Predictive factors for obstructive sleep apnea in adults with severe asthma receiving biologics: a single-center cross-sectional study.

PURPOSE: To evaluate the prevalence and potential predictors of obstructive sleep apnea (OSA) in a cohort of adults with severe asthma. METHODS: From March 2021 to December 2021, this cross-sectional study enrolled patients with severe asthma receiving biologics, who were consecutively referred for sleep evaluation irrespective of sleep-related symptoms. Clinical and functional data, including three OSA screening instruments (GOAL, STOP-Bang, and NoSAS) were recorded. All participants underwent a portable sleep test (ApneaLink Air™). OSA diagnosis was based on the respiratory disturbance index ≥ 5.0/h and subclassified according to severity thresholds. Data were subjected to logistic regression tests to identify possible predictors for OSA. Discrimination was estimated from the area under the curve (AUC). RESULTS: Overall, 56 outpatients were included (80% females): 54% with any OSA, 13% with moderate-to-severe OSA, and 4% with severe OSA. In the multivariate analysis, no parameter emerged as an independent predictor for OSA: age (p = 0.080), body mass index (p = 0.060), loud snoring (p = 0.130), and hypertension (p = 0.848). No screening instrument was useful to predict any OSA: GOAL (AUC: 0.714; 95% confidence interval (CI): 0.579-0.849), NoSAS (AUC: 0.645; 95% CI: 0.497-0.793), and STOP-Bang (AUC: 0.640; 95% CI: 0.493-0.788). Similarly, no screening tool was also useful for predicting moderate-to-severe OSA or severe OSA. CONCLUSION: Patients with evere asthma receiving biologics exhibit a high prevalence of OSA. However, no clinical, functional, or OSA screening instrument showed acceptable discriminatory ability to predict the presence of OSA in these patients with severe asthma.

Sleep-Disordered Breathing in Adults with Precapillary Pulmonary Hypertension: Prevalence and Predictors of Nocturnal Hypoxemia.

PURPOSE: To evaluate the frequency of sleep-disordered breathing (SDB) and predictors of the presence of nocturnal desaturation in adults with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. METHODS: Outpatients with a hemodynamic diagnosis of precapillary pulmonary hypertension who underwent portable polysomnography were evaluated. Diagnosis and severity of SDB were assessed using three well-established respiratory disturbance index (RDI) thresholds: 5.0/h, 15.0/h, and 30.0/h, while nocturnal hypoxemia was defined by the average oxygen saturation (SpO2) < 90%. Multiple linear regression analysis evaluated the potential relationships among explanatory variables with the dependent variable (average SpO2 values), with comparisons based on the standardized regression coefficient (ß). The R-squared (R2; coefficient of determination) was used to evaluate the goodness-of-fit measure for the linear regression model. RESULTS: Thirty-six adults were evaluated (69.4% females). The majority of the participants (75.0%) had SDB (26 with obstructive sleep apnea [OSA] and one with central sleep apnea [CSA]); while 50% of them had nocturnal hypoxemia. In the linear regression model (R2 = 0.391), the mean pulmonary artery pressure [mPAP] (ß - 0.668; p = 0.030) emerged as the only independent parameter of the average SpO2. CONCLUSION: Our study found that the majority of the participants had some type of SDB with a marked predominance of OSA over CSA, while half of them had nocturnal desaturation. Neither clinical and hemodynamic parameters nor the RDI was a predictor of nocturnal desaturation, except for mPAP measured during a right heart catheterization, which emerged as the only independent and significant predictor of average SpO2.

Predicting Obstructive Sleep Apnea in Patients with Insomnia: A Comparative Study with Four Screening Instruments.

