risk of poor tuberculosis-related outcomes, to develop treatment-decision algorithms. Interpretation: We adopted an evidence-based approach to develop pragmatic algorithms to guide tuberculosis treatment decisions in children, irrespective of the resources locally available. This approach will empower health workers in primary health-care settings with high tuberculosis incidence and limited resources to initiate tuberculosis treatment in children to improve access to care and reduce tuberculosis-related mortality. These algorithms have been included in the operational handbook accompanying the latest WHO guidelines on the management of tuberculosis in children and adolescents. Future prospective evaluation of algorithms, including those developed in this work, is necessary to investigate clinical performance. Funding: WHO, US National Institutes of Health

Améliorer le diagnostic de la tuberculose chez l'enfant dans un contexte de prévalence élevée de la tuberculose et du VIH

The world health organization estimates that in 2017, close to 1 million children below 15 years developed tuberculosis but only half of them were notified. Difficulty to obtain sputum in children and the paucibacillary nature of intrathoracic childhood tuberculosis challenge the diagnosis of tuberculosis in children. This leads to the common use of empirical treatment with a high risk of over or under diagnosis. Besides that, few facilities in low resource settings have adequate laboratory capacity to diagnose tuberculosis. Samples must be transported to a reference laboratory, which can effect performance of the tests, especially in the absence of cold chain.Three studies were conducted in Mbarara (Uganda) to evaluate non-respiratory samples and specimen preservation methods to improve diagnosis of pediatric tuberculosis. In the first study, we assessed the performance of XpertMTB/RIF on sputum and stool in children with presumptive tuberculosis and documented outcomes of children according to the tuberculosis treatment decision. In the second study, we assessed the performance of stool XpertMTB/RIF and urine lipoarabinomanann (LAM) among children admitted with severe illness. In the 3rd study, we determined XpertMTB/RIF and MGIT culture recovery rates of smear positive sputum specimen kept untreated at room temperature and treated with either Omnigene or ethanol over different time periods.Of 392 children (median age 3.9 years, 45.4% female and 31% HIV infected) enrolled in the 1st study, 4.3% (17/392) were microbiologically confirmed tuberculosis. Using a microbiological reference standard, sputum XpertMTB/RIF had a 90.9% sensitivity and specificity of 99.1%. The sensitivity and specificity of stool XpertMTB/RIF was 55.6% and 98.2%. The study reported mortality of 6.9% within three months with a higher proportion (10.7%) among children treated for tuberculosis compared to the non-treated children (4.5%). None of treated children with bacteriologically confirmed tuberculosis died compared to 12.3% of those treated empirically.Of 234 patients (median age 16.5 months, 48.3% female, 31.6% HIV infected, 58.5% severely malnourished) enrolled in the 2nd study, 5.1% were microbiologically confirmed tuberculosis. Stool XpertMTB/RIF had a sensitivity of 50% and specificity of 99.1%. For the urine LAM test, it was 50% and 74.1%, respectively. False positive LAM results were more common among low grade positive LAM results and occurred more frequently when urine samples had bacterial contamination.The 3rd study documented that by 15th day, there was no difference of XpertMTB/RIF recovery rate between samples treated with Omnigene or ethanol and untreated samples, meaning that in the study conditions there was no benefit of adding any preservative for samples stored at room temperature up to 15 days. We observed a substantial loss of viability of Mycobacterium tuberculosis on samples treated with Omnigene, which does not support the use of Omnigene for sample transportation before MGIT testing.In conclusion, XpertMTB/RIF on stool gave promising results for the use in children unable to provide sputum and could be an interesting alternative to more complex methods such as sputum induction and gastric aspirate for primary health care centers of