A Controlled Study of Tuberculosis Diagnosis in HIV-Infected and Uninfected Children in Peru.

BACKGROUND: Diagnosing tuberculosis in children is challenging because specimens are difficult to obtain and contain low tuberculosis concentrations, especially with HIV-coinfection. Few studies included well-controls so test specificities are poorly defined. We studied tuberculosis diagnosis in 525 children with and without HIV-infection. METHODS AND FINDINGS: 'Cases' were children with suspected pulmonary tuberculosis (n = 209 HIV-negative; n = 81 HIV-positive) and asymptomatic 'well-control' children (n = 200 HIV-negative; n = 35 HIV-positive). Specimens (n = 2422) were gastric aspirates, nasopharyngeal aspirates and stools analyzed by a total of 9688 tests. All specimens were tested with an in-house hemi-nested IS6110 PCR that took <24 hours. False-positive PCR in well-controls were more frequent in HIV-infection (P≤0.01): 17% (6/35) HIV-positive well-controls versus 5.5% (11/200) HIV-negative well-controls; caused by 6.7% (7/104) versus 1.8% (11/599) of their specimens, respectively. 6.7% (116/1719) specimens from 25% (72/290) cases were PCR-positive, similar (P>0.2) for HIV-positive versus HIV-negative cases. All specimens were also tested with auramine acid-fast microscopy, microscopic-observation drug-susceptibility (MODS) liquid culture, and Lowenstein-Jensen solid culture that took ≤6 weeks and had 100% specificity (all 2112 tests on 704 specimens from 235 well-controls were negative). Microscopy-positivity was rare (0.21%, 5/2422 specimens) and all microscopy-positive specimens were culture-positive. Culture-positivity was less frequent (P≤0.01) in HIV-infection: 1.2% (1/81) HIV-positive cases versus 11% (22/209) HIV-negative cases; caused by 0.42% (2/481) versus 4.7% (58/1235) of their specimens, respectively. CONCLUSIONS: In HIV-positive children with suspected tuberculosis, diagnostic yield was so low that 1458 microscopy and culture tests were done per case confirmed and even in children with culture-proven tuberculosis most tests and specimens were false-negative; whereas PCR was so prone to false-positives that PCR-positivity was as likely in specimens from well-controls as suspected-tuberculosis cases. This demonstrates the importance of control participants in diagnostic test evaluation and that even extensive laboratory testing only rarely contributed to the care of children with suspected TB. TRIAL REGISTRATION: This study did not meet Peruvian and some other international criteria for a clinical trial but was registered with the ClinicalTrials.gov registry: ClinicalTrials.gov NCT00054769.

Antiviral resistance and correlates of virologic failure in the first cohort of HIV-infected children gaining access to structured antiretroviral therapy in Lima, Peru: a cross-sectional analysis.

BACKGROUND: The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru. METHODS: Between 2002-5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·105 RNA/ml (range: 2.1·10(3) - 1.2·10(6)), and a median CD4-count of 232 cells/µL (range: 1-1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots. RESULTS: During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure. CONCLUSIONS: Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/- NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.

Risk factors predisposing to pedestrian road traffic injury in children living in Lima, Peru: a case-control study.

OBJECTIVE: To describe the epidemiology of pedestrian road traffic injury in Lima and to identify associated child-level, family-level, and school travel-related variables. DESIGN: Case-control study. SETTING: The Instituto Nacional de Salud del Niño, the largest paediatric hospital in the city. PARTICIPANTS: Cases were children who presented because of pedestrian road traffic injury. Controls presented with other diagnoses and were matched on age, sex and severity of injury. RESULTS: Low socioeconomic status, low paternal education, traffic exposure during the trip to school, lack of supervision during outside play, and duration of outside play were all statistically significantly associated with case-control status. In multivariate logistic regression, a model combining the lack of supervision during outside play and the number of the streets crossed walking to school best predicted case-control status (p<0.001). CONCLUSIONS: These results emphasise that an assessment of children's play behaviours and school locations should be considered and integrated into any plan for an intervention designed to reduce pedestrian road traffic injury. A child-centred approach will ensure that children derive maximum benefit from sorely needed public health interventions.

Evaluation of tuberculosis diagnostics in children: 2. Methodological issues for conducting and reporting research evaluations of tuberculosis diagnostics for intrathoracic tuberculosis in children. Consensus from an expert panel.

Confirming the diagnosis of childhood tuberculosis is a major challenge. However, research on childhood tuberculosis as it relates to better diagnostics is often neglected because of technical difficulties, such as the slow growth in culture, the difficulty of obtaining specimens, and the diverse and relatively nonspecific clinical presentation of tuberculosis in this age group. Researchers often use individually designed criteria for enrollment, diagnostic classifications, and reference standards, thereby hindering the interpretation and comparability of their findings. The development of standardized research approaches and definitions is therefore needed to strengthen the evaluation of new diagnostics for detection and confirmation of tuberculosis in children. In this article we present consensus statements on methodological issues for conducting research of Tuberculosis diagnostics among children, with a focus on intrathoracic tuberculosis. The statements are complementary to a clinical research case definition presented in an accompanying publication and suggest a phased approach to diagnostics evaluation; entry criteria for enrollment; methods for classification of disease certainty, including the rational use of culture within the case definition; age categories and comorbidities for reporting results; and the need to use standard operating procedures. Special consideration is given to the performance of microbiological culture in children and we also recommend for alternative methodological approaches to report findings in a standardized manner to overcome these limitations are made. This consensus statement is an important step toward ensuring greater rigor and comparability of pediatric tuberculosis diagnostic research, with the aim of realizing the full potential of better tests for children.

Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and PCR: a prospective case-control study.

BACKGROUND: The diagnosis of pulmonary tuberculosis presents challenges in children because symptoms are non-specific, specimens are difficult to obtain, and cultures and smears of Mycobacterium tuberculosis are often negative. We assessed new diagnostic approaches for tuberculosis in children in a resource-poor country. METHODS: Children with symptoms suggestive of pulmonary tuberculosis (cases) were enrolled from August, 2002, to January, 2007, at two hospitals in Lima, Peru. Age-matched and sex-matched healthy controls were enrolled from a low-income shanty town community in south Lima. Cases were grouped into moderate-risk and high-risk categories by Stegen-Toledo score. Two specimens of each type (gastric-aspirate, nasopharyngeal-aspirate, and stool specimens) taken from each case were examined for M tuberculosis by auramine smear microscopy, broth culture by microscopic-observation drug-susceptibility (MODS) technique, standard culture on Lowenstein-Jensen medium, and heminested IS6110 PCR. Specimens from controls consisted of one nasopharyngeal-aspirate and two stool samples, examined with the same techniques. This study is registered with ClinicalTrials.gov, number NCT00054769. FINDINGS: 218 cases and 238 controls were enrolled. 22 (10%) cases had at least one positive M tuberculosis culture (from gastric aspirate in 22 cases, nasopharyngeal aspirate in 12 cases, and stool in four cases). Laboratory confirmation of tuberculosis was more frequent in cases at high risk for tuberculosis (21 [14.1%] of 149 cases with complete specimen collection were culture positive) than in cases at moderate risk for tuberculosis (one [1.6%] of 61). MODS was more sensitive than Lowenstein-Jensen culture, diagnosing 20 (90.9%) of 22 patients compared with 13 (59.1%) of 22 patients (p=0.015), and M tuberculosis isolation by MODS was faster than by Lowenstein-Jensen culture (mean 10 days, IQR 8-11, vs 25 days, 20-30; p=0.0001). All 22 culture-confirmed cases had at least one culture-positive gastric-aspirate specimen. M tuberculosis was isolated from the first gastric-aspirate specimen obtained in 16 (72.7%) of 22 cases, whereas in six (27.3%), only the second gastric-aspirate specimen was culture positive (37% greater yield by adding a second specimen). In cases at high risk for tuberculosis, positive results from one or both gastric-aspirate PCRs identified a subgroup with a 50% chance of having a positive culture (13 of 26 cases). INTERPRETATION: Collection of duplicate gastric-aspirate specimens from high-risk children for MODS culture was the best available diagnostic test for pulmonary tuberculosis. PCR was insufficiently sensitive or specific for routine diagnostic use, but in high-risk children, duplicate gastric-aspirate PCR provided same-day identification of half of all culture-positive cases.

Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method.

OBJECTIVE: The diagnosis of pulmonary tuberculosis presents challenges in children, because symptoms are nonspecific, sputa are not accessible, and Mycobacterium tuberculosis cultures and smears often are negative. The Microscopic Observation Drug Susceptibility technique is a simple, inexpensive method for Mycobacterium tuberculosis isolation with superior speed and sensitivity over Lowenstein-Jensen culture in studies of adults with pulmonary tuberculosis. The objective of this study was to determine whether Microscopic Observation Drug Susceptibility culture can improve the sensitivity and the speed of Mycobacterium tuberculosis recovery among Peruvian children with symptoms suggestive of pulmonary tuberculosis. METHODS: Two specimens of each type (gastric aspirate, nasopharyngeal aspirate, and stool specimens) were collected from each patient, examined by auramine stain, and cultured by Microscopic Observation Drug Susceptibility and Lowenstein-Jensen techniques. Patients (n = 165) were enrolled between April 2002 and February 2004 at the Instituto de Salud del Niño, the major pediatric hospital in Lima, Peru. Inclusion criteria were age < or = 12 years, Stegen-Toledo clinical score > or = 5 points, and absence of antituberculous therapy. The main outcome measurements were (1) proportion of specimens that were culture positive by Microscopic Observation Drug Susceptibility versus Lowenstein-Jensen and (2) days required for positive culture result, stratified by specimen type and auramine stain result. RESULTS: Fifteen (9%) patients had at least 1 positive Mycobacterium tuberculosis culture (from stool in 3 cases, nasopharyngeal aspirate in 8 cases, and gastric aspirate in 15 cases). Thirty-eight culture-positive specimens were obtained (22 gastric aspirate, 12 nasopharyngeal aspirates, and 4 stools). Microscopic Observation Drug Susceptibility provided significantly more positive cultures than Lowenstein-Jensen (33 of 38 specimens culture positive by Microscopic Observation Drug Susceptibility vs 21 of 38 by Lowenstein-Jensen). This was attributed to enhanced recovery of Mycobacterium tuberculosis from auramine-negative specimens (19 of 23 by Microscopic Observation Drug Susceptibility vs 9 of 23 by Lowenstein-Jensen), in contrast to similar detection rates for the 2 tests with auramine-positive samples. Similar results were found for analyses that were limited to gastric aspirates. Isolation was faster by Microscopic Observation Drug Susceptibility than Lowenstein-Jensen. CONCLUSIONS: Isolation of Mycobacterium tuberculosis from children with suspected pulmonary tuberculosis by Microscopic Observation Drug Susceptibility demonstrated greater yield and faster recovery than by Lowenstein-Jensen method, significantly improving local capabilities to detect pediatric tuberculosis in resource-poor settings.