PCR-based approach for qualitative molecular analysis of six neurotropic pathogens.

In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.

Direct PCR: a new pharmacogenetic approach for the inexpensive testing of HLA-B*57:01.

One of the most successful applications of pharmacogenetics research is the genetic screening for HLA-B*57:01, strongly associated with an increased risk to develop hypersensitivity reaction in HIV-positive patients following abacavir administration. Taking into consideration the limits of current genotyping methodologies, we have developed and validated (150 buccal swabs) an inexpensive pharmacogenetic approach for HLA-B*57:01 typing. In our assay DNA extraction and amplification are combined in one single step (direct PCR protocol), which is performed directly on the biological sample without the need of extraction and sequencing passages. The amplicons obtained by direct PCR can be easily separated on the agarose gel under ultraviolet. As per our results, the direct PCR represents a good alternative to the traditional methods of HLA-B*57:01 pharmacogenetic test, especially for those laboratories or countries where currently available approaches are often not available or not affordable. Furthermore it is an innovative approach, promoting a personalized, safer and cost-effective therapy.

Another patient with 12q13 microduplication.

Interstitial duplication of the long arm of chromosome 12 is a rare cytogenetic condition. While several reports describe distal 12q duplication, only one case report of homogeneous, non-mosaic interstitial 12q13 duplication has been documented to date. The authors of that observation proposed that the associated phenotype represented a phenocopy of Wolf-Hirschhorn syndrome [Dallapiccola et al., 2009]. Only a few other recorded patients with deletion 12q13 → 12q21 involved mosaicism. We describe a new patient with homogeneous 12q13 duplication in a 6-year-old girl who, in early infancy, presented with dysmorphic features suggesting Wolf-Hirschhorn syndrome. What is potentially significant about this patient is that her facial phenotype evolved with age, suggesting a different gestalt in older patients.