PURPOSE: Obstructive sleep apnea (OSA) and insomnia are very prevalent disorders, especially in sleep-lab setting, and insomnia may be the presenting complaint of OSA. Here, we aimed to validate No-Apnea as screening tool for OSA in patients with self-reported insomnia complaints and to compare its performance with other models. METHODS: This cross-sectional study involved evaluation of No-Apnea as well as STOP-Bang, NoSAS and Epworth Sleepiness Scale (ESS) in subjects with insomnia being evaluated with full in-lab polysomnography. Discrimination was assessed by area under the curve (AUC), while predictive parameters were calculated by contingency tables. OSA severity was classified based on the apnea/hypopnea index: ≥ 5.0/h as any OSA (OSA≥5), ≥ 15.0/h as moderate/severe OSA (OSA≥15), and ≥ 30.0/h as severe OSA (OSA≥30). RESULTS: Overall, 2591 patients with a clinical diagnosis of insomnia were included. Diagnosis of OSA≥5, OSA≥15, and OSA≥30 was of 76.3%, 53.1%, and 32.6%, respectively. At all levels of OSA severity, No-Apnea had sensitivity ranging from 84.5 to 94.1% and specificity ranging from 58.2 to 35.1%. For screening of OSA≥5, OSA≥15, and OSA≥30, discriminatory ability (AUC) of No-Apnea was: 0.790 [95% confidence interval (CI) 0.770-0.810], 0.758 (95% CI 0.740-0.777), and 0.753 (95% CI 0.734-0.772), respectively. Based on AUCs, No-Apnea, STOP-Bang, and NoSAS performed similar at all levels of OSA severity. The ESS did not present satisfactory discrimination as OSA screening model. CONCLUSIONS: In a large sample of patients with insomnia, No-Apnea, STOP-Bang, and NoSAS, but not ESS, enable satisfactory and similar discrimination at all levels of OSA severity.

Comparative performance of screening instruments for obstructive sleep apnea in morbidly obese patients referred to a sleep laboratory: a prospective cross-sectional study.

PURPOSE: Obstructive sleep apnea (OSA) is very common occurrence among morbidly obese patients. Our main objectives were to validate the No-Apnea, a 2-item screening tool, in morbidly obese patients and compare its performance with three other instruments: STOP-Bang questionnaire, NoSAS score, and Epworth Sleepiness Scale (ESS). METHODS: A cross-sectional analysis of morbidly obese patients (body mass index [BMI] ≥ 35.0 kg/m2) grouped into two independent samples: bariatric surgery patients (BS) and non-bariatric surgery patients (NBS). All patients underwent overnight polysomnography. Discriminatory ability was assessed by area under the curve (AUC). OSA severity was defined by apnea/hypopnea index cut-off points: ≥ 5.0/h (OSA≥5), ≥ 15.0/h (OSA≥15), and ≥ 30.0/h (OSA≥30). RESULTS: A total of 1017 subjects (40.4% in BS cohort and 59.6% in NBS cohort) were evaluated. In the BS cohort, No-Apnea had similar discrimination to STOP-Bang and NoSAS for predicting OSA≥5 (p = 0.979 and p = 0.358, respectively), OSA≥15 (p = 0.158 and p = 0.399, respectively), and OSA≥30 (p = 0.388 and p = 0.903, respectively). In the NBS cohort, No-Apnea had similar discrimination to STOP-Bang and NoSAS for predicting OSA≥5 (p = 0.528 and p = 0.428, respectively), OSA≥15 (p = 0.825 and p = 0.108, respectively), and OSA≥30 (p = 0.458 and p = 0.186, respectively). Moreover, No-Apnea performed significantly better than ESS in both BS and NBS cohorts (p < 0.001). CONCLUSIONS: No-Apnea is a useful and practical tool for screening of OSA in morbidly obese patients, with non-inferior performance to STOP-Bang questionnaire and NoSAS score.

Fractional Exhaled Nitric Oxide Measurements and Screening of Obstructive Sleep Apnea in a Sleep-Laboratory Setting: A Cross-Sectional Study.

