Point-of-care tests for malaria: speeding up the diagnostics at the bedside and challenges in malaria cases detection.

Malaria remains as one of the major public health problems worldwide. About 228 million cases occurred in 2018 only, with Africa bearing about 93% of the cases. Asymptomatic population carrying the various forms of the parasite Plasmodium in endemic areas plays an important role in the spread of the disease. To tackle this battle, more sensitive and precise detection kits for malaria are crucial to better control the number of new malaria cases. In this review, we not only discuss some of the available approaches to rapidly detect new malaria cases in endemic areas but also shed light on parallel problems that may affect the detection of individuals infected with the parasite, covering kelch 13 mutation, glucose 6-phosphate dehydrogenase deficiency, and hemoglobin disorders. Available approaches for malaria detection covered in this review are focused on point-of-care tests, including portable polymerase chain reaction and aptamers.

Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review.

BACKGROUND: Fungal infections have increased during the last years due to the AIDS epidemic and immunosuppressive therapies. The available diagnostic methods, such as culture, histopathology and serology, have several drawbacks regarding sensitivity, specificity and time-consuming, while molecular methods are still expensive and dependent on many devices. In order to overcome these challenges, isothermal nucleic acid amplification techniques (INAT) arose as promising diagnostic methods for infectious diseases. OBJECTIVE: This review aimed to present and discuss the main contributions of the isothermal nucleic acid amplification techniques applied in medical mycology. METHODS: Papers containing terms for each INAT (NASBA, RCA, LAMP, CPA, SDA, HAD or PSR) and the terms 'mycoses' or 'disease, fungal' were obtained from National Center for Biotechnology Information database until August 2019. RESULTS: NASBA, RCA, LAMP and PSR are the INAT reported in the literature for detection and identification of pathogenic fungi. Despite the need of a previous conventional PCR, the RCA technique might also be used for genotyping or cryptic species differentiation, which may be important for the treatment of certain mycoses; nevertheless, LAMP is the most used INAT for pathogen detection. CONCLUSION: Among all INATs herein reviewed, LAMP seems to be the most appropriate method for fungal detection, since it is affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free and deliverable to end-users, fulfilling all ASSURED criteria of the World Health Organization for an ideal diagnostic method.

CODIV-19 et détection moléculaire du SARS-CoV-2 chez les individus asymptomatiques / CODIV-19 and molecular detection of SARS-CoV-2 in asymptomatic individuals

