Optimización de recursos para determinar la carga viral de HIV-1 en un país con pocos recursos / Optimizing resources to reduce costs to determine HIV viral load in limited resources settings

Resumen Introducción. Las metas globales para controlar la epidemia de HIV contemplan que la carga viral sea indetectable en 90 % de las personas en tratamiento. El costo de la medición de la carga viral en lotes de muestras puede reducirse y, así, aumentar la cobertura cuando los recursos son limitados; sin embargo, su eficacia disminuye al aumentar la prevalencia del fracaso del tratamiento antirretroviral. Objetivo. Evaluar estrategias para disminuir la proporción de pacientes con fracaso del tratamiento antirretroviral en los lotes de muestras y, de esta manera, aumentar el ahorro en las pruebas de carga viral. Materiales y métodos. Las estrategias evaluadas fueron: a) la organización de los lotes de muestras según el esquema de tratamiento antirretroviral, y b) la exclusión de aquellos pacientes con antecedente reciente de fracaso del tratamiento antirretroviral, aquellos con menos de 12 meses de tratamiento antirretroviral y aquellos sin tratamiento antirretroviral previo. Los resultados de los lotes se compararon con los resultados individuales. Resultados. El valor diagnóstico negativo fue similar para los pacientes con esquema de primera línea (100,0 %; IC95% 99,5-100,0) o de segunda línea de tratamiento (99,4 %; IC95% 96,9-99,9). La incidencia del fracaso del tratamiento antirretroviral fue menor en los pacientes con tratamiento de primera línea (p<0,01), lo cual permitió un mayor ahorro en las pruebas de laboratorio en este grupo (74,0 %; IC95% 71,0-76,7) que en los pacientes con tratamiento de segunda línea (50,9 %; IC95% 44,4-57,3) (p<0,01). Conclusión. La selección de las muestras que se incluyeron en los lotes para determinar la carga viral del HIV según el tipo de esquema de tratamiento, permitió maximizar el porcentaje de ahorro en pruebas de laboratorio.

Abstract Introduction: HIV viral load testing is a key factor to evaluate the accomplishment of the UNAIDS target of 90% of viral suppression among people receiving antiretroviral therapy. Pooled samples are a potentially accurate and economic approach in resource-constrained settings, but efficiency can be negatively affected by high prevalence rates of virological failure. Objective: Strategies were assessed to increase the relative efficiency of pooled HIV viral load testing in resource-constrained settings. Materials and methods: We evaluated two strategies: a) plasma samples were not included in pools if patients had <12 months on antiretroviral therapy, patients had previous viral load >1,000 copies/ml, or were antiretroviral therapy naïve patients, and b) plasma pools were organized separately for first and second-line antiretroviral therapy regimens. Individual viral load tests were used to compare pooled results. Results: Negative predictive values were similar for patients on first (100.0%; 95% CI 99.5 to 100.0) and second-line antiretroviral therapy regimens (99.4%; 95% CI 96.9 to 99.9). However, the incidence of virological failure among individuals on first-line antiretroviral therapy was lower than second-line antiretroviral therapypatients (p <0.01), resulting in greater savings in laboratory tests in patients on first-line antiretroviral therapy (74.0%; 95% CI 71.0 to 76.7) compared with the group of patients on second-line antiretroviral therapy (50.9%; 95% CI 44.4 to 57.3) (p<0.01). Conclusion: Selecting the samples to be included in the pools and selecting the pools according to ART regimens are criteria that could lead to decreased spending on laboratory tests for HIV viral load determination in resource-constrained settings.

Use of "Parasep filter fecal concentrator tubes" for the detection of intestinal parasites in stool samples under routine conditions.

Parasitic gastrointestinal infections are a major cause of morbidity and mortality in the developing world, with stool microscopy being the mainstay of diagnostic practice. Both direct microscopy and concentration techniques can be utilized; direct microscopy may be time consuming and tedious; however clinical laboratories in developing countries lack trained staff who can effectively use concentration methods. In our practice we used the Parasep O and P filter concentrator tubes (manufactured by DiaSys Ltd, Berkshire, England. Product Code 146000) along with direct microscopic techniques and found that Parasep filters enhanced the ability to detect intestinal parasites that would have been missed on routine microscopy. We found the Parasep filter concentration method to be easy, cost-effective and reliable for routine stool examinations.

