Carlton A. Evans

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

BACKGROUNDnThe 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.nnnMETHODSnChildren 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.nnnFINDINGSn218 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).nnnINTERPRETATIONnCollection 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 ≤12 years, Stegen-Toledo clinical score ≥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.

The Innovative Socio-economic Interventions Against Tuberculosis (ISIAT) project: An operational assessment

SETTINGnTuberculosis (TB) affected households in impoverished shantytowns, Lima, Peru.nnnOBJECTIVEnTo evaluate socio-economic interventions for strengthening TB control by improving uptake of TB care and prevention services.nnnDESIGNnBarriers to TB control were characterised by interviews with TB-affected families. To reduce these barriers, a multidisciplinary team offered integrated community and household socio-economic interventions aiming to: 1) enhance uptake of TB care by education, community mobilisation and psychosocial support; and 2) reduce poverty through food and cash transfers, microcredit, microenterprise and vocational training. An interim analysis was performed after the socio-economic interventions had been provided for 2078 people in 311 households of newly diagnosed TB patients for up to 34 months.nnnRESULTSnPoverty (46% earned <US

Cash transfer and microfinance interventions for tuberculosis control: review of the impact evidence and policy implications

1 per day), depression (40%), stigmatisation (77%), and perceived isolation (39%) were common among TB patients (all P < 0.05 vs. non-patients). The project had 100% recruitment, and involved 97% of TB-affected households in regular visits, 71% in community groups, 78% in psychosocial support and 77% in poverty-reduction interventions. The socio-economic interventions were associated with increases in household contact TB screening (from 82% to 96%); successful TB treatment completion (from 91% to 97%); patient human immunodeficiency virus testing (from 31% to 97%); and completion of preventive therapy (from 27% to 87%; all P < 0.0001).nnnCONCLUSIONSnSocio-economic interventions can strengthen TB control activities.nnnKEYWORDSntuberculosis; control; microcredit; poverty;social determinants.

Defining Catastrophic Costs and Comparing Their Importance for Adverse Tuberculosis Outcome with Multi-Drug Resistance: A Prospective Cohort Study, Peru

OBJECTIVEnTo quantify the impact of cash transfer and microfinance interventions on a selected list of tuberculosis (TB) risk factors and assess their potential role in supporting TB control.nnnDATA SOURCEnPublished and unpublished references identified from clinical and social electronic databases, grey literature and web sites.nnnMETHODSnEligible interventions had to be conducted in middle- or low-income countries and document an impact evaluation on any of the following outcomes: 1) TB or other respiratory infections; 2) household socio-economic position; and 3) factors mediating the association between low household socio-economic position and TB, including inadequate health-seeking behaviours, food insecurity and biological TB risk factors such as human immunodeficiency virus (HIV) and adult malnutrition. Interventions targeting special populations were excluded.nnnRESULTSnFifteen cash transfer schemes (four unconditional and 11 conditional) and seven microfinance programmes met the eligibility criteria. No intervention addressed TB or any other respiratory infection. Of 11 cash transfer and four microfinance interventions, respectively seven and four reported a positive impact on indicators of economic well-being. A positive impact on household food security was documented in respectively eight of nine and three of five cash transfer and microfinance interventions. Improved health care access was documented respectively in 10 of 12 cash transfer and four of five microfinance interventions. The only intervention evaluating impact on HIV incidence was a microfinance project that found no effect. No cash transfer or microfinance interventions had an impact on adult malnutrition.nnnCONCLUSIONSnCash transfer and microfinance interventions can positively impact TB risk factors. Evaluation studies are urgently needed to assess the impact of these social protection interventions on actual TB indicators.

Analysis of nucleotide diversity of NAT2 coding region reveals homogeneity across Native American populations and high intra-population diversity

Tom Wingfield and colleagues investigate the relationship between catastrophic costs and tuberculosis outcomes for patients receiving free tuberculosis care in Peru. Please see later in the article for the Editors Summary

Discriminating Active from Latent Tuberculosis in Patients Presenting to Community Clinics

N-acetyltransferase 2 (NAT2), an important enzyme in clinical pharmacology, metabolizes antibiotics such as isoniazid and sulfamethoxazole, and catalyzes the transformation of aromatic and heterocyclic amines from the environment and diet into carcinogenic intermediates. Polymorphisms in NAT2 account for variability in the acetylator phenotype and the pharmacokinetics of metabolized drugs. Native Americans, settled in rural areas and large cities of Latin America, are under-represented in pharmacogenetics studies; therefore, we sequenced the coding region of NAT2 in 456 chromosomes from 13 populations from the Americas, and two from Siberia, detecting nine substitutions and 11 haplotypes. Variants *4 (37%), *5B (23%) and *7B (24%) showed high frequencies. Average frequencies of fast, intermediate and slow acetylators across Native Americans were 18, 56 and 25%, respectively. NAT2 intra-population genetic diversity for Native Americans is higher than East Asians and similar to the rest of the world, and NAT2 variants are homogeneously distributed across native populations of the continent.