CONTEXTE: Le présent document ainsi que les constats qu'il énonce ont été rédigés en soutien aux travaux du ministère de la Santé et des Services sociaux (MSSS) et de l'Institut national de santé publique du Québec (INSPQ) dans le contexte de l'urgence sanitaire liée à la maladie à coronavirus (COVID-19) au Québec. L'objectif est de réaliser une recension sommaire des données publiées et de mobiliser les savoirs clés afin d'informer les décideurs publics et les professionnels de la santé et des services sociaux. Vu la nature rapide de cette réponse, les constats ou les positions qui en découlent ne reposent pas sur un repérage exhaustif des données publiées, une évaluation de la qualité méthodologique des études avec une méthode systématique ou sur un processus de consultation élaboré. Dans les circonstances d'une telle urgence de santé publique, l'INESSS reste à l'affût de toutes nouvelles données susceptibles de lui faire modifier cette réponse rapide. CONSTATS DE L'INESSS À CE JOUR: Considérant la situation actuelle de pandémie au Québec et ailleurs dans le monde, et à la lumière des informations présentées dans cet état des connaissances sur le diagnostic moléculaire de la COVID-19, l'INESSS dégage les constats suivants: -La charge virale du SARS-CoV-2 évolue dans le temps en fonction du stade de la maladie et influence le résultat du test par RT-PCR. Généralement, les faux négatifs sont attribuables à une charge virale trop faible au moment du prélèvement, surtout si celui-ci a été effectué au tout début ou à la toute fin de l'infection virale. -Un ou plusieurs résultats négatifs n'excluent pas la possibilité d'une infection par le SARS-CoV-2. -Plusieurs enquêtes épidémiologiques suggèrent que des individus auraient été infectés par des personnes pré-symptomatiques, mais aussi par des asymptomatiques. -Des données fragmentaires issues de différentes sources comportant plusieurs limites rapportent que la proportion d'individus asymptomatiques serait importante. Les chiffres disponibles varient en fonction de l'épidémiologie locale, de la population à l'étude et des conditions et critères d'accès au test. -Bien que ne faisant pas consensus, le dépistage systématique de certains groupes de personnes, comme les travailleurs de la santé et les résidents de milieux de soins de longue durée, a été recommandé par quelques organisations et experts afin de contenir la propagation de la maladie et protéger les personnes vulnérables. PRÉSENTATION DE LA DEMANDE: Dans le contexte actuel de pandémie de la COVID-19, des efforts massifs sont investis dans la détection de cette maladie. Dans un premier temps, le présent document traite de la détection moléculaire du virus SARS-CoV-2 par technique d'amplification des acides nucléiques (TAAN), et ce, en ce qui a trait aux situations cliniques à prioriser, aux performances diagnostiques attendues et aux éléments à considérer pour en assurer la fiabilité. Ce document aborde également la situation des cas asymptomatiques, notamment, leur potentiel infectieux, leur proportion et leur contribution à la chaîne de transmission et à la pertinence de les détecter, s'il y a lieu. MÉTHODOLOGIE: 1) Quelles sont les positions et orientations d'organisations savantes et d'autorités de santé en matière de priorisation de l'accès aux tests de détection du SARS-CoV-2? 2) Quelle est la performance de détection du SARS-CoV-2 par RT-PCR relativement au diagnostic de la COVID-19? a) Quels sont les proportions de résultats faussement négatifs? b) Quels sont les éléments susceptibles de nuire à la fiabilité des résultats du test par RT-PCR pour la détection du SARS-CoV-2? 3) Le dépistage systématique des asymptomatiques peut-il être pertinent et si oui, dans quelles situations? a) Quel est le potentiel infectieux des asymptomatiques (et pré-symptomatiques) et ceux présentant des formes légères de la maladie? b) Quelles sont les proportions d'asymptomatiques et de cas subcliniques dans la communauté? c) Dans quels milieux ou dans quelles situations serait-il bénéfique d'effectuer le dépistage systématique de la COVID-19? SOMMAIRE DES RÉSULTATS: 1. Positions et orientations de sociétés savantes et d'autorités de santé concernant l'utilisation du test moléculaire pour la détection du SARS-CoV-2. En date du 22 avril, vingt-et-un (21) documents en lien avec la pertinence et les critères de détection moléculaire de la COVID-19 adoptés par les autorités de santé d'autres juridictions ont été recensés. Les principaux renseignements extraits de ces documents sont les suivants: -Personnes devant systématiquement être testées ou testées en priorité; -Particularités pour les travailleurs de la santé; -Limites du test à considérer. Ces éléments sont résumés dans les paragraphes suivants. 2. Fiabilité des tests moléculaires pour la détection du SARS-CoV-2 et le diagnostic de la COVID-19 Onze (11) publications traitant de la fiabilité de la détection moléculaire du SARS-CoV-2 ont été retenues, dont : -cinq (5) présentant des proportions de faux négatifs dans le temps [Li Y.; Li D.; Long; Xie; Ai, 2020] et; -six (6) abordant certains éléments susceptibles de nuire à la qualité des résultats de la RT-PCR [Pan; Loeffelholz & Tang; Lippi; Wölfel; Lo; Wikramaratna, 2020]. 3. Potentiel infectieux des individus asymptomatiques et pré-symptomatiques Quinze (15) publications traitant du potentiel infectieux des asymptomatiques, pré-symptomatiques et de formes légères de la maladie ont été retenues.