PURPOSE: Obstructive sleep apnea (OSA) is a common condition characterized by repetitive collapse of the upper airways and intermittent oxygen desaturation, which may lead to airway inflammation. Here, we explored whether fractional exhaled nitric oxide (FeNO) levels provide a non-invasive screening tool of OSA. METHODS: Over a 3-month period, FeNO levels were measured in consecutive non-smoking patients referred for a sleep laboratory. All patients underwent full polysomnography. OSA severity was classified based on the apnea/hypopnea index: ≥ 5.0/h as any OSA, ≥ 15.0/h as moderate/severe OSA, and ≥ 30.0/h as severe OSA. FeNO was measured by a portable device (NIOX-MINO®; Aerocrine AB, Solna, Sweden) and expressed as parts per billion (ppb). Discrimination by area under the curve (AUC) and binary logistic regression were performed. RESULTS: A total of 229 subjects were evaluated. Mean FeNO values were similar among subjects without OSA or with OSA: 16.9 ± 10.6 ppb versus 20.2 ± 14.5 ppb, p = 0.221; respectively. FeNO was not an inclusionary parameter to predict any OSA, moderate/severe OSA, and severe OSA: odds ratio (OR) 1.023 (95% confidence interval [CI]: 0.986-1.062); OR 1.012 (95% CI: 0.991-1.034); and OR 0.999 (95% CI: 0.980-1.018), respectively. The AUC values for FeNO in the diagnosis of any OSA, moderate/severe OSA, and severe OSA showed no discriminatory properties: AUC: 0.567 (95% CI: 0.464-0.670), AUC: 0.541 (95% CI: 0.465-0.618), and AUC: 0.535 (95% CI: 0.459-0.610); respectively. CONCLUSIONS: In a sleep-lab setting, our findings suggest that FeNO measurements are inconsequential in the screening of OSA in adults.

Simplifying the Screening of Obstructive Sleep Apnea With a 2-Item Model, No-Apnea: A Cross-Sectional Study.

STUDY OBJECTIVES: To develop and validate a practical model for obstructive sleep apnea (OSA) screening in adults based on objectively assessed criteria, and then compare it with two widely used tools, namely STOP-BANG and NoSAS. METHODS: This is a retrospective study of an existing database of consecutive outpatients who were referred for polysomnography for suspected sleep-disordered breathing by their primary care physicians. Area under the curve (AUC) and 2 × 2 contingency tables were employed to obtain the performance of the new instrument. RESULTS: A total of 4,072 subjects were randomly allocated into two independent cohorts: one for derivation (n = 2,037) and one for validation (n = 2,035). A mnemonic model, named No-Apnea, with two variables (neck circumference and age) was developed (total score: 0-9 points). We used the cutoff ≥ 3 to classify patients at high risk of having OSA. OSA severity was categorized by apnea-hypopnea index (AHI): any OSA (AHI 5 ≥ events/h; OSA-5), moderate/ severe OSA (AHI 15 ≥ events/h; OSA-15); and severe OSA (AHI 30 ≥ events/h; OSA-30). In the derivation cohort, the AUCs for screening of OSA-5, OSA-15, and OSA-30 were: 0.784, 0.758, and 0.754; respectively. The rate of subjects correctly screened was 78.1%, 68.8%, and 54.4%, respectively for OSA-5, OSA-15, and OSA-30. Subsequently, the model was validated confirming its reproducibility. In both cohorts, No-Apnea discrimination was similar to STOP-BANG or NoSAS. CONCLUSIONS: The No-Apnea, a 2-item model, appears to be a useful and practical tool for OSA screening, mainly when limited resources constrain referral evaluation. Despite its simplicity when compared to previously validated tools (STOP-BANG and NoSAS), the instrument exhibits similar performance characteristics.

Performance of the TB-LAMP diagnostic assay in reference laboratories: Results from a multicentre study.

OBJECTIVE: To evaluate the diagnostic performance of TB-LAMP, a manual molecular tuberculosis (TB) detection method, and provide comparison to the Xpert MTB/RIF assay. METHODS: In a large multicentre study, two sputum samples were collected from participants with TB symptoms in reference laboratories in Peru, South Africa, Brazil, and Vietnam. Each sample was tested with TB-LAMP. The reference standard consisted of four direct smears, four cultures, and clinical and radiological findings. Individuals negative on conventional tests were followed up after 8 weeks. The Xpert MTB/RIF assay was performed on fresh or frozen samples as a molecular test comparison. RESULTS: A total of 1036 adults with suspected TB were enrolled. Among 375 culture-confirmed TB cases with 750 sputum samples, TB-LAMP detected 75.6% (95% confidence interval (CI) 71.8-79.4%), including 97.9% (95% CI 96.4-99.4%) of smear-positive TB samples and 46.6% (95% CI 40.6-52.7%) of smear-negative TB samples. Specificity in 477 culture-negative participants not treated for TB (954 sputum samples) was 98.7% (95% CI 97.9-99.6%). TB-LAMP test results were indeterminate in 0.3% of cases. CONCLUSIONS: TB-LAMP detects nearly all smear-positive and half of smear-negative TB cases and has a high specificity when performed in reference laboratories. Performance was similar to the Xpert MTB/RIF assay.

