René Minnaar

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

ABSTRACT The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.

Detection of human enterovirus and human parechovirus (HPeV) genotypes from clinical stool samples: polymerase chain reaction and direct molecular typing, culture characteristics, and serotyping

Molecular (polymerase chain reaction [PCR]) methods are increasingly used to detect and type human enteroviruses (HEVs) and parechoviruses (HPeV). Here, we assessed their value in comparison to virus culture and serotyping for detection and typing of HEV and HPeV in stool samples from hospitalized patients. By use of real-time PCR, 221/1174 patients (18.8%) were found positive for HEV/HPeV. By cell culture, a virus could be isolated from 107 of the HEV/HPeV PCR-positive samples. Culture efficiency was correlated to the Ct value, (geno)type, and cell lines used. Of the HEV/HPeV PCR-positive samples, 47% could be genotyped by VP1 genotyping and 25% by serotyping. In conclusion, PCR detection of HEV/HPeV from stool is more sensitive than virus culture, particularly for coxsackieviruses A and HPeVs. However, the genotyping method used here could identify only 47% of the HEV/HPeV strains. Further optimization and validation of direct genotyping are needed, and clinical relevance of HEV/HPeV detection in stool needs to be determined.

Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination

Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes

ABSTRACT The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed.

Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBPβ isoforms

During genital human papillomavirus (HPV) infection several cytokines are released, such as interleukin‐1 (IL‐1), tumor necrosis factorα (TNFα), IL‐6, and IL‐8. These cytokines may play a role in the immune surveillance against viral infection. Two of these cytokines, IL‐1 and TNFα, suppress the transcription of the HPV16 early genes. CAATT/enhancer binding proteinβ (C/EBPβ), which is activated by IL‐1 and TNFα, has been suggested to act as a mediator of this transcriptional downregulation. C/EBPβ contains three different translation initiation sites that can lead probably by leaky ribosome scanning to the generation of three isoforms of C/EBPβ, namely full‐length C/EBPβ, liver enriched transcriptional activator protein (LAP), and liver enriched inhibitory protein (LIP). When transiently expressed in C33A and HeLa cells, the first two C/EBPβ isoforms activate the HPV16 long control region (LCR). LIP, which acts as an antagonist of C/EBPβ, represses the HPV16 LCR activity. Our observation that treatment of HeLa cells with IL‐1 leads to induction of LIP supports the hypothesis that the LCR downregulation by IL‐1 is mediated by LIP. Mol. Carcinog. 28:42–50, 2000.

Presence of human non-polio enterovirus and parechovirus genotypes in an Amsterdam hospital in 2007 to 2011 compared to national and international published surveillance data: a comprehensive review.

Enteroviruses (EV) and human parechoviruses (HPeV) are endemic worldwide. These infections are a constant cause of hospitalisation and severe disease, predominantly in young children and infants. Coordinated monitoring and surveillance are crucial to control these infections. We have monitored EV and HPeV epidemiology in Amsterdam from 2007 to 2011 with real-time RT-PCR and direct genotyping, facilitating highly sensitive surveillance. Moreover, we conducted a literature survey of existing surveillance data for comparison. Only 14 studies were identified. While HPeV1 was most frequently detected in Amsterdam, EV-B viruses dominated nationally and internationally. Furthermore, the top 10 strains detected differed yearly and per study. However, detection and typing methods were too varied to allow direct comparison and comprehension of the worldwide distribution and circulation patterns of the different genotypes. This limited a direct response to anticipate peaks. Uniform European monitoring programmes are essential to aid prediction of outbreaks and disease management.

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.