Solicitud de incorporación de nuevo equipamiento para diagnóstico microbiológico de la tuberculosis / Application for the incorporation of new equipment for the microbiological diagnosis of tuberculosis

INTRODUCIÓN: La tuberculosis (en adelante TB) es una enfermedad causada por Mycobacterium tuberculosis, una bacteria transmitida principalmente por la inhalación de microgotas expelidas al toser, hablar y respirar. Según su localización anatómica puede clasificarse en TB pulmonar y extrapulmonar, también se divide según la co-infección con VIH y la resistencia a medicamentos. La TB es una de las principales causas de mortalidad en el mundo. En 2016, 10,4 millones de personas enfermaron de TB y 1,7 millones murieron por esta enfermedad (entre ellas 0,4 millones de personas con VIH). Más del 95% de las muertes ocurre en países de ingresos bajos y medianos. En Argentina se considera que la enfermedad tiene una carga moderada. En 2016 se notificaron 11560 casos con aumento de la tasa de notificación de 24,9 a 26,5 por 100000 habitantes. Esto determinó el aumento sostenido de la enfermedad, a lo que se sumó un incremento de casos de TB en grupos jóvenes. La provincia de Neuquén es una población de baja endemia con 50 casos incidentes anuales y con un 5 % de casos nuevos o recaídas. Casi no hay reportes de casos de multi-resistencia a drogas. El rango etario más afectado es el de 45 a 64 años, quienes podrían diseminar la enfermedad en caso de ser bacilíferos. EVALUACIÓN DE TECNOLOGÍAS: A partir de un pedido de incorporación se analizaron dos tecnologías principales y se revisaron las recomendaciones y factibilidad de incorporar otras tecnologías en distintos niveles de complejidad para mejorar la detección precoz de Mycobacterium tuberculosis. Se trabajó en la priorización de tecnologías, en la distribución actual con enfoque de mapa sanitario y analizando la organización del sistema de salud y la red prestacional de laboratorio. Las principales tecnologías sanitarias solicitadas fueron: Cultivo en medio líquido, BACTEC MGIT de BECKTON DICKINSON: tecnología fluorescente basada en el consumo de oxígeno para la detección del crecimiento bacteriano del complejo M. tuberculosis. Xpert MTB/RIF de Cepheid: tecnología de amplificación de ácidos nucleicos para la detección del complejo M. tuberculosis y la resistencia a la rifampicina. METODOLOGÍA: Se conformó un equipo multidisciplinario e independiente que luego de diversas reuniones definió preguntas de investigación que abarcó el informe. BÚSQUEDA Y ANÁLISIS DE LA EVIDENCIA CIENTÍFICA: Se realizó una búsqueda no sistemática de bibliografía científica priorizando la inclusión de revisiones sistemáticas y meta-análisis, evaluaciones de tecnologías sanitarias e informes de seguridad, guías OMS y otras Guías de Práctica Clínica basadas en la evidencia. CONSENSO: Se mantuvieron reuniones y rondas de preguntas por mail para lograr consenso sobre los aspectos donde variaban las opiniones. CONCLUSIONES: 1. Se recomienda mejorar la calidad del sistema de información clínica, de laboratorio y epidemiológica. En el 2019 se planifica la mejora del sistema informático junto com proyectos de capacitaciones a personal de salud de toda la provincia para impulsar la notificación de todos los casos. 2. Se recomienda implementar microscopía de fluorescencia en los laboratorios de los hospitales cabeceras de zona según factibilidad. 3. Realizar capacitaciones al personal de salud de las diversas zonas para estimular el pedido de baciloscopías, el adecuado seguimiento de contactos y de tratamientos. 4. A pesar de que la tecnología Gene Xpert muestre numerosas ventajas con respecto al BACTEC MGIT, se trata de un método muy costoso; Teniendo en cuenta que nuestra provincia no es una zona endémica ni presenta resistencia a fármacos se recomienda a favor de la implementación del cultivo en medio líquido automatizado, BACTEC MGIT de BECKTON DICKINSON.

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10-1 CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5 × 103 CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

Evaluación de técnicas de amplificación de ácido nucleico (NAT) para detección del virus de la inmunodeficiencia humana, hepatitis C y hepatitis B en bancos de sangre / Assessment on nucleic acid amplification techniques (nat) for human immunodeficiency, hepatitis C and hepatitis B virus screening in blood banks