Transcriptomic Biomarkers for Tuberculosis: Evaluation of DOCK9. EPHA4, and NPC2 mRNA Expression in Peripheral Blood.

Lately, much effort has been made to find mRNA biomarkers for tuberculosis (TB) disease/infection with microarray-based approaches. In a pilot investigation, through RNA sequencing technology, we observed a prominent modulation of DOCK9, EPHA4, and NPC2 mRNA abundance in the blood of TB patients. To corroborate these findings, independent validations were performed in cohorts from different areas. Gene expression levels in blood were evaluated by quantitative real-time PCR (Brazil, n = 129) or reanalysis of public microarray data (UK: n = 96; South Africa: n = 51; Germany: n = 26; and UK/France: n = 63). In the Brazilian cohort, significant modulation of all target-genes was observed comparing TB vs. healthy recent close TB contacts (rCt). With a 92% specificity, NPC2 mRNA high expression (NPC2high) showed the highest sensitivity (85%, 95% CI 65%-96%; area under the ROC curve [AUROC] = 0.88), followed by EPHA4 (53%, 95% CI 33%-73%, AUROC = 0.73) and DOCK9 (19%, 95% CI 7%-40%; AUROC = 0.66). All the other reanalyzed cohorts corroborated the potential of NPC2high as a biomarker for TB (sensitivity: 82-100%; specificity: 94-97%). An NPC2high profile was also observed in 60% (29/48) of the tuberculin skin test positive rCt, and additional follow-up evaluation revealed changes in the expression levels of NPC2 during the different stages of Mycobacterium tuberculosis infection, suggesting that further studies are needed to evaluate modulation of this gene during latent TB and/or progression to active disease. Considering its high specificity, our data indicate, for the first time, that NPC2high might serve as an accurate single-gene biomarker for TB.

A Phase 2 Randomized Trial of a Rifapentine plus Moxifloxacin-Based Regimen for Treatment of Pulmonary Tuberculosis.

BACKGROUND: The combination of rifapentine and moxifloxacin administered daily with other anti-tuberculosis drugs is highly active in mouse models of tuberculosis chemotherapy. The objective of this phase 2 clinical trial was to determine the bactericidal activity, safety, and tolerability of a regimen comprised of rifapentine, moxifloxacin, isoniazid, and pyrazinamide administered daily during the first 8 weeks of pulmonary tuberculosis treatment. METHODS: Adults with sputum smear-positive pulmonary tuberculosis were randomized to receive either rifapentine (approximately 7.5 mg/kg) plus moxifloxacin (investigational arm), or rifampin (approximately 10 mg/kg) plus ethambutol (control) daily for 8 weeks, along with isoniazid and pyrazinamide. The primary endpoint was sputum culture status at completion of 8 weeks of treatment. RESULTS: 121 participants (56% of accrual target) were enrolled. At completion of 8 weeks of treatment, negative cultures using Löwenstein-Jensen (LJ) medium occurred in 47/60 (78%) participants in the investigational arm vs. 43/51 (84%, p = 0.47) in the control arm; negative cultures using liquid medium occurred in 37/47 (79%) in the investigational arm vs. 27/41 (66%, p = 0.23) in the control arm. Time to stable culture conversion was shorter for the investigational arm vs. the control arm using liquid culture medium (p = 0.03), but there was no difference using LJ medium. Median rifapentine area under the concentration-time curve (AUC0-24) was 313 mcg*h/mL, similar to recent studies of rifapentine dosed at 450-600 mg daily. Median moxifloxacin AUC0-24 was 28.0 mcg*h/mL, much lower than in trials where rifapentine was given only intermittently with moxifloxacin. The proportion of participants discontinuing assigned treatment for reasons other than microbiological ineligibility was higher in the investigational arm vs. the control arm (11/62 [18%] vs. 3/59 [5%], p = 0.04) although the proportions of grade 3 or higher adverse events were similar (5/62 [8%] in the investigational arm vs. 6/59 [10%, p = 0.76] in the control arm). CONCLUSION: For intensive phase daily tuberculosis treatment in combination with isoniazid and pyrazinamide, a regimen containing moxifloxacin plus low dose rifapentine was at least as bactericidal as the control regimen containing ethambutol plus standard dose rifampin. TRIAL REGISTRATION: www.ClinicalTrials.gov NCT00728507.

