A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis.

INTRODUCTION: Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. METHODS: A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. RESULTS: With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. CONCLUSION: Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Validating a scoring system for the diagnosis of smear-negative pulmonary tuberculosis in HIV-infected adults.

BACKGROUND: The challenge of diagnosing smear-negative pulmonary TB (tuberculosis) in people living with HIV justifies the use of instruments other than the smear test for diagnosing the disease. Considering the clinical-radiological similarities of TB amongst HIV-infected adults and children, the proposal of this study was to assess the accuracy of a scoring system used to diagnose smear-negative pulmonary TB in children and adolescents, in HIV-infected adults suspected of having smear-negative pulmonary TB. METHODS: A Phase III validation study aiming to assess the diagnostic accuracy of a scoring system for diagnosing smear-negative pulmonary TB in HIV-infected adults. The study assessed sensitivity, specificity, positive and negative likelihood ratios, and positive and negative predictive values of the scoring system. Three versions of the scoring system were tested. RESULTS: From a cohort of 2,382 (HIV-infected adults), 1276 were investigated and 128 were diagnosed with pulmonary TB. Variables associated with the diagnosis of TB were: coughing, weight loss, fever, malnutrition, chest X-ray, and positive tuberculin test. The best diagnostic performance occurred with the scoring system with new scores, with sensitivity = 81.2% (95%-CI 74.5% -88%), specificity = 78% (75.6% -80.4%), PPV = 29.2% (24.5% -33.9%) and NPV = 97.4% (96.4% -98.4%), LR+ = 3.7 (3.4-4.0) and LR- = 0.24 (0.2-0.4). CONCLUSION: The proposed scoring system (with new scores) presented a good capacity for discriminating patients who did not have pulmonary TB, in the studied population. Further studies are necessary in order to validate it, thus permitting the assessment of its use in diagnosing smear-negative pulmonary TB in HIV-infected adults.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost. .

Factors related to changes in CD4+ T-cell counts over time in patients living with HIV/AIDS: a multilevel analysis.

BACKGROUND: The measurement of CD4+ T-cell (CD4) counts is a strong predictor of progression to AIDS and a means of monitoring antiviral therapy (ART). The success or failure of controlling virus levels in untreated patients or those taking ART may be associated with treatment adherence, habits, correlated infections unrelated to HIV, cancer, immunosuppressive drugs; as well as socio-economic and psychosocial aspects and access to healthcare. The aim of the present study was to identify, using a multilevel model, the factors related to the variations of CD4 counts over time, in patients living with HIV. METHODOLOGY: A cohort study was conducted with patients living with HIV, selected from July 2007 to December 2010. Patients were monitored from records of their first CD4 count after being diagnosed with HIV. A multilevel model with 3 levels of aggregation was applied to analyze the associations of predictor variables and the behavior of CD4 over time. PRINCIPAL FINDINGS: A total of 1870 patients were enrolled. The mean number of CD4 at the beginning of the cohort was 393.1 cells/mm³, and there was a mean increase of 1.529 cells/mm³ per month. Patient's age, smoking, use of illicit drugs, hospital treatment, changing doctors and the use of ART, were factors that affected the kinetics of the CD4 count during the follow-up period. CONCLUSION/SIGNIFICANCE: The results of this study indicated increased levels of CD4 over time in a cohort of patients living with HIV/AIDS and identified factors that may influence this increase and are liable to intervention.

Predictive factors for repetition of the tuberculin test after a nonreactive test in patients with HIV/AIDS.

OBJECTIVE: The outcome of interest was repetition of the tuberculin skin test (TST) and the objectives were to estimate the rate of TST repetition, the probability of no TST repetition after 1 year, and the probability of no TST repetition at the end of the follow-up period in patients whose initial test was nonreactive. The study also set out to analyze factors associated with the time until TST repetition at two HIV/AIDS referral services that carry out the TST on a routine basis in Recife, Pernambuco, Brazil. METHODS: A cohort of HIV-positive patients who initially tested nonreactive on the TST were followed from November 2007 to February 2010. The Kaplan-Meier method was used to estimate the probability of not repeating the TST, and Cox's regression analysis was used to analyze the factors associated with time until repeating the TST. Cox's multivariate analysis was stratified according to each hospital where patients were followed, because this variable did not respect the principle of proportionality of risk. RESULTS: The probability of not repeating the TST for 1 year was 80.0% and at the end of the follow-up period it was 42.0%. The variables that remained associated with TST repetition in the final Cox multivariate model were an age of 40 years or older, body mass index between 18.0 and 24.9, being female, and years of schooling. CONCLUSIONS: This study encountered a very low TST repetition rate after 1 year of follow-up and identified groups of individuals who should be the target of interventions aimed at repeating the TST.