CONTEXTO CLÍNICO: Las transfusiones de sangre y hemoderivados constituyen una terapéutica frecuente en la actualidad. Sin embargo, si no se toman las medidas necesarias, quienes reciben las donaciones pueden contraer ciertas enfermedades. Disminuir los riesgos de la transmisión de enfermedades infecciosas es un requisito indispensable para los servicios de medicina transfusional.Para tender a la eliminación de este riesgo, se pueden implementar tres tipos de estrategias: a) tratar los componentes con métodos para inactivación de patógenos, con la finalidad de eliminar agentes infecciosos, b) implementar políticas para la captación del donante no relacionado, habitual y responsable y c) aplicar métodos para detección de infecciones transmisibles con una mayor sensibilidad. En relación a esto último, la detección de marcadores serológicos para infecciones transmisibles por transfusión es muy eficiente y ha reducido de manera importante el impacto de esta complicación transfusional en las últimas décadas.1, 2 Sin embargo, existe un período de ventana, en el cual un donante infectado y potencialmente infectante, no presenta niveles dosables de marcadores serológicos; por este motivo se ha postulado el uso de técnicas que permitan la detección en este período de ventana. Un ejemplo de ellos es la técnica NAT (por sus siglas en inglés: nucleic acid amplification Technology), la cual es una prueba de biología molecular que se ha postulado a nivel mundial desde 1999, para la detección del material genético de los virus de la inmunodeficiencia humana (VIH), hepatitis C (VHC) y hepatitis B (VHB) con el fin de disminuir el período de ventana de no-detección, en las muestras provenientes de donantes infectados. TECNOLOGÍA: NAT se realiza mediante la realización de los siguientes pasos básicos: la preparación de la muestra, la amplificación de secuencias de ácido nucleico, y la detección de productos de amplificación (amplicons). OBJETIVO: Evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura de la técnica de amplificación de ácido nucleico en forma rutinaria para la detección del virus de la inmunodeficiencia humana, el virus de la hepatitis C y el de la hepatitis B en muestras de sangre de donantes. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas (incluyendo Medline, Cochrane y CRD), en buscadores genéricos de Internet, agencias de evaluación de tecnologías sanitarias y financiadores de salud utilizando la siguiente estrategia: (Nucleic Acid Amplification Techniques[Mesh:NoExp] OR Amplification Tech*[tiab] OR Nucleic-Acid Amplificat*[tiab] OR DNA Amplificat*[tiab] OR RNA Amplificat*[tiab] OR NAT[tiab]) AND (Blood Donors[Mesh] OR Blood Don*[tiab] OR Blood Banks[Mesh] OR Blood Bank*[tiab]). Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias y económicas, guías de práctica clínica y políticas de cobertura de otros sistemas de salud cuando estaban disponibles. RESULTADOS: Para el siguiente informe se incluyeron dos estudios observacionales, una revisión no sistemática, cuatro GPC, una ETS, seis normas y dos evaluaciones económicas. CONCLUSIONES: Si bien existe escasa evidencia de moderada calidad metodológica que muestra que la utilización de la técnica de amplificación de ácido nucleico (NAT) es efectiva en la detección de los virus de la inmunodeficiencia humana, hepatitis C y hepatitis B en el período ventana serológico, la magnitud del beneficios de su aplicación en forma rutinaria en muestras de sangre de donantes no ha sido aún aclarada. Si bien el uso de NAT se encuentra muy extendido en el mundo y muchas normativas y guías internacionales lo recomiendan; su implementación no es mandatorio en muchos otros países. La Organización Mundial de la Salud aún no lo recomienda en forma rutinaria para todos los países, sobre todo debido a que se estima que los potenciales beneficios de su aplicación son bajos a expensas de un alto costo. Su uso estaría más justificado en contextos con una mayor prevalencia e incidencia de infecciones transmisibles por la sangre.

HIV-1 Genetic Variability in Cuba and Implications for Transmission and Clinical Progression.

