Distribution of HCV genotypes in Belgium from 2008 to 2015.

BACKGROUND: The knowledge of circulating HCV genotypes and subtypes in a country is crucial to guide antiviral therapy and to understand local epidemiology. Studies investigating circulating HCV genotypes and their trends have been conducted in Belgium. However they are outdated, lack nationwide representativeness or were not conducted in the general population. METHODS: In order to determine the distribution of different circulating HCV genotypes in Belgium, we conducted a multicentre study with all the 19 Belgian laboratories performing reimbursed HCV genotyping assays. Available genotype and subtype data were collected for the period from 2008 till 2015. Furthermore, a limited number of other variables were collected: some demographic characteristics from the patients and the laboratory technique used for the determination of the HCV genotype. RESULTS: For the study period, 11,033 unique records collected by the participating laboratories were used for further investigation. HCV genotype 1 was the most prevalent (53.6%) genotype in Belgium, with G1a and G1b representing 19.7% and 31.6%, respectively. Genotype 3 was the next most prevalent (22.0%). Further, genotype 4, 2, and 5 were responsible for respectively 16.1%, 6.2%, and 1.9% of HCV infections. Genotype 6 and 7 comprise the remaining <1%. Throughout the years, a stable distribution was observed for most genotypes. Only for genotype 5, a decrease as a function of the year of analysis was observed, with respectively 3.6% for 2008, 2.3% for 2009 and 1.6% for the remaining years. The overall M:F ratio was 1.59 and was mainly driven by the high M:F ratio of 3.03 for patients infected with genotype 3. Patients infected with genotype 3 are also younger (mean age 41.7 years) than patients infected with other genotypes (mean age above 50 years for all genotypes). The patients for whom a genotyping assay was performed in 2008 were younger than those from 2015. Geographical distribution demonstrates that an important number of genotyped HCV patients live outside the Belgian metropolitan cities. CONCLUSION: This national monitoring study allowed a clear and objective view of the circulating HCV genotypes in Belgium and will help health authorities in the establishment of cost effectiveness determinations before implementation of new treatment strategies. This baseline characterization of the circulating genotypes is indispensable for a continuous surveillance, especially for the investigation of the possible impact of migration from endemic regions and prior to the increasing use of highly potent direct-acting antiviral (DAA) agents.

Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae.

Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20M. pneumoniae genomes per reaction.

Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control.

Polymerase chain reaction (PCR) with real-time detection using two adjacent fluorescent probes in a Lightcycler instrument was applied for detection of the Mycoplasma pneumoniae P1 protein gene. To monitor inhibition in each sample an internal control was constructed that can be amplified by the same primers but detected by different probes and dual color detection. The real-time PCR was applied on 115 respiratory samples from 82 patients and compared to a conventional PCR. There was 100% agreement between the assays, but the real-time PCR proved to be highly superior in speed with a much lower risk of false positives by laboratory contamination.

Systemic inflammation, Chlamydia pneumoniae DNA in circulating leukocytes and coronary atherosclerosis.

OBJECTIVE: Earlier studies have suggested that C. pneumoniae may be involved in the progression of atherosclerosis by contributing to the pathogenesis of inflammation in the vessel wall. The aim of the present study was to determine the prevalence of C. pneumoniae DNA in circulating white blood cells of patients with ischaemic heart disease and to correlate these findings with the extent of coronary atherosclerosis and serum markers of inflammation. METHODS AND RESULTS: In 203 consecutive patients undergoing diagnostic coronary angiography for different coronary syndromes, presence of C. pneumoniae DNA in circulating leukocytes could not be demonstrated by the polymerase chain reaction. Serum concentrations of CRP were significantly higher in patients with significant coronary artery disease compared to those with normal coronary arteries. In addition, patients with a three-vessel disease had significantly higher serum CRP compared to patients with diffuse, non-critical coronary atherosclerosis. A positive correlation was found between serum fibrinogen and serum CRP. CONCLUSION: In spite of a significant relation between serum CRP and the extent of atherosclerotic coronary artery disease, we were unable to detect C. pneumoniae DNA in circulating white blood cells. This observation suggests that there is no relation between circulating C. pneumoniae, systemic inflammation and extent of coronary atherosclerosis.

Cytomegalovirus DNA detection in Guthrie cards: role in the diagnostic work-up of childhood hearing loss.

INTRODUCTION: Cytomegalovirus (CMV) infection is the leading cause of congenital nongenetic sensorineural hearing loss (SNHL) and a major cause of prelingual SNHL that is not present at birth. Polymerase chain reaction (PCR) analysis of dried blood samples on the Guthrie card has been proposed as a sensitive and specific method to screen for congenital CMV infection. METHODS: Prospectively, consecutive infants who failed universal neonatal hearing screening and children referred for a noncongenital SNHL (NCHL) were included and underwent a standard audiometric and etiologic work-up. DNA was extracted from dried blood spots on neonatal Guthrie cards and amplified by real-time PCR. Data were available for 96 cases. RESULTS: Mean age of the universal neonatal hearing screening group was 3.8 +/- 2.4 months (n = 41). Auditory brain stem response thresholds were 72.9 +/- 20.2 dB nHL. A CMV-positive PCR was obtained in 4 babies. One test was considered false-positive. This resulted in a 7.3% prevalence of congenital CMV infections.Mean age of the NCHL group was 4.9 +/- 3.2 years (n = 55). Hearing loss was moderate in 37, severe in 5, and profound in 13 children. A CMV-positive PCR was obtained in 4 children (7.3%). Other causes of SNHL were excluded in the PCR positive cases of both study groups. CONCLUSION: We advocate PCR for CMV DNA detection on Guthrie cards in the etiologic work-up of childhood SNHL and recommend serologic confirmation to exclude false-positive PCR results. 7.3% of SNHL in babies with congenital hearing loss and children with NCHL could be attributed with this technique to congenital CMV infection.

Immunohistostaining assays for detection of Chlamydia pneumoniae in atherosclerotic arteries indicate cross-reactions with nonchlamydial plaque constituents.

Detection of Chlamydia pneumoniae antigens in PCR-negative atheromata by immunohistochemistry assays has given rise to controversies regarding a link between the bacterium and atherosclerosis. One hundred ninety-seven human arterial segments removed surgically were examined for C. pneumoniae DNA by conventional PCR with three different primer pairs and by real-time PCR in two different laboratories. No C. pneumoniae DNA was detected. Eighty atherosclerotic lesions were studied by immunohistochemistry assays. Immunoreactivity for C. pneumoniae was frequently present but was not related to the extent of atherosclerosis. Mammary arteries showed immunoreactivity. Serial sections of 17 atheromata were analyzed by Western blotting, histological staining, and UV fluorescence microscopy. Chlamydial proteins were not detected. The sites with positive results by C. pneumoniae immunohistostaining assays precisely matched the sites with autofluorescent ceroid deposits. Immunoblotting and antigenic staining for C. pneumoniae were negative in tests with fetal aortas. The absence of C. pneumoniae DNA in human atherosclerotic lesions, together with negative results for C. pneumoniae proteins by Western blotting analysis, and the perfect matching of C. pneumoniae immunoreactive sites with sites with autofluorescent ceroid deposits suggest a nonspecific reactivity of antichlamydial antibodies with plaque constituents. On the basis of the results of the present study, there are no arguments for an etiologic role of C. pneumoniae in atherosclerosis.