Predictive factors for repetition of the tuberculin test after a nonreactive test in patients with HIV/AIDS / Factores predictivos para la repetición de la prueba de la tuberculina después de un resultado no reactivo en pacientes con infección por el VIH/sida

OBJECTIVE: The outcome of interest was repetition of the tuberculin skin test (TST) and the objectives were to estimate the rate of TST repetition, the probability of no TST repetition after 1 year, and the probability of no TST repetition at the end of the follow-up period in patients whose initial test was nonreactive. The study also set out to analyze factors associated with the time until TST repetition at two HIV/AIDS referral services that carry out the TST on a routine basis in Recife, Pernambuco, Brazil. METHODS: A cohort of HIV-positive patients who initially tested nonreactive on the TST were followed from November 2007 to February 2010. The Kaplan-Meier method was used to estimate the probability of not repeating the TST, and Cox's regression analysis was used to analyze the factors associated with time until repeating the TST. Cox's multivariate analysis was stratified according to each hospital where patients were followed, because this variable did not respect the principle of proportionality of risk. RESULTS: The probability of not repeating the TST for 1 year was 80.0 percent and at the end of the follow-up period it was 42.0 percent. The variables that remained associated with TST repetition in the final Cox multivariate model were an age of 40 years or older, body mass index between 18.0 and 24.9, being female, and years of schooling. CONCLUSIONS: This study encountered a very low TST repetition rate after 1 year of follow-up and identified groups of individuals who should be the target of interventions aimed at repeating the TST.

OBJETIVO: El resultado principal de interés fue la repetición de la prueba de la tuberculina (PT) y los objetivos fueron calcular la tasa de repetición de la PT, la probabilidad de no repetir la PT después de un año y la probabilidad de no repetir la PT al final del período de seguimiento en los pacientes cuya prueba inicial fue no reactiva. En el estudio también se analizaron los factores asociados con el tiempo hasta la repetición de la PT en dos servicios de referencia de infección por el VIH/sida que llevan a cabo la PT de manera sistemática en Recife, Pernambuco, Brasil. MÉTODOS: Entre noviembre del 2007 y febrero del 2010 se siguió a una cohorte de pacientes seropositivos para el VIH con una PT inicialmente no reactiva. Para calcular la probabilidad de no repetir la PT se empleó el método de Kaplan-Meier, y para analizar los factores asociados con el tiempo hasta la repetición de la PT se usó el análisis de regresión de Cox. El análisis multifactorial de Cox se estratificó conforme a cada hospital donde se seguía a los pacientes, dado que esta variable no respetaba el principio de proporcionalidad del riesgo. RESULTADOS: La probabilidad de no repetir la PT durante un año fue de 80,0 por ciento y hacia el final del período de seguimiento fue de 42,0 por ciento. Las variables asociadas con la repetición de la PT en el modelo multifactorial de Cox final fueron una edad de 40 años o más, un índice de masa corporal de 18,0 a 24,9, el sexo femenino y los años de escolaridad. CONCLUSIONES: En este estudio se encontró una tasa de repetición de la PT muy baja después de un año de seguimiento y se identificó a los grupos de individuos que deben ser los destinatarios de las intervenciones dirigidas a repetir la prueba de la tuberculina.

Comparison between the Framingham and prospective cardiovascular of Münster scores for risk assessment of coronary heart disease in human immunodeficiency virus-positive patients in Pernambuco, Brazil.

BACKGROUND: The Framingham score is used in most studies on human immunodeficiency virus (HIV)-positive patients to estimate the risk for coronary heart disease; however, it may have some limitations for detecting risk among these individuals. OBJECTIVE: The aim of this study was to evaluate the agreement between the Framingham and Prospective Cardiovascular of Münster (PROCAM) scores among HIV-positive individuals and to investigate the factors associated with disagreement between the two scores. METHOD: A cross-sectional study was conducted in a population of HIV/acquired immunodeficiency syndrome (AIDS) patients attending the outpatient's clinics of two reference centers for HIV/AIDS in Pernambuco, Brazil. Agreement between the Framingham and PROCAM scores was evaluated using the kappa index. From this analysis, a variable called "disagreement between scores" was created, and univariate and multivariate analysis were performed to investigate the factors associated with this variable. RESULTS: The prevalence of low, moderate, and high risk were, respectively, 78.7%, 13.5%, and 7.8% by Framingham score and 88.5%, 4.3%, and 7.2% by PROCAM (kappa = 0.64, P ≤ 0.0001). Agreement in the subgroup with metabolic syndrome by the International Diabetes Federation (IDF) (kappa = 0.51, P ≤ 0.0001) and the National Cholesterol Education Program (NCEP) (kappa = 0.59, P ≤ 0.0001) criteria was moderate. The Framingham score identified greater proportion of women with moderate risk. Factors independently associated with disagreement were: smoking, sex, age, low-density lipoprotein cholesterol, diastolic blood pressure, and metabolic syndrome. CONCLUSION: There was a good agreement between the Framingham and PROCAM scores in HIV-positive patients, but a higher proportion of moderate-high risk was identified by the Framingham score. This disagreement should be evaluated in cohort studies to observe clinical outcomes over the course of time.