INTRODUCTION Serological and molecular HIV-1 studies in Cuba have shown very low prevalence of seropositivity, but an increasing genetic diversity attributable to introduction of many HIV-1 variants from different areas, exchange of such variants among HIV-positive people with several coinciding routes of infection and other epidemiologic risk factors in the seropositive population. The high HIV-1 genetic variability observed in Cuba has possible implications for transmission and clinical progression. OBJECTIVE Study genetic variability for the HIV-1 env, gag and pol structural genes in Cuba; determine the prevalence of B and non-B subtypes according to epidemiologic and behavioral variables and determine whether a relationship exists between genetic variability and transmissibility, and between genetic variability and clinical disease progression in people living with HIV/AIDS. METHODS Using two molecular assays (heteroduplex mobility assay and nucleic acid sequencing), structural genes were characterized in 590 people with HIV-1 (480 men and 110 women), accounting for 3.4% of seropositive individuals in Cuba as of December 31, 2013. Nonrandom sampling, proportional to HIV prevalence by province, was conducted. Relationships between molecular results and viral factors, host characteristics, and patients' clinical, epidemiologic and behavioral variables were studied for molecular epidemiology, transmission, and progression analyses. RESULTS Molecular analysis of the three HIV-1 structural genes classified 297 samples as subtype B (50.3%), 269 as non-B subtypes (45.6%) and 24 were not typeable. Subtype B prevailed overall and in men, mainly in those who have sex with men. Non-B subtypes were prevalent in women and heterosexual men, showing multiple circulating variants and recombinant forms. Sexual transmission was the predominant form of infection for all. B and non-B subtypes were encountered throughout Cuba. No association was found between subtypes and transmission or clinical progression, although the proportion of deaths was higher for subtype B. Among those who died during the study period, there were no differences between subtypes in the mean time from HIV or AIDS diagnosis to death. CONCLUSIONS Our results suggest that B and non-B HIV-1 subtypes found in Cuba do not differ in transmissibility and in clinical disease progression. KEYWORDS HIV-1, AIDS, molecular epidemiology, transmissibility, clinical progression, subtypes, circulating recombinant forms, pathogenesis, Cuba.

Cervical cytology, HPV DNA and mRNA - a comparative study in 162 patients / Citologia cervicovaginal, DNA HPV e RNAm - estudo comparativo em 162 pacientes

In contrast to the general improvement of the socioeconomic status of the Brazilian population, pathologies that are characteristic of poor health assistance persist. Among those, cervical cancer (CC) is emblematic; it still presents a persistently high incidence. Objective: to compare the performance of cervical cytology to HPV DNA and mRNA detection methods in 162 patients undergoing routine gynecological clinical practice. Methods:a total of 162 patients attended during routine gynecological examination in a private clinic in Florianópolis, Santa Catarina, Brazil, had cervical samplescollected and processed for cytopathological and molecular tests, conventional PCR and NASBA. Positive samples positive for HPV DNA were submittedto Type-Specific PCR (TS-HPV PCR). Patients with altered smears were submitted to colposcopy and biopsy. Results: among the 162 samples, 19.8%(32/162) had altered smears, being 4/32 classified as ASC-H, 9/32 as ASC-US, 9/32 as LSIL and 10/32 as HSIL. Biopsies revealed nine cases of CIN I,nine CIN II and one CIN III, while seven were negative for cervical neoplasia. Overall, HPV DNA was detected in 38.3% (62/162) of the samples and HPV E6/E7 mRNA expression was found in 13.6% (22/162). Using TS-HPV PCR, HPV 16 was the most frequent type, found in 8% of the samples (5/62).Considering CIN2+ the gold-standard, cytology had 38.5% of specificity. Sensitivity and specificity of HPV-DNA PCR and NASBA were, respectively,100% and 60%; 18.7% and 68.7%. Conclusion: mRNA E6/E7 expression was not a highly specific or sensitive marker for prevalent cervical disease while HPV DNA may be used for cervical cancer screening only in conjunction to more specific adjuvant tests.