Gender-related factors influencing tuberculosis control in shantytowns: a qualitative study

Background Because of the high global prevalence of latent TB infection (LTBI), a key challenge in endemic settings is distinguishing patients with active TB from patients with overlapping clinical symptoms without active TB but with co-existing LTBI. Current methods are insufficiently accurate. Plasma proteomic fingerprinting can resolve this difficulty by providing a molecular snapshot defining disease state that can be used to develop point-of-care diagnostics. Methods Plasma and clinical data were obtained prospectively from patients attending community TB clinics in Peru and from household contacts. Plasma was subjected to high-throughput proteomic profiling by mass spectrometry. Statistical pattern recognition methods were used to define mass spectral patterns that distinguished patients with active TB from symptomatic controls with or without LTBI. Results 156 patients with active TB and 110 symptomatic controls (patients with respiratory symptoms without active TB) were investigated. Active TB patients were distinguishable from undifferentiated symptomatic controls with accuracy of 87% (sensitivity 84%, specificity 90%), from symptomatic controls with LTBI (accuracy of 87%, sensitivity 89%, specificity 82%) and from symptomatic controls without LTBI (accuracy 90%, sensitivity 90%, specificity 92%). Conclusions We show that active TB can be distinguished accurately from LTBI in symptomatic clinic attenders using a plasma proteomic fingerprint. Translation of biomarkers derived from this study into a robust and affordable point-of-care format will have significant implications for recognition and control of active TB in high prevalence settings.

Sputum PCR-Single-Strand Conformational Polymorphism Test for Same-Day Detection of Pyrazinamide Resistance in Tuberculosis Patients

BackgroundThere is evidence that female gender is associated with reduced likelihood of tuberculosis diagnosis and successful treatment. This study aimed to characterize gender-related barriers to tuberculosis control in Peruvian shantytowns.MethodsWe investigated attitudes and experiences relating gender to tuberculosis using the grounded theory approach to describe beliefs amongst key tuberculosis control stakeholders. These issues were explored in 22 semi-structured interviews and in four focus group discussions with 26 tuberculosis patients and 17 healthcare workers.ResultsWe found that the tuberculosis program was perceived not to be gender discriminatory and provided equal tuberculosis diagnostic and treatment care to men and women. This contrasted with stereotypical gender roles in the broader community context and a commonly expressed belief amongst patients and healthcare workers that female health inherently has a lower priority than male health. This belief was principally associated with mens predominant role in the household economy and limited employment for women in this setting. Women were also generally reported to experience the adverse psychosocial and economic consequences of tuberculosis diagnosis more than men.ConclusionsThere was a common perception that womens tuberculosis care was of secondary importance to that of men. This reflected societal gender values and occurred despite apparent gender equality in care provision. The greatest opportunities for improving womens access to tuberculosis care appear to be in improving social, political and economic structures, more than tuberculosis program modification.

Tuberculosis Diagnosis and Multidrug Resistance Testing by Direct Sputum Culture in Selective Broth without Decontamination or Centrifugation

ABSTRACT Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis PCR-single-strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and Mycobacterium tuberculosis isolates. The reliability of PCR-SSCP tests for sputum samples (n = 65) and Mycobacterium tuberculosis isolates (n = 185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated culture, the Wayne biochemical test, DNA sequencing for pncA mutations, and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputum samples, and 100% of Mycobacterium tuberculosis isolates. There was 100% agreement between PCR-SSCP results from sputum samples and Mycobacterium tuberculosis isolates and 100% concordance between 50 blinded PCR-SSCP rereadings by three observers. PCR-SSCP agreement with the four other tests for pyrazinamide resistance varied from 89 to 97%. This was similar to how frequently the four other tests for pyrazinamide resistance agreed with each other: 90 to 94% for Bactec-460, 90 to 95% for Wayne, 92 to 95% for sequencing, and 91 to 95% for broth culture. PCR-SSCP took less than 24 hours and cost approximately