EBV-encoded microRNAs profile evaluation in pediatric liver transplant recipients

BackgroundEpstein–Barr virus (EBV) infects 90% of the world population, commonly causing self-limiting infectious mononucleosis or rarely inciting a range of malignancies. EBV microRNAs (miRNAs) were discovered by sequencing libraries of small RNAs generated from several EBV-positive cell lines. Little is known about their roles, but their high stability and easy quantification make these molecules potential biomarkers.ObjectivesIn this study a stem-loop MGB real-time RT-PCR has been used to detect and quantify miR-BART2-5p, miR-BART15 and miR-BART22 EBV miRNAs levels.Study designsThe profiles of EBV miRNAs levels were evaluated in 51 serum samples of 37 pediatric liver transplant patients subdivided in 3 study groups: EBV seronegative, EBV seropositive and PCR negative, EBV seropositive and PCR positive.ResultsmiR-BART22 serum levels in patients with positive EBV PCR were significantly higher than those in patients with negative EBV PCR (p = 0.0005). On the contrary, miR-BART2-5p and miR-BART15 did not exhibit significant difference in positive and negative EBV PCR patients (p = 0.5511 and p = 0.3523, respectively).ConclusionThis study described a method for quantitative detection of miR-BART 22, miR-BART2-5p and miR-BART15 EBV miRNAs in liver transplanted patients, and suggests the use of miR-BART22 as a potential biomarker for EBV reactivation.     

Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis

There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work‐up of two internally controlled multiplex, probe‐based PCR assays and reports on the clinical validation over a 3‐year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan? and minor groove binder (MGB?) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel‐based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe‐based assays detected 16 more positive specimens than the nested gel‐based assay. Post‐introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3‐year study period. This clinically validated, probe‐based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis. J. Med. Virol. 83:1650–1656, 2011. © 2011 Wiley‐Liss, Inc.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.