Em contraste com a melhora geral da situação socioeconômica da população brasileira, patologias que são características de uma deficiente assistência à saúde persistem. Entre elas, o câncer cervical (CC) é emblemático, ainda apresentando uma persistente alta incidência. Objetivo: avaliaro desempenho da citologia e de métodos de detecção de DNA e RNAm de HPV em 162 pacientes submetidas a prática clínica ginecológica de rotina.Métodos: cento e sessenta e duas pacientes atendidas em uma clínica particular de Florianópolis, Santa Catarina, Brasil, tiveram amostras cervicais coletadas e processadas para estudo citopatológico e molecular; PCR convencional e NASBA. Amostras positivas para o DNA do HPV foram submetidas àPCR tipo-específica (PCR HPV-TE). Resultados: entre as 162 amostras, 19,8% (32/162) apresentaram esfregaços alterados, sendo 4/32 classificadas comoASC-H, 9/32 como ASC-US, 9/32 como LSIL e 10/32 como HSIL. Biópsias revelaram nove casos de NIC I, nove casos de NIC II e um caso de NIC III. ODNA do HPV foi detectado em 38,3% (62/162) das amostras. Expressão de E6/E7 (RNAm) foi encontrada em 13,6% (22/162) das amostras. Utilizando a PCR tipo-específica (HPV-TE), o HPV 16 foi o tipo mais frequente, encontrado em 8% (5/62) das amostras HPV+. Considerando NIC 2+ o padrão-ouro,a especificidade da citologia foi de apenas 38,5%, enquanto a sensibilidade e a especificidade da PCR DNA e RNAm foram, respectivamente, 100% e 60%;18,7% e 68,7%. Conclusão: a expressão de E6/E7 RNAm não se mostrou um marcador altamente específico ou sensível para doença cervical prevalente,enquanto o DNA HPV pode ser utilizado para rastreamento apenas em conjunto com testes adjuvantes mais específicos.

Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites.

DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

Effectiveness of dual and triple antiretroviral therapy in the treatment of HIV-infected children.

OBJECTIVE: The use of antiretroviral therapy in HIV-infected children has been a widely discussed issue. The aim of this study was to compare the effectiveness of dual nucleoside analogue reverse transcriptase inhibitor (NRTI) regimens and three-drug regimens [2NRTI+ non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI)] in a cohort of HIV-infected children. METHODS: The study was carried out in a referral center for the management of infected children, which is affiliated with the School of Medicine of Universidade Federal de Minas Gerais (UFMG). Those children whose antiretroviral therapy was implemented between January 1998 and December 2000 and who were followed until December 2001 were included in the study. Therapeutic failure or death was regarded as the endpoint in our analysis. RESULTS: A total of 101 patients were assessed, 58 (57.4%) on dual therapy and 43 (42.6%) on triple therapy. No statistically significant difference was observed between the groups in terms of gender, age, CD4+ count and baseline viral load. The average duration of dual therapy was 26.3 months (95%CI 21.3-31.3) and that of triple therapy was 34.3 months (95%CI 29.2-39.5%). There was therapeutic failure in 33 (56.9%) patients on dual therapy and in 11 (25.6%) patients on triple therapy (log rank = 5.03; p = 0.025). The relative risk of therapeutic failure of the dual therapy was 2.2 times higher (95%CI 1.3-3.9). The percentage of initial CD4+ T cells was a predictor of risk for therapeutic failure (p = 0.001). Patients on triple therapy showed a more remarkable reduction in their viral load (p = 0.001). CONCLUSION: Triple therapy was efficient for a longer time period and showed better virologic response than dual therapy in this cohort of HIV-infected children. Therefore, triple therapy should be the treatment of choice.

Effectiveness of dual and triple antiretroviral therapy in the treatment of HIV-infected children

OBJETIVOS: Como iniciar a terapia anti-retroviral é uma questão amplamente discutida no manejo de crianças infectadas pelo HIV. O objetivo deste estudo foi comparar a efetividade da terapia dupla e tríplice em uma coorte de crianças infectadas pelo HIV. MÉTODO: Este estudo foi realizado em um serviço de referência para assistência à criança infectada da Faculdade de Medicina da UFMG. Foram incluídas crianças que iniciaram o primeiro regime anti-retroviral entre janeiro de 1998 e dezembro de 2000, com seguimento até dezembro de 2001. O evento final para análise foi a primeira falha terapêutica ou óbito. RESULTADOS: Foram analisados 101 pacientes, sendo 58 (57,4 por cento) e 43 (42,6 por cento) com terapia dupla e tríplice, respectivamente. Não houve diferença entre os grupos quanto ao sexo, idade, contagem de linfócitos CD4+ e carga viral basal. A média de duração da terapia dupla foi de 26,3 meses (IC95 por cento 21,3-31,3) e da terapia tríplice, de 34,3 meses (IC95 por cento 29,2-39,5 por cento). Falha terapêutica ocorreu em 33 (56,9 por cento) pacientes em terapia dupla e 11 (25,6 por cento) em terapia tríplice (log rank 5,03; p = 0,025). O risco relativo de falha para terapia dupla foi 2,2 vezes maior (IC = 1,3-3,9). O percentual de linfócitos T CD4+ inicial foi preditor de risco para falha terapêutica (p = 0,001). Pacientes em terapia tríplice apresentaram maior redução da carga viral (p = 0,001). CONCLUSÃO: A terapia tríplice permaneceu eficaz por mais tempo e apresentou melhor resposta virológica do que a terapia dupla nesta coorte de crianças infectadas pelo HIV, justificando a sua escolha como regime preferencial de tratamento.