Clinical, virological and epidemiological characteristics of rhinovirus infections in early childhood: A comparison between non-hospitalised and hospitalised children

Abstract Background Several studies have been published regarding the epidemiology and clinical significance of the different rhinovirus (RV) species (-A, -B and -C). However, data on RV types and the associations with clinical outcome in young children are limited. Here, we investigated the clinical, virological and epidemiological characteristics of RV infections in young children with mild or asymptomatic infection (non-hospitalised children) and in symptomatic young children admitted to the hospital. Objectives The aim of this study was to evaluate associations between different characteristics of RV infections and clinical outcome in young children. Study design RV-infected children were retrospectively selected from a Dutch birth cohort (EUROPA-study) and from hospitalised children admitted to the hospital because of respiratory symptoms. In total 120 RV-typed samples could be selected from 65 non-hospitalised and 49 hospitalised children between November 2009 and December 2012. Results RV-A was the predominant species in both study populations, followed closely by RV-C. RV-B was observed only sporadically. The distribution of the RV species was comparable in non-hospitalised and hospitalised children. In children with respiratory distress who required ICU-admission the distribution of RV species did not differ significantly from the non-hospitalised children. No predominant RV type was present in non-hospitalised nor hospitalised children. However, hospitalised children were younger, had more often an underlying illness, a higher RV load and more frequently a bacterial co-infection. Conclusions Clinical outcome of RV infected young children was not related to RV species or types, but may more likely be influenced by multiple (host-specific) factors.

A molecular epidemiological perspective of rhinovirus types circulating in Amsterdam from 2007 to 2012

Abstract Rhinoviruses (RVs) are frequently detected respiratory viruses that cause mild common cold symptoms, but may also lead to more severe respiratory tract infections. The large number of RV types, classified into species A, B and C, hampers clear insights into the epidemiology and clinical significance of each RV type. The aim of this study was to map the circulation of RV types in the Amsterdam area. RV-positive nasopharyngeal and oropharyngeal samples, collected from 2007 to 2012 in the Academic Medical Centre (Amsterdam, the Netherlands), were typed based on the sequence of the region coding for capsid proteins VP4 and VP2. RV-A, RV-B and RV-C were found in proportions of of 52.4% (334/637), 11.3% (72/637), and 36.2% (231/637), respectively. We detected 129 of the 167 currently classified types. RVs circulated throughout the entire year with a peak in the autumn and a decline in the summer. Some RV types were observed throughout the entire sampling period and others had a more seasonal pattern. Nine RV-A and four RV-B novel provisionally assigned types were identified. This study provides an insight into the molecular epidemiology of RVs in the Amsterdam area. The RVs circulating are diverse and include several provisionally new types.

Prevalence of rhinoviruses in young children of an unselected birth cohort from the Netherlands

Abstract Rhinovirus (RV) is a frequent pathogen in young children, eliciting symptoms ranging from common colds to wheezing illnesses and lower respiratory tract infections. The recently identified RV-C seems to be associated with asthma exacerbations and more severe disease, but results vary. We studied the prevalence and severity of infection with RV in an unselected birth cohort. Children with respiratory symptoms entered the symptomatic arm of the cohort and were compared with asymptomatic children. Severity of wheezing and other respiratory symptoms was registered. Respiratory viruses were evaluated using throat and nasopharyngeal swabs on first presentation and after recovery (wheezing children). RV genotyping was performed on RV-PCR positive samples. RV was the most prevalent respiratory virus and was found in 58/140 symptomatic children (41%), 24/96 (25%) control children and 19/74 (26%) wheezing symptomatic children after recovery (p <0.05) and did not differ between wheezing and non-wheezing symptomatic children—respectively, 42% (38/90) and 40% (20/50). RV-A was the most commonly detected species (40/68, 59%), followed by RV-C (22/68, 32%) and RV-B (6/68, 9%). RV-B was more frequently detected in asymptomatic children (5/6, p <0.05). There was no significant difference in the frequency of RV species between wheezing and non-wheezing symptomatic children. Children with RV mono-infection had more severe symptoms, but no association between RV species and severity of disease was seen. In an unselected birth cohort from the Netherlands with mild respiratory disease RV was the most prevalent respiratory virus. RV(-C) infection was not associated with more severe disease or wheezing.