limited resource countries. The low specificity of the urine LAM requires further investigation before its use for diagnosis of tuberculosis in children. Despite the encouraging XpertMTB/RIF results from specimen preserved either with Omnigene or ethanol further evaluation under routine field conditions is necessary.L’Organisation Mondiale de la Santé estime qu’en 2017 près d’un million d’enfants de moins de 15 ans ont développé la tuberculose mais seulement la moitié des cas ont été notifiés. Les difficultés pour recueillir des échantillons de crachat chez les enfants et la nature paucibacillifère de la tuberculose pédiatrique représentent de véritables challenges diagnostiques. Cela aboutit à la prescription fréquente de traitement empirique avec un risque de sur- ou sous-diagnostic. De plus, peu de laboratoires dans les pays à ressources limitées ont les capacités du diagnostic de la tuberculose. Les échantillons doivent être transportés vers des laboratoires de référence pouvant affecter les performances des tests, notamment en l’absence de chaine de froid.Trois études ont été menées à Mbarara (Ouganda) pour évaluer des échantillons non-respiratoires et des méthodes de conservation des échantillons pour améliorer le diagnostic de la tuberculose de l’enfant. Dans la première étude, nous avons évalué les performances de l’XpertMTB/RIF sur les expectorations et les selles d’enfants avec présomption de tuberculose et nous avons documenté le devenir des enfants selon la décision thérapeutique. Dans la deuxième étude, nous avons évalué les performances de l’XpertMTB/RIF dans les selles et du test lipoarabinomanann (LAM) dans les urines chez des enfants admis dans un état critique. Dans la troisième étude, nous avons déterminé le taux de détection avec XpertMTB/RIF et la culture MGIT d’échantillons de crachats frottis-positifs conservés à température ambiante sans traitement, ou traités avec Omnigène ou éthanol à différents périodes de temps.Sur 392 enfants (âge médian 3,9 ans, 45,5% de filles et 31% VIH positifs) inclus dans la 1e étude, 4,3% ont été confirmés microbiologiquement. L’XpertMTB/RIF dans le crachat avait une sensibilité de 90,9% et une spécificité de 99,1% contre un test de référence microbiologique. La sensibilité et la spécificité de l’Xpert dans les selles étaient de 55,6% et 98,2%. La mortalité était de 6,9% à trois mois, et était plus importante chez les enfants traités (10,7%) que chez les enfants non-traités (4,5%). Aucun des enfants traités pour une tuberculose microbiologiquement confirmée n’est décédé contre 12,3% de ceux traités de façon empirique.Parmi les 234 enfants (âge médian 16,5 mois, 48,3% de filles, 31,6% VIH positifs et 58,5% sévèrement malnutris) inclus dans la 2e étude, 5,1% avaient une tuberculose microbiologiquement confirmée. XpertMTB/RIF dans les selles avait une sensibilité de 50% et une spécificité de 99,1%. La sensibilité du test urinaire LAM était de 50% et la spécificité de 74,1%. Les faux positifs LAM étaient plus fréquents parmi les résultats positifs LAM de bas grade et dans les urines avec une contamination bactérienne.Dans la 3e étude, après 15jours, il n’y avait pas de différence de détection par XpertMTB/RIF entre les échantillons traités avec Omnigène ou éthanol et les échantillons non traités, ne montrant pas de bénéfice de l’ajout d’un conservateur. Nous avons décrit une baisse substantielle de viabilité de Mycobacterium tuberculosis dans les échantillons traités par Omnigène, ce qui n’est pas en faveur de l’utilisation de l’Omnigène pour le transport des échantillons avant culture MGIT.En conclusion, XpertMTB/RIF dans les selles a montré des résultats prometteurs chez les enfants ne pouvant pas cracher et pourrait être une alternative intéressante à des méthodes plus complexes comme l’induction du crachat et l’aspiration gastrique pour les centres de santé primaire des pays à ressources limitées. La faible spécificité du LAM dans les urines nécessite des investigations complémentaires avant son utilisation pour le diagnostic de la tuberculose de l’enfant. En dépit des résultats encourageants de l’XpertMTB/RIF sur les échantillons conservés avec Omnigène ou l’éthanol, des investigations complémentaires dans des conditions programmatiques sont nécessaires