OBJECTIVE: The use of antiretroviral therapy in HIV-infected children has been a widely discussed issue. The aim of this study was to compare the effectiveness of dual nucleoside analogue reverse transcriptase inhibitor (NRTI) regimens and three-drug regimens [2NRTI+ non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI)] in a cohort of HIV-infected children. METHODS: The study was carried out in a referral center for the management of infected children, which is affiliated with the School of Medicine of Universidade Federal de Minas Gerais (UFMG). Those children whose antiretroviral therapy was implemented between January 1998 and December 2000 and who were followed up until December 2001 were included in the study. Therapeutic failure or death was regarded as the endpoint in our analysis. RESULTS: A total of 101 patients were assessed, 58 (57.4 percent) on dual therapy and 43 (42.6 percent) on triple therapy. No statistically significant difference was observed between the groups in terms of gender, age, CD4+ count and baseline viral load. The average duration of dual therapy was 26.3 months (95 percentCI 21.3-31.3) and that of triple therapy was 34.3 months (95 percentCI 29.2-39.5 percent). There was therapeutic failure in 33 (56.9 percent) patients on dual therapy and in 11 (25.6 percent) patients on triple therapy (log rank = 5.03; p = 0.025). The relative risk of therapeutic failure of the dual therapy was 2.2 times higher (95 percentCI = 1.3-3.9). The percentage of initial CD4+ T cells was a predictor of risk for therapeutic failure (p = 0.001). Patients on triple therapy showed a more remarkable reduction in their viral load (p = 0.001). CONCLUSION: Triple therapy was efficient for a longer time period and showed better virologic response than dual therapy in this cohort of HIV-infected children. Therefore, triple therapy should be the treatment of choice.

Evaluation of a new NASBA assay for the qualitative detection of hepatitis C virus based on the NucliSens Basic Kit reagents.

BACKGROUND: Direct detection of HCV RNA by nucleic acid amplification methods is an essential tool in the diagnosis of HCV infections. In-house developed methods based on reverse transcribed polymerase chain reaction (RT-PCR) are widely used but they are laborious and usually lack the standardization required by clinical laboratories. OBJECTIVES: To evaluate the sensitivity and the clinical performance of an HCV specific nucleic acid sequence based amplification (NASBA) assay based on the commercially available, NucliSens Basic Kit (bioMérieux) reagents. STUDY DESIGN: The analytical sensitivity of the Basic Kit-based HCV assay (BK-HCV) was determined using dilutions of the First World Health Organization International Standard for HCV RNA. The performance of the BK-HCV was evaluated at two study sites in comparison with in-house RT-nested PCR (RT-nPCR) by testing a total of 77 plasma specimens. Additional HCV laboratory tests such as Amplicor HCV v2.0 (Roche Diagnostics) and genotype were also included in the comparative analysis. RESULTS: The sensitivity of the BK-HCV was 100-150 IU/ml HCV RNA (85-100% hit rate). When evaluating the clinical performance, we found 96-100% correlation between BK-HCV and RT-nPCR, and 85-91% correlation between BK-HCV and Amplicor. The level of efficiency of the BK-HCV for detecting prevalent HCV genotypes was equal to in house RT-nPCR and Amplicor. CONCLUSIONS: The BK-HCV offers adequate sensitivity for diagnostic purposes and equivalent clinical performance to in-house RT-nPCR assays. The BK-HCV could become a suitable alternative to the in-house amplification methods, providing standardized reagents and procedures, plus rapid results to clinical laboratories.