Development of two artificial neural network models to support the diagnosis of pulmonary tuberculosis in hospitalized patients in Rio de Janeiro, Brazil.

Pulmonary tuberculosis (PTB) remains a worldwide public health problem. Diagnostic algorithms to identify the best combination of diagnostic tests for PTB in each setting are needed for resource optimization. We developed one artificial neural network model for classification (multilayer perceptron-MLP) and another risk group assignment (self-organizing map-SOM) for PTB in hospitalized patients in a high complexity hospital in Rio de Janeiro City, using clinical and radiologic data collected from 315 presumed PTB cases admitted to isolation rooms from March 2003 to December 2004 (TB prevalence = 21.5 %). The MLP model included 7 variables-radiologic classification, age, gender, cough, night sweats, weight loss and anorexia. The sensitivity of the MLP model was 96.0 % (95 % CI ±2.0), the specificity was 89.0 % (95 % CI ±2.0), the positive predictive value was 72.5 % (95 % CI ±3.5) and the negative predictive value was 98.5 % (95 % CI ±0.5). The variable with the highest discriminative power was the radiologic classification. The high negative predictive value found in the MLP model suggests that the use of this model at the moment of hospital admission is safe. SOM model was able to correctly assign high-, medium- and low-risk groups to patients. If prospective validation in other series is confirmed, these models can become a tool for decision-making in tertiary health facilities in countries with limited resources.

The functional assessment of patients with pulmonary multidrug-resistant tuberculosis.

BACKGROUND: Tuberculosis (TB) remains an important public health problem worldwide, as its residual lesions result in functional and quality of life impairments. Few studies have investigated multiple-drug-resistant pulmonary TB (MDR-TB), and the literature regarding the functional parameters of this group of patients is scarce. Functional characterization may point to the need for post-treatment intervention measures that optimize the quality of life in patients with MDR-TB. Thus, this study sought to analyze the respiratory function, functional capacity, and quality of life of patients who were treated for MDR pulmonary TB. METHODS: This study investigated a cross-sectional cohort of MDR-TB patients who underwent drug treatment for at least 18 months. Patients who had associated diseases (human immunodeficiency virus [HIV], severe heart disease, and hypertension) or disabilities that prevented them from walking were excluded. The subjects underwent the following assessments: forced spirometry, a chest radiograph, the 6-min walk test, a bioelectrical impedance analysis, maximal inspiratory and expiratory pressures, and a health-related quality of life questionnaire. RESULTS: Eighteen patients who met the eligibility criteria were enrolled. Spirometric evaluation showed that 78% of the subjects had abnormal patterns. The maximal respiratory pressures were significantly decreased in all subjects, despite the fact that their nutritional status was within the normal range. The distance completed in the 6-min walk test was less than expected in 72% of the subjects. All of the subjects who were evaluated had residual lesions, and 78% reported a worsening in their quality of life. CONCLUSIONS: In conclusion MDR-TB cured subjects exhibit impaired respiratory function and a mildly reduced functional capacity and quality of life, suggesting that a portion of these patients may require a pulmonary rehabilitation approach.

Effect of continuous positive airway pressure on fluid absorption among patients with pleural effusion due to tuberculosis.