Clinical Conditions of Hospitalized Older Adult Patients and Their Outcomes in a Regional Referral Hospital in Southwestern Uganda

Background. Recent advances in medicine have caused positive impact on the life expectancy of most countries, resulting in increased older adult population. Aging comes with a number of health challenges. This study investigated health conditions of older adults at admission and clinical outcomes in a regional referral hospital in southwestern Uganda. Methods. A retrospective study reviewed clinical data of older adult patients admitted between January 2016 and December 2017. Demographic data, cause of admission, length, and outcomes of hospitalization are described. Results. Up to 813 patient files were reviewed. The patients had been hospitalized to emergency, 371 (45.6%); medical, 355 (43.7%); surgical, 84 (10.3%); psychiatry, 2 (0.3%); and obstetrics and gynecology, 1 (0.1%) wards. The majority, 427 (52.5%), of the patients were females. Cancer was the most common reason for hospitalization, 130/889 (14.6%), followed by stroke, 94/889 (10.6%); heart failure, 76/889 (8.6%); chronic obstructive pulmonary disease, 56/889 (6.3%); pneumonia, 47/889 (5.3%); and head injury, 45/889 (5.1%), whilst 560 (68.9%) of the hospitalized patients were discharged, 197 (24.2%) died, 18 (2.2%) were referred for advanced care, and 38 (4.7%) escaped from the facility. The emergency ward had the highest deaths, 101 (51.3%), then medical, 56 (28.4%), and surgical, 39 (19.8%), wards. Mortality of those who died was admitted with stroke, 30 (15.2%), cancer, 21 (10.7%), head injury, 16 (8.1%), heart failure, 14 (7.1%), sepsis, 14 (7.1%), and renal disease, 12 (6.1%). On average, patients were admitted for 5 days (IQR: 3–8). Conclusions. The high proportion of mortality in this group is worrying and requires further investigations

A review of antimicrobial resistance in East Africa

Background and objectives: Knowledge of local and regional antimicrobial resistance (AMR) is important for clinical decision making. However, surveillance capacity for AMR is lacking throughout East Africa, and current AMR data are sparse. We sought to address this gap by summarising all available high-quality data on AMR in the East Africa region. Method: We searched the PubMed database and African Journals Online archives in April and May 2015 using the search term ‘antimicrobial resistance AND sub-Saharan Africa’ to find articles published from 2005 onwards. Only full-text articles in English were included. Results: We included 12 published articles in our analysis. Most articles were on bloodstream infections, hospital-based and cross-sectional in design; a majority described either community- or hospital-acquired infections. High levels of AMR to commonly-used antibiotics were reported, including 50% – 100% resistance to ampicillin and cotrimoxazole infections, emerging resistance to gentamicin (20% – 47%) and relatively high levels of resistance to ceftriaxone (46% – 69%) among Gram-negative infections. Much of the resistance was reported to be in Klebsiella species and Escherichia coli. Among Gram-positive infections, extensive resistance was reported to ampicillin (100%), gentamicin and ceftriaxone (50% – 100%), with methicillin-resistant Staphylococcus aureus prevalence ranging from 2.6% – 4.0%. Conclusion: Overall, bacterial resistance was reported among commonly-used antibiotics (ampicillin, gentamicin and ceftriaxone), raising concern that these antibiotics may no longer be useful for treating moderate or severe bacterial infections in East Africa. Thus, empirical treatment of bacterial infections needs to be reconsidered and guided by local assessment of AMR. Improvements in the limited amount of quality data and lack of harmonisation in assessing the burden of AMR are also needed

Evaluation of the Deki Reader™, an automated RDT reader and data management device, in a household survey setting in low malaria endemic southwestern Uganda

Abstract Background Early diagnosis of suspected malaria cases with a rapid diagnostic test (RDT) has been shown to be an effective malaria control tool used in many resource-constrained settings. However, poor quality control and quality assurance hinder the accurate reporting of malaria diagnoses. Recent use of a portable, battery operated RDT reader (Deki Reader™, Fio Corporation) has shown to have high agreement with visual inspection across diverse health centre settings, however evidence of its feasibility and usability during cross sectional surveys are limited. This study aimed to evaluate the performance of the Deki Reader™ in a cross-sectional survey of children from southwestern Uganda. Methods A two-stage, stratified cluster sampling survey was conducted between July and October 2014 in three districts of southwestern Uganda, with varying malaria transmission intensities. A total of 566 children aged 6–59 months were included in the analysis. Blood samples were collected and tested for malaria using: the SD Bioline Malaria Ag Pf/Pan RDT and microscopy. Results were compared between visual inspection of the RDT and by the Deki Reader™. Diagnostic performance of both methods were compared to gold-standard microscopy. Results The sensitivity and specificity of the Deki Reader™ was 94.1% (95% CI 69.2–99.6%) and 95.6% (95% CI 93.4–97.1%), respectively. The overall percent agreement between the Deki Reader™ and visual RDT inspection was 98.9% (95% CI 93.2–99.8), with kappa statistic of 0.92 (95% CI 0.85–0.98). Conclusions The findings from this study suggest that the Deki Reader™ is comparable to visual inspection and performs well in detecting microscopy-positive Plasmodium falciparum cases in a household survey setting. However, the reader’s performance was highly dependent on ensuring adequate battery life and a work environment free of dirt particles