Risk-factors for non-adherence to antiretroviral therapy.

Cross-sectional study analyzed as case-control to identify risk factors for non-adherence to antiretroviral therapy. We studied 412 out-clinics HIV infected subjects of three public hospitals of Recife, Pernambuco. The objective was to examine the association between non-adherence to the antiretroviral therapy and biological, social-behavior and demographics and economic factors, factors related to the disease and/or treatment, factors related to life habits and depression symptoms. Variables significantly associated with non-adherence to antiretroviral therapy were: time elapsed since HIV diagnosis (p = 0.002), daily dose (p = 0.046), use of alcohol (p = 0.030) and past drug use (p = 0.048), and borderline p-values were found for educational level (p = 0.093) and family monthly income (p = 0.08). In the multivariable analysis, the factors that remained in the final model were family monthly income, time period with HIV infection and use of alcohol. No association was observed between non-adherence to antiretroviral therapy and gender, age, sexual orientation, marital status, educational level and place of residence. Based on our results and the local situation we suggest: assessment of social needs; training of partners and/or families on supporting adherence, creation of 'adherence groups' to motivate and to reassure patients on the benefits of treatment; counseling and/or psychotherapy for alcohol drinkers.

Risk-factors for non-adherence to antiretroviral therapy / Fatores preditivos de não-adesão à terapia antiretroviral

Cross-sectional study analyzed as case-control to identify risk factors for non-adherence to antiretroviral therapy. We studied 412 out-clinics HIV infected subjects of three public hospitals of Recife, Pernambuco. The objective was to examine the association between non-adherence to the antiretroviral therapy and biological, social-behavior and demographics and economic factors, factors related to the disease and/or treatment, factors related to life habits and depression symptoms. Variables significantly associated with non-adherence to antiretroviral therapy were: time elapsed since HIV diagnosis (p = 0.002), daily dose (p = 0.046), use of alcohol (p = 0.030) and past drug use (p = 0.048), and borderline p-values were found for educational level (p = 0.093) and family monthly income (p = 0.08). In the multivariable analysis, the factors that remained in the final model were family monthly income, time period with HIV infection and use of alcohol. No association was observed between non-adherence to antiretroviral therapy and gender, age, sexual orientation, marital status, educational level and place of residence. Based on our results and the local situation we suggest: assessment of social needs; training of partners and/or families on supporting adherence, creation of "adherence groups" to motivate and to reassure patients on the benefits of treatment; counseling and/or psychotherapy for alcohol drinkers.

Estudo transversal com análise tipo caso-controle, que avaliou 412 pacientes de hospitais públicos do Recife - PE, com o objetivo de identificar fatores preditivos de não adesão à terapia antiretroviral. Verificou-se associação entre não adesão à terapia antiretroviral e aspectos biológicos, sócio-comportamentais e demográficos, econômicos, relacionados à doença e ao tratamento, aos hábitos de vida e aos distúrbios do humor. Variáveis com associação estatisticamente significante com não adesão na análise univariada foram: tempo de diagnóstico (p = 0,002), maior número diário de comprimidos (p = 0,046), uso de álcool (p = 0,030) e uso de drogas no passado (p = 0,048). Observou-se associação limítrofe com grau de instrução (p = 0,093) e renda mensal familiar (p = 0,08). Após análise multivariada, permaneceram no modelo final: renda mensal familiar, tempo de diagnóstico e uso de álcool. Não houve associação entre gênero, idade, comportamento sexual, estado civil, grau de instrução, município de residência e não adesão à terapia antiretroviral. Baseados nos nossos resultados e nas características da população estudada sugerimos: valorização das necessidades sociais, sensibilização de parceiros e/ou familiares para estimular a adesão, criação de grupos de adesão para motivar e persuadir os pacientes sobre os benefícios do tratamento, aconselhamento e/ou psicoterapia para alcoolistas.