Diagnostic Performance of Different Laboratory Methods for the Detection of Extrapulmonary Tuberculosis.

Accurate and appropriate extrapulmonary tuberculosis (EPTB) diagnosis remains challenging due to its paucibacillary nature, requirement of invasive collection procedures, and lack of sensitive tests. This study investigated the diagnostic performance of different methods for the diagnosis of EPTB. A total of 1340 EPTB specimens were collected from presumptive EPTB patients from four different hospitals between November 2015 and March 2017. The collected specimens were tested with AFB microscopy, culture, Xpert MTB/RIF assay (Xpert), and MTBDRplus assay. Among the 1340 EPTB specimens, 49 (3.66%), 141 (10.52%), 166 (12.39%), and 154 (11.49%) were positive in AFB microscopy, culture, Xpert MTB/RIF, and MTBDRplus assay, respectively. A total of 194 (14.9%) cases were found positive in at least one of these methods. Using culture as a reference standard, the sensitivity and specificity of AFB microscopy, Xpert MTB/RIF, and MTBDRplus assay were: 27.0%/99.1%, 83.7%/96.0%, and 79.4%/96.5%, respectively. Compared to the composite reference standard, the sensitivity of culture, AFB microscopy, Xpert MTB/RIF, and MTBDRplus assay was 72.7%, 25.3%, 85.6%, and 79.4%, respectively, with a specificity of 100% for all the methods. The Xpert MTB/RIF assay showed the highest sensitivity compared to other methods. Considering the short turnaround time and promising findings, Xpert MTB/RIF assay should be integrated into national TB guidelines as a routine diagnostic test.

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.

Xpert MTB/RIF Ultra assay for the detection of Mycobacterium tuberculosis in people with negative conventional Xpert MTB/RIF but chest imaging suggestive of tuberculosis in Dhaka, Bangladesh.

BACKGROUND: The World Health Organization is considering substituting Xpert MTB/RIF (Xpert) with Xpert MTB/RIF Ultra (Ultra) for tuberculosis (TB) diagnosis, but supportive evidence is scarce, particularly among people more likely (presumptive) to have paucibacillary pulmonary TB (PTB). METHODS: During January-July 2018, presumptive PTB patients visiting TB Screening and Treatment Centres of Dhaka for routine chest X-ray (CXR) and conventional Xpert were enrolled. Sputum specimens were additionally tested with microscopy, culture, and Ultra. Specimens with "Trace call" by Ultra (Ultra-trace) were retested. Yield and diagnostic accuracy using various approaches to Ultra-trace and concordance of Ultra with bacteriological-positive PTB were assessed. RESULTS: Altogether, 1,083 participants (104 'Xpert-positive'; 979 'Xpert-negative and CXR-suggestive') were enrolled. All Xpert-positives and 900 (92%) Xpert-negatives were concordant with Ultra, however, seventy-nine (8.1%) Xpert-negative specimens tested positive with Ultra; 37 (46.8%) were categorically positives, and 42 (53.2%) were Ultra-trace. Sixteen of the 42 were retested, of whom eight (50.1%) Ultra-trace turned categorically positive, leading to 45 (4.6%) additionally detected by Ultra. Ultra sensitivity and specificity were 93.9% and 94.6%, and it additionally detected 5.4% more TB patients with a concordance of 94.6% (kappa, □=0.78) compared to any bacteriologically positive specimen (microscopy, culture, or Xpert). CONCLUSION: Ultra exhibited improved detection and accuracy among Xpert-negatives in a cohort with a high likelihood of PTB.

Genetic diversity and characterization of M. tuberculosis isolates causing extrapulmonary tuberculosis in Bangladesh.