BACKGROUND: Tuberculosis (TB) remains as an important public health problem worldwide. The most common type is pulmonary TB, and the most prevalent form of extra-pulmonary disease among HIV-negative patients is pleural disease. OBJECTIVE: The objective of the present study was to determine the effect of continuous positive airway pressure (CPAP) on fluid absorption among patients with pleural effusion due to TB. METHODS: Twenty patients were randomized into two groups. The interventional group (n=10) received CPAP three times a week during the initial four weeks of anti-TB treatment, and the control group (n=10) received anti-TB drugs only. The primary endpoint was the volume of pleural fluid after four weeks of treatment. Both groups were submitted to thoracic computed tomography using three-dimensional image reconstruction. The Mann-Whitney test for independent samples and the Wilcoxon paired samples test were used for statistical analysis. The normal distribution samples were analyzed using the unpaired t test. RESULTS: The reduction of pleural effusion volume was significantly greater in the intervention group (83.5%+/-SD 3.6) than in the control group (36.9%+/-SD 2.9; p<0.001), and the final dyspnea index was lower in the Intervention group than in the control group (p=0.002). CONCLUSION: Our findings indicate that CPAP during the first month of TB treatment accelerates the absorption of pleural effusion, however, additional studies are needed to confirm these findings and evaluate the impact of CPAP on pleural sequelae after the end of anti-TB treatment. Article registered in the Clinical Trials under the number NCT00560521.

Effect of continuous positive airway pressure on fluid absorption among patients with pleural effusion due to tuberculosis / Efeito da pressão positiva nas vias aéreas sobre a absorção do derrame pleural em pacientes devido à tuberculose

BACKGROUND: Tuberculosis (TB) remains as an important public health problem worldwide. The most common type is pulmonary TB, and the most prevalent form of extra-pulmonary disease among HIV-negative patients is pleural disease. OBJECTIVE: The objective of the present study was to determine the effect of continuous positive airway pressure (CPAP) on fluid absorption among patients with pleural effusion due to TB. METHODS: Twenty patients were randomized into two groups. The interventional group (n=10) received CPAP three times a week during the initial four weeks of anti-TB treatment, and the control group (n=10) received anti-TB drugs only. The primary endpoint was the volume of pleural fluid after four weeks of treatment. Both groups were submitted to thoracic computed tomography using three-dimensional image reconstruction. The Mann-Whitney test for independent samples and the Wilcoxon paired samples test were used for statistical analysis. The normal distribution samples were analyzed using the unpaired t test. RESULTS: The reduction of pleural effusion volume was significantly greater in the intervention group (83.5 percent±SD 3.6) than in the control group (36.9 percent±SD 2.9; p<0.001), and the final dyspnea index was lower in the Intervention group than in the control group (p=0.002). CONCLUSION: Our findings indicate that CPAP during the first month of TB treatment accelerates the absorption of pleural effusion, however, additional studies are needed to confirm these findings and evaluate the impact of CPAP on pleural sequelae after the end of anti-TB treatment.

CONTEXTUALIZAÇÃO: A tuberculose (TB) permanece como um importante problema de saúde pública no mundo. A forma mais comum de apresentação é a pulmonar e, em pacientes soronegativos, a forma de doença extrapulmonar mais prevalente é a pleural. OBJETIVO: O objetivo deste estudo foi determinar o efeito da pressão positiva contínua em vias aéreas (CPAP) na absorção do derrame pleural em pacientes com tuberculose. MÉTODOS: Vinte pacientes foram randomizados em dois grupos. O grupo intervenção (n=10) recebeu CPAP três vezes por semana durante as quatro primeiras semanas do tratamento anti-TB, e o grupo controle (n=10) recebeu somente droga anti-TB. O ponto final de avaliação foi o volume de líquido pleural após quatro semanas de tratamento. Ambos os grupos foram submetidos à tomografia computadorizada, usando a reconstrução tridimensional (3D) da imagem. A análise estatística foi realizada por meio do teste de Mann-Whitney para amostras independentes e Wilcoxon para amostras pareadas, e as que apresentaram distribuição normal foram analisadas por meio do teste t de Student não pareado. RESULTADOS: A redução do volume de derrame pleural foi significativamente maior no grupo intervenção (83,5 por cento±DP 3,6) que no grupo controle (36,9 por cento±DP 2,9) (p<0,001), e o índice de dispnéia final foi menor no grupo CPAP que no grupo controle (p=0,002). CONCLUSÃO: Tais achados indicam que a CPAP, durante o primeiro mês de tratamento anti-TB, acelera a absorção do derrame pleural, no entanto estudos adicionais são necessários para confirmar estes achados e avaliar o impacto da CPAP na sequela pleural após o término do tratamento anti-TB.

