Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6 percent (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75 percent for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30 percent for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

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.