Tuberculosis (TB) remains one of the leading causes of death and Bangladesh ranks 7th among the highest TB burden countries. Though molecular epidemiological data for pulmonary TB (PTB) have previously been described in Bangladesh, data on the molecular characterization and clinical association with different lineages among extrapulmonary TB (EPTB) is lacking. The aim of the study was to investigate the molecular characterization and lineage distribution of M. tuberculosis isolates obtained from patients with EPTB in Bangladesh. Between November 2015 and March 2017, a total of 1,340 EPTB specimens including lymph node, pus, tissue, ascitic fluid, cerebrospinal fluid, pleural fluid, abscess wall, urine etc. were collected from four tertiary care hospitals in Dhaka city, Bangladesh. Among the specimens, 141 were found positive on solid culture. Molecular characterization of the 141 isolates was done by deletion analysis, spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR) analysis. Among the 141 isolates, 80 (56.7%) were found as 'modern' and the remaining 61 (43.3%) were 'ancestral' type. Spoligotyping results revealed 91 distinct patterns of which 74 isolates were unique and the remaining 67 were divided into 17 distinct clusters. East African- Indian (EAI) lineage was the most predominant, comprising 26 (18.4%) isolates, followed by the Beijing lineage (14.2%). 15-loci MIRU-VNTR analysis revealed that 132 isolates (93.5%) had unique patterns, whereas only 9 (6.5%) isolates were grouped into 4 distinct clusters. In conclusion, the study findings provide a first insight into genetic diversity of EPTB isolates in Bangladesh. The present study demonstrated that 'modern' strains were more prevalent among the EPTB cases, while EAI lineages were predominantly circulating in this region.

Xpert Ultra Assay on Stool to Diagnose Pulmonary Tuberculosis in Children.

BACKGROUND: The World Health Organization recommends the Xpert MTB/RIF Ultra assay for diagnosing pulmonary tuberculosis (PTB) in children. Though stool is a potential alternative to respiratory specimens among children, the diagnostic performance of Xpert Ultra on stool is unknown. Thus, we assessed the diagnostic performance of Xpert Ultra on stool to diagnose PTB in children. METHODS: We conducted a cross-sectional study among consecutively recruited children (< 15 years of age) with presumptive PTB admitted in 4 tertiary care hospitals in Dhaka, Bangladesh, between January 2018 and April 2019. Single induced sputum and stool specimens were subjected to culture, Xpert, and Xpert Ultra. We considered children as bacteriologically confirmed on induced sputum if any test performed on induced sputum was positive for Mycobacterium tuberculosis and bacteriologically confirmed if M. tuberculosis was detected on either induced sputum or stool. RESULTS: Of 447 children, 29 (6.5%) were bacteriologically confirmed on induced sputum and 72 (16.1%) were bacteriologically confirmed. With "bacteriologically confirmed on induced sputum" as a reference, the sensitivity and specificity of Xpert Ultra on stool were 58.6% and 88.1%, respectively. Xpert on stool had sensitivity and specificity of 37.9% and 100.0%, respectively. Among bacteriologically confirmed children, Xpert Ultra on stool was positive in 60 (83.3%), of whom 48 (80.0%) had "trace call." CONCLUSIONS: In children, Xpert Ultra on stool has better sensitivity but lesser specificity than Xpert. A high proportion of Xpert Ultra assays positive on stool had trace call. Future longitudinal studies on clinical evolution are required to provide insight on the management of children with trace call.

Social Enterprise Model (SEM) for private sector tuberculosis screening and care in Bangladesh.