Sequence analysis of NAT2 gene in Brazilians: identification of undescribed single nucleotide polymorphisms and molecular modeling of the N-acetyltransferase 2 protein structure.

N-Acetyltransferase 2 (NAT2) metabolizes a variety of xenobiotics that includes many drugs, chemicals and carcinogens. This enzyme is genetically variable in human populations and polymorphisms in the NAT2 gene have been associated with drug toxicity and efficacy as well as cancer susceptibility. Here, we have focused on the identification of NAT2 variants in Brazilian individuals from two different regions, Rio de Janeiro and Goiás, by direct sequencing, and on the characterization of new haplotypes after cloning and re-sequencing. Upon analysis of DNA samples from 404 individuals, six new SNPs (c.29T>C, c.152G>T, c.203G>A, c.228C>T, c.458C>T and c.600A>G) and seven new NAT2 alleles were identified with different frequencies in Rio de Janeiro and Goiás. All new SNPs were found as singletons (observed only once in 808 genes) and were confirmed by three independent technical replicates. Molecular modeling and structural analysis suggested that p.Gly51Val variant may have an important effect on substrate recognition by NAT2. We also observed that amino acid change p.Cys68Tyr would affect acetylating activity due to the resulting geometric restrictions and incompatibility of the functional group in the Tyr side chain with the admitted chemical mechanism for catalysis by NATs. Moreover, other variants, such like p.Thr153Ile, p.Thr193Met, p.Pro228Leu and p.Val280Met, may lead to the presence of hydrophobic residues on NAT2 surface involved in protein aggregation and/or targeted degradation. Finally, the new alleles NAT2*6H and NAT2*5N, which showed the highest frequency in the Brazilian populations considered in this study, may code for a slow activity. Functional studies are needed to clarify the mechanisms by which new SNPs interfere with acetylation.

Role of IFN-gamma +874 T/A single nucleotide polymorphism in the tuberculosis outcome among Brazilians subjects.

Several genetic cytokine gene variants have been associated with host susceptibility to infectious diseases, including tuberculosis. Based upon the importance of IFN-gamma in protective immunity against Mycobacterium tuberculosis, and the functional role of the IFN-gamma + 874T/A single nucleotide polymorphism in IFN-gamma production, we genotyped 93 Brazilian tuberculosis patients and 266 asymptomatic health care workers, including 150 individuals with a positive tuberculin skin test, and analyzed the possible association of the +874A low IFN-gamma producer allele with tuberculosis occurrence. Using multivariable logistic regression models, genotype and allele frequencies of the mutant + 874A (low IFN-gamma producer) allele were significantly associated with tuberculosis disease. Heterozygous carriers had a 25% increased chance, while individuals presenting the A/A homozygous genotype had an over two-fold risk of having active tuberculosis (95% CI, 1.16-5.91, P = 0.03). Despite the mixed ethnicity observed in Brazilian populations, the present data agree with observations reported in other populations and thus demonstrate that the functional +874T/A IFN-gamma gene polymorphism is associated with tuberculosis in different populations.

Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil.

The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in approximately 30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RD(Rio). Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RD(Rio) locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RD(Rio) strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RD(Rio) lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RD(Rio) strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RD(Rio) strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RD(Rio) M. tuberculosis.

Clinical evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to isoniazid or rifampin.

