Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae.

Rapid, accurate and inexpensive diagnosis of bacterial meningitis is critical for patient management. This study describes the development and evaluation of a multiplex PCR assay for the detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b, which globally account for 90% of cases of bacterial meningitis. The single-tube assay, based on the ctrA, ply and bex targets, respectively, enabled detection of 5-10 pg DNA. When the assay was tested with clinical samples (n = 425), its sensitivity for the three targets was 93.9%, 92.3% and 88%, respectively, while the overall specificity and positive predictive value of the assay was 100%. The negative predictive value was 99.1-99.5%. The methodology permits rapid and accurate detection of the three main pathogens that cause bacterial meningitis.

Detection of hepatitis C virus in sera and genotyping according to the 5' non-coding region.

A reverse transcription (RT)-polymerase chain reaction (PCR) method was used for detection of the RNA of hepatitis C virus (HCV) in 120 samples of sera from Crete, which were positive for HCV-specific antibodies, by ELISA and Western blot analyses. A segment of 255 bp, located in the most conserved region of the HCV genome (the 5' untranslated region, 5' UTR), was amplified. For the identification of sequence variation from the HCV-1 strain, twenty of these samples were sequenced and compared to prototype strain (HCV-1) according to current genotypic classification. We were able to identify fourteen of the twenty as type 1a (i.e. similar to the prototype), two as type 1b, two as type 3a and two as type 4a. These findings generally agree with the geographic distribution of the already identified genotypes, though 3a type has not been reported previously in Crete (Greece).

Allelic loss in chromosomal region 1q21-23 in breast cancer is associated with peritumoral angiolymphatic invasion and extensive intraductal component.

AIMS: Breast cancer is the most frequent cancer in the female population and the involvement of chromosomal alterations is often implicated in the development of cancer. The aim of our study was to assess loss of heterozygosity (LOH) in chromosome 1 in relation to clinical and pathological parameters. METHODS: Tumours, corresponding normal tissues and peripheral blood samples from 50 women with operable breast cancer, were analysed by polymerase chain reaction (PCR) at 16 polymorphic DNA markers, on both the long and short arm of chromosome 1. RESULTS: There was a significant correlation between chromosomal region 1q21-23 and the presence of extensive intraductal component (EIC) and peritumoral angiolymphatic (PALI) invasion, both independent markers of local recurrence. CONCLUSIONS: Allelic loss in region 1q21-23 may be a valuable prognostic biological marker for the detection of local relapse in breast cancer, in combination with other histological and clinical parameters.

Evaluation of western blot in routine diagnosis of hepatitis C virus.

Hepatitis C virus (HCV) is among the major causes of parenterally transmitted hepatitis. Detection of infected persons would greatly diminish transmission rates and is therefore a significant parameter for prevention. Current assays are not able to resolve all cases and sometimes the results are controversial. The present study outlines problems that arise during routine testing. Two ELISA tests and three confirmatory tests were used and Polymerase Chain Reaction (PCR) data were available for some of the samples. The results of this study show that only 77.4% of samples positive for both ELISAs were confirmed as being positive. Controversial ELISA results remained controversial, depending on the confirmatory test used. PCR results, though not complete, point to the major problem of Western blot (WB) negative sera that prove positive for the viral genome and have to be excluded from screening for blood and organ donation. Since PCR cannot be used as a routine screening procedure, improvement of the routine assays is needed to minimize ambiguous results.

Loss of heterozygosity and microsatellite instability in human non-neoplastic hepatic lesions.

AIMS/BACKGROUND: Carcinogenesis is thought to be a multistage process that occurs as a result of mutations in oncogenes and tumor suppressor genes. One way to monitor a vast range of these changes is by microsatellite PCR amplification that detects loss of heterozygosity and microsatellite instability between normal and tumor specimens of the same subject. Viral cirrhosis is considered a strong predisposing factor for the development of liver cancer. The aim of the study therefore was to examine precancerous hepatic lesions and compare them with others not considered as high risk for hepatocellular carcinoma. METHODS: We examined 43 subjects for 19 microsatellite markers spanning chromosomes 1, 9 and 17. Normal specimens were blood samples that were compared to liver needle biopsies. Samples were classified according to histological features as non-cancerous (10 cases) and pre-cancerous (33 cases, chronic hepatitis and cirrhosis). RESULTS: Our results indicate that there is a tendency of increased chromosomal alteration as lesions become chronic. Samples from patients with antibodies to antibodies for hepatitis C virus show more alterations than hepatitis B positive samples. Steatohepatitis, a disease of unknown etiology, appears to have a high number of microsatellite abnormalities. CONCLUSIONS: Microsatellite APOA2 located on chromosome 1, shows a statistically significant increase in the rate of loss of heterozygosity as liver lesions become more severe, indicating the presence of tumor suppressor genes which may be involved in the development of these lesions.