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 µg (median 15 µg, mean 20.71 µg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Células bucais são fontes convenientes de DNA para diagnóstico e estudos epidemiológicos. O objetivo deste trabalho foi desenvolver um método simples e prático para obter células epiteliais, através de bochechos, a fim de serem usadas como fonte de DNA e avaliar a estabilidade do DNA na solução de bochecho no decorrer do tempo. Os procedimentos usados neste estudo evitam o uso de solventes orgânicos permitindo uma pratica laboratorial mais segura. Isto é alcançado pela remoção das proteínas celulares por desidratação e precipitação com uma solução saturada de acetato de amônio. Este protocolo permite a extração de maneira rápida, simples, econômica e garante o processamento de várias amostras ao mesmo tempo, agilizando assim os procedimentos laboratoriais. Nossas análises forneceram evidências consistentes de que o DNA extraído por esta metodologia é suficiente para diversas amplificações por PCR (polymerase chain reaction - reação em cadeia pela polimerase). O produto total de DNA variou de 5 a 93 µg (mediana 15 µg; média 20,71 µg). Além disso, o DNA mostrou-se eficientemente preservado na solução de bochecho, a qual pode ser estocada em temperatura ambiente por até trinta dias.

Detección de Chlamydia trachomatis en orina por LCR: aplicación del método de mezcla de muestras biológicas / Ligase chain reaction testing of pooled urine specimens to diagnose Chlamydia trachomatis infection

Objective. The purpose of this study was to assess the utility and validity of pooling urine samples for molecular diagnosis of Chlamydia trachomatis infection. Material and methods. Of 1,220 urine samples collected from Mexican female and male adolescents, 305 pools were composed of fourth individual samples each, based on a calculation of optimal pool size. These were processed by ligase chain reaction (LCR) for the detection of C. trachomatis. Positive and gray-zone pools were reanalyzed individually. Cost savings were calculated comparing actual costs of testing to the cost that would have been incurred testing all 1,220 samples individually. Results.Pools results were: 56 positive, 19 gray-zones and 230 negative. Following individual retesting of positive and gray-zone pools, 59 cases of C. trachomatis infection were identified (4.8% prevalence). Thus, a total of 601 LCR tests were performed, for a 50.4% savings considering only the direct cost of the test. Conclusions.Our experience shows that sample pooling is both a reliable and convenient tool for CT surveillance in our setting. It should be considered in other similar settings where limited resources constraint surveillance of STIs.

Objetivo. Evaluar la validez y conveniencia de la estrategia de la mezcla de muestras de orinas para el diagnóstico molecular de Chlamydia trachomatis (CT). Material y métodos. A partir de 1,220 muestras de orina recolectadas de jóvenes de uno y otro sexos, se conformaron 305 mezclas con cuatro alícuotas de muestras individuales, previo cálculo del tamaño óptimo de la mezcla. A continuación se determinó la presencia de ácidos nucleicos de clamidia en esas mezclas, mediante el método de reacción en cadena de la ligasa. Las mezclas positivas o en zona gris fueron reanalizadas de manera individual (cuatro pruebas adicionales). El número final de pruebas realizadas se comparó con el total de pruebas que se habrían efectuado individualmente. Resultados. Del total de mezclas analizadas, 230 resultaron negativas, 56 fueron positivas y 19 más se ubicaron en zona gris. Una vez reanalizadas de manera individual las mezclas positivas y las de zona gris, se obtuvieron 59 muestras de orina positivas a clamidia (prevalencia de 4.81%). De esta manera, el número total de pruebas efectuadas fue de 605 en contraste con las 1,220 que tendrían que haberse hecho si se hubieran procesado las muestras individualmente, es decir, que se logró un ahorro de 50.5% del costo directo del reactivo de diagnóstico. Conclusiones. La metodología aplicada mostró ser tanto confiable como conveniente en el entorno mexicano para llevar a cabo vigilancia epidemiológica de la infección por CT. Dado lo anterior, esta metodología podría ser considerada en otros entornos en los que la falta de recursos limita la vigilancia de las infecciones de transmisión sexual.

Reduction of the cost of testing for antibody to human immunodeficiency virus, without losing sensitivity, by pooling sera.

The sensitivity of testing pooled sera instead of individual sera for antibody against human immunodeficiency virus (HIV) was evaluated using a non-competitive enzyme-linked immunosorbent assay (ELISA). For this purpose, 42 HIV antibody positive sera were titrated and introduced into 42 sets of pools of 2, 4, 8, 16, 32 or 64 sera in such a manner that each pool had one positive sample and the rest, HIV antibody negative sera. When the pools were tested in ELISA, all pools with high titred antibody positive sera were reactive irrespective of pool size, while some of the pools containing medium or low titred sera were non-reactive when pool size exceeded 16. Subsequently the pool size was limited to 16. When 208 previously unscreened samples were tested in 52 pools of 4, 26 pools of 8 or 13 pools of 16 sera, or individually, 6 antibody positive sera were correctly identified. Thus, it was found that the pooling method did not reduce the sensitivity of ELISA test, whereas the cost was reduced to less than half of that of individual testing.