This prospective study evaluated the performance of the microscopic observation drug susceptibility (MODS) assay for the direct detection of Mycobacterium tuberculosis drug resistance. MODS assay sensitivity, specificity, and positive and negative predictive values were 96.7% (95% confidence interval [95% CI], 92.1 to 98.8%), 78.4% (95% CI, 73.5 to 80.6%), 82.4% (95% CI, 78.4 to 84.2%), and 95.8% (95% CI, 89.9 to 98.5%), respectively, for isoniazid resistance and 96.0% (95% CI, 90.3 to 98.6%), 82.9% (95% CI, 78.8 to 84.7%), 80.0% (95% CI, 75.2 to 82.1%), and 96.7% (95% CI, 91.9 to 98.8%), respectively, for rifampin resistance. For both rifampin and isoniazid testing, the likelihood ratio for a negative test was < or =0.05, indicating that the MODS assay may be useful for ruling out drug resistance.

Genetic profile of the arylamine N-acetyltransferase 2 coding gene among individuals from two different regions of Brazil.

Arylamine N-acetyltranferase 2 is the main enzyme responsible for the isoniazid metabolization into hepatotoxic intermediates and the degree of hepatotoxicity severity has been attributed to genetic variability in the NAT2 gene. The main goal of this study was to describe the genetic profile of the NAT2 gene in individuals from two different regions of Brazil: Rio de Janeiro and Goiás States. Therefore, after preparation of DNA samples from 404 individuals, genotyping of the coding region of NAT2 was performed by direct PCR sequencing. Thirteen previously described SNPs were detected in these Brazilian populations, from which seven: 191 G>A; 282 C>T; 341 T>C; 481 C>T; 590 G>A; 803 A>G and 857 G>A are the most frequent in other populations. The presence of so-called ethnic-specific SNPs in our population is in accordance with the Brazilians' multiple ancestry. Upon allele and genotype analysis, the most frequent NAT2 alleles were respectively NAT2*5B (33%), NAT2*6A (26%) and NAT2*4 (20%) being NAT2*5/*5 the more prevalent genotype (31.7%). These results clearly demonstrate the predominance in the studied Brazilian groups of NAT2 alleles associated with slow over the fast and intermediate acetylator genotypes. Additionally, in Rio de Janeiro, a significantly higher frequency of intermediate acetylation status was found when compared to Goiás (42.5% versus 25%) (p=0.05), demonstrating that different regions of a country with a population characterized by a multi-ethnic ancestry may present a large degree of variability in NAT2 allelic frequencies. This finding has implications in the determination of nationwide policies for use of appropriate anti-TB drugs.

Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis.

BACKGROUND: There is an urgent need for low-cost methods for rapid, accurate detection of Mycobacterium tuberculosis in clinical specimens. The microscopic-observation drug-susceptibility (MODS) assay is a relatively low-cost and simple liquid culture method that has been proposed for use in resource-limited environments. METHODS: This prospective study evaluated the performance of the MODS assay for detection of M. tuberculosis in persons undergoing evaluation for pulmonary tuberculosis in Brazil and Honduras. Respiratory specimens were evaluated using smear microscopy, culture on Lowenstein-Jensen medium, and culture using the MODS assay. A subset of specimens was also cultured using the Mycobacterial Growth Indicator Tube (MGIT) 960 automated system (Becton Dickinson). A study subject was considered to have tuberculosis if at least 1 culture on Lowenstein-Jensen medium was positive for M. tuberculosis. FINDINGS: A total of 1639 respiratory specimens obtained from 854 study subjects were analyzed. On a per-subject basis, MODS sensitivity was 97.5% (95% confidence interval [CI], 95.7-98.6), and specificity was 94.4% (95% CI, 93.1-95.2). Median times to detection were 21 days (interquartile range [IQR], 17-25 days) and 7 days (IQR, 5-10) for culture on Lowenstein-Jensen medium and for the MODS assay, respectively (P<.01). For 64 specimens cultured using the MGIT 960 automated system, median time to growth was similar for the MODS assay (7 days; IQR, 7-10 days) and the MGIT 960 automated system (8 days; IQR, 6-11.5 days; P=.16). The percentage of contaminated cultures was lower for the MODS assay than for culture on Lowenstein-Jensen medium (3.8% vs. 5.8%; P<.01). CONCLUSIONS: The MODS assay is a relatively simple test whose good performance characteristics for detection of pulmonary tuberculosis may make it suitable for resource-limited environments.

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR-restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S-23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.