BACKGROUND: In Bangladesh, about 80% of healthcare is provided by the private sector. Although free diagnosis and care is offered in the public sector, only half of the estimated number of people with tuberculosis are diagnosed, treated, and notified to the national program. Private sector engagement strategies often have been small scale and time limited. We evaluated a Social Enterprise Model combining external funding and income generation at three tuberculosis screening centres across the Dhaka Metropolitan Area for diagnosing and treating tuberculosis. METHODS AND FINDINGS: The model established three tuberculosis screening centres across Dhaka Metropolitan Area that carried the icddr,b brand and offered free Xpert MTB/RIF tests to patients visiting the screening centres for subsidized, digital chest radiographs from April 2014 to December 2017. A network of private and public health care providers, and community recommendation was formed for patient referral. No financial incentives were offered to physicians for referrals. Revenues from radiography were used to support screening centres' operation. Tuberculosis patients could choose to receive treatment from the private or public sector. Between 2014 and 2017, 1,032 private facilities networked with 8,466 private providers were mapped within the Dhaka Metropolitan Area. 64, 031 patients with TB symptoms were referred by the private providers, public sector and community residents to the three screening centres with 80% coming from private providers. 4,270 private providers made at least one referral. Overall, 10,288 pulmonary and extra-pulmonary tuberculosis cases were detected and 7,695 were bacteriologically positive by Xpert, corresponding to 28% of the total notifications in Dhaka Metropolitan Area. CONCLUSION: The model established a network of private providers who referred individuals with presumptive tuberculosis without financial incentives to icddr,b's screening centres, facilitating a quarter of total tuberculosis notifications in Dhaka Metropolitan Area. Scaling up this approach may enhance national and international tuberculosis response.

Multicenter analysis of sputum microbiota in tuberculosis patients.

The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment.

Consequences of Lack of Neonatal Breastfeeding in Infants Hospitalized for Diarrhea at an Urban Hospital, Bangladesh: A Case Control Design.

Background. Exclusive breastfeeding is important for immunity and lack of breastfeeding during the neonatal age impedes that. Our aim was to assess the consequences of lack of breastfeeding on infants with diarrhea in the neonatal period. Methods. In this design, infants from DDSS (Diarrheal Disease Surveillance System) from 2009 to 2013 were studied. Those who did not have breastfeeding or had cessation of breastfeeding at the neonatal age constituted the cases, whereas infants who continued breastfeeding since birth or for at least 6 months since birth constituted the controls. Results. The cases more often presented at a younger age, had an illiterate mother, frequently presented with complicated diarrhea, had severe wasting, and had abnormal mental status compared with the controls. In logistic regression, after adjusting for potential confounders, infants who lacked breastfeeding at the neonatal period had an independent association with dehydrating diarrhea. Conclusions. The results of our analyses suggest that infants with diarrhea who did not have breastfeeding at the neonatal age are prone to develop some or severe dehydration.

Evaluation of Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in stool samples of adults with pulmonary tuberculosis.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay technology allows rapid and sensitive diagnosis of pulmonary tuberculosis (PTB) from sputum specimens. However, diagnosis of PTB is difficult for patients who cannot produce sputum. The objective of this study was to investigate the use of Xpert assay for successful detection of PTB using stool samples from adult subjects. METHODS: Both stool and sputum samples from known smear and Xpert positive PTB patients were collected from a TB hospital in Dhaka. Stool samples were collected from healthy individuals without TB symptoms from a slum area of Dhaka. Stool and sputum samples were decontaminated and concentrated using NALC-NaOH-Na-citrate solution and the resultant sediment was used for Xpert, acid-fast bacilli (AFB) microscopy and culture. RESULTS: A total of 102 stool samples were collected from PTB patients and another 50 stool samples from healthy individuals without TB. The sensitivity of the Xpert assay for detection of M. tuberculosis in stool samples of PTB patients was 90.2% (95% CI, 82.9-95.0). All 50 stool samples from healthy individuals were negative by the assay (Specificity 100%; 95% CI, 92.9-100). Compared with the sputum culture positive results the sensitivity of the stool Xpert assay was 94.8% (95% CI, 88.5-97.8). Moreover, stool Xpert demonstrated full concordant results with the sputum culture for detection of rifampicin susceptibility. The cycle threshold values of rpoB probes obtained from Xpert assay correlated significantly with the bacilli load in the corresponding stool (Spearman correlation = -0.40, P < 0.01) and sputum (Spearman correlation = -0.77, P < 0.01) samples as determined by microscopy. CONCLUSIONS: Stool Xpert can be applied as a potential alternative of sputum testing for detection of M. tuberculosis and accurate determination of RIF susceptibility in adult PTB patients. The assay would be beneficial for rapid diagnosis of PTB for those adult patients who cannot expectorate sputum.

Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh.

OBJECTIVE: Better methods are needed for the accurate detection of child tuberculosis (TB). This study compared different laboratory tests and evaluated IS6110 PCR for the detection of Mycobacterium tuberculosis (MTB) among clinically diagnosed child TB patients. METHODS: A total of 102 paediatric patients (<15 years old) with clinically diagnosed TB were enrolled in this study. The patients were admitted to the icddr,b hospital in Dhaka between 2003 and 2005. Sputum/gastric lavage samples were collected for smear microscopy, culture (solid/Lowenstein-Jensen medium and liquid/MGIT), and IS6110 PCR testing. The sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of smear microscopy and PCR were compared to the two culture methods. RESULTS: Three patients were positive on smear microscopy (2.9%). MTB was detected by conventional culture in 15.7% (16/102), liquid culture in 14% (14/100), and IS6110 PCR in 61.8% (63/102). PCR detected an additional 45 patients who were undetected with the three other tests. Compared to conventional and liquid culture, respectively, smear microscopy showed sensitivity of 18.8% and 21.4%, specificity of 100% individually, PPV of 100% individually, and NPV of 86.9% and 88.7%, whereas PCR had sensitivity of 87.5% and 92.9%, specificity of 43% individually, PPV of 22.2% and 21%, and NPV of 94.9% and 97.4%. CONCLUSIONS: PCR can be useful compared to smear microscopy and culture methods and is applicable as a rapid screening test for child TB. A larger scale study is required to determine its diagnostic efficacy in improving the detection of child TB in the presence and absence of severe malnutrition.

Factors Associated with Streptococcal Bacteremia in Diarrheal Children under Five Years of Age and Their Outcome in an Urban Hospital in Bangladesh.

BACKGROUND: Although Streptococcal bacteremia is common in diarrheal children with high morbidity and mortality, no systematic data are available on Streptococcal bacteremia in diarrheal children. We sought to evaluate the factors associated with Streptococcal bacteremia in diarrheal children under five years of age and their outcome. METHODS: We used an unmatched case-control design to investigate the associated factors with Streptococcal bacteremia in all the diarrheal children under five years of age through electronic medical record system of Dhaka hospital of International Centre for Diarrhoeal Disease Research, Bangladesh. We had simultaneously used a retrospective cohort design to further evaluate the outcome of our study children. All the enrolled children had their blood culture done between January 2010 and December 2012. Comparison was made among the children with (cases = 26) and without Streptococcal bacteremia (controls = 78). Controls were selected randomly from hospitalized diarrheal children under five years of age. RESULTS: Cases had proportionately higher deaths compared to controls, but it was statistically insignificant (15% vs. 10%, p = 0.49). The cases more often presented with severe dehydration, fever, respiratory distress, severe sepsis, and abnormal mental status compared to the controls (for all p<0.05). In the logistic regression analysis, after adjusting for potential confounders, it has been found that Streptococcal bacteremia in diarrheal children under five years of age was independently associated with nutritional edema (OR: 5.86, 95% CI = 1.28-26.80), hypoxemia (OR: 19.39, 95% CI = 2.14-175.91), fever (OR: 4.44, 95% CI = 1.13-17.42), delayed capillary refill time (OR: 7.00, 95% CI = 1.36-35.93), and respiratory distress (OR: 2.69, 95% CI = 1.02-7.12). CONCLUSIONS AND SIGNIFICANCE: The results of our analyses suggest that diarrheal children under five years of age presenting with nutritional edema, hypoxemia, fever, delayed capillary refill time, and respiratory distress may be at risk of Streptococcal bacteremia. It underscores the importance of identification of these simple clinical parameters for the prompt recognition and management in order to reduce the morbidity and death of such children especially in resource limited settings.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.