Analysis of eight polymorphic human genetic markers in a well-defined Greek population.

The use of DNA in forensic science has become a basic tool for person identification or parentage testing. The use of polymorphic markers permits the formation of a unique profile for each individual. The knowledge of allele frequencies in a given population allows the scientist to estimate the probability of a particular allele combination. For this task, allele databanks are essential. In this report, the authors estimate the frequencies of eight polymorphic markers (namely, HumFES, HumF13A1, HumTHO1, HumVWA, HumFABP2, HumLIPOL, D1S80, and D17S5) in a randomly selected sample from Crete, Greece. The allele profile of all markers, with the exception of D17S5 and HumFABP2, concurs with previous reports and international data.

Mutations of retinoblastoma gene (Rb-1) as a prognostic factor in children with acute leukemia and neuroblastoma.

Rb-1 is a tumor suppressor gene encoding for a nuclear phosphoprotein acting as a cell cycle regulator, normally expressed in hematopoietic cells and more often inactivated by point mutations with predominance for exons 20-24. The aim of this study is to correlate the retinoblastoma-1 (Rb-1) gene mutations with the prognosis and progression of childhood acute leukemia and neuroblastoma. Bone marrow slides from 26 children with leukemia (18 acute lymphoblastic leukemia [ALL] and 8 acute myeloid leukemia [AML]) and 4 children with neuroblastoma were studied. Exons 20, 21, and 22 were amplified using the polymerase chain reaction technique. Single strand conformational polymorphism (SSCP) and heterodoublex analysis were performed to detect mutations. In ALL cases, two samples in exon 20 (11.11%), one in exon 21 (5.56%), and four in exon 22 (22.22%) had altered conformation. All but one of these cases were classified as high-risk leukemia patients who either relapsed or never achieved remission. Two of the AML cases who did not achieve remission and one of the neuroblastoma cases with concomitant bone marrow infiltration had altered conformation as well. The SSCP and heterodoublex analysis showed that all but one who did not belong to the high-risk group had the same altered conformation. These data suggest that Rb-1 gene could possibly be used as an independent prognostic factor for the acute leukemia of childhood and result in the intensification of chemotherapy. In solid tumors with bone marrow involvement it could play a role as a marker of aggressive disease.

A multistep process for the dispersal of a Y chromosomal lineage in the Mediterranean area.

In this work we focus on a microsatellite-defined Y-chromosomal lineage (network 1.2) identified by us and reported in previous studies, whose geographic distribution and antiquity appear to be compatible with the Neolithic spread of farmers. Here, we set network 1.2 in the Y-chromosomal phylogenetic tree, date it with respect to other lineages associated with the same movements by other authors, examine its diversity by means of tri- and tetranucleotide loci and discuss the implications in reconstructing the spread of this group of chromosomes in the Mediterranean area. Our results define a tripartite phylogeny within HG 9 (Rosser et al. 2000), with the deepest branching defined by alleles T (Haplogroup Eu10) or G (Haplogroup Eu9) at M172 (Semino et al. 2000), and a subsequent branching within Eu9 defined by network 1.2. Population distributions of HG 9 and network 1.2 show that their occurrence in the surveyed area is not due to the spread of people from a single parental population but, rather, to a process punctuated by at least two phases. Our data identify the wide area of the Balkans, Aegean and Anatolia as the possible homeland harbouring the largest variation within network 1.2. The use of recently proposed tests based on the stepwise mutation model suggests that its spread was associated to a population expansion, with a high rate of male gene flow in the Turkish-Greek area.