Richard Molenkamp

Frequent Detection of Respiratory Viruses without Symptoms: Toward Defining Clinically Relevant Cutoff Values

ABSTRACT Highly sensitive techniques, such as PCR, have greatly improved the detection of respiratory viruses. However, the sensitivity of PCR tests also complicates clinical interpretation, as the presence of small amounts of viral targets may not necessarily have clinical relevance. We performed a prospective case-control study in asymptomatic and symptomatic young children. PCR detection of 14 respiratory viruses was performed in nasal washes, and results were quantified in copies per milliliter. A total of 141 cases and 157 controls were included. In 72% of the cases and 28% of the controls, at least one virus was identified. When stratified for age, at least one virus was identified in 47% of the controls younger than 1 year old. Rhinovirus (RV) was frequently detected in both symptomatic and asymptomatic individuals. Receiver operating characteristic analysis for quantitative rhinovirus detection showed that cutoff values for clinical relevance are feasible for RV. In contrast to rhinovirus, respiratory syncytial virus (RSV) was rarely detected in controls, suggesting that a positive RSV test result is almost always of clinical relevance, independent of viral quantity. In conclusion, our study shows that asymptomatic carriage of a respiratory virus occurs frequently in young children. However, significant differences in the amount of virus present were observed between cases and controls. This suggests that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.

Human Parechoviruses as an Important Viral Cause of Sepsislike Illness and Meningitis in Young Children

BACKGROUND Enteroviruses (EVs) belong to the family Picornaviridae and are a well-known cause of neonatal sepsis and viral meningitis. Human parechoviruses (HPeVs) type 1 and 2, previously named echovirus 22 and 23, have been associated with mild gastrointestinal or respiratory symptoms in young children. Six HPeV genotypes are currently known, of which HPeV3 is associated with neonatal sepsis and meningitis. METHODS Cerebrospinal fluid samples from children aged <5 years previously tested by EV-specific polymerase chain reaction (PCR) during 2004-2006 were selected (N= 761). Samples from 716 of those children were available for retrospective testing by HPeV-specific real-time PCR. The prevalence of EV and HPeV in these samples was compared. Data on clinical presentation of children infected with HPeV were retrospectively documented. RESULTS HPeV was found in cerebrospinal fluid samples from 33 (4.6%) of the children. Yearly prevalence of HPeV in cerebrospinal fluid varied remarkably: 8.2% in 2004, 0.4% in 2005, and 5.7% in 2006. EV was detected in 14% (108 of 761 samples), with no variation in yearly prevalence. Children with HPeV in cerebrospinal fluid presented with clinical symptoms of sepsislike illness and meningitis, which led to hospitalization and antibiotic treatment. CONCLUSION EV-specific PCRs do not detect HPeVs. The addition of an HPeV-specific PCR has led to a 31% increase in detection of a viral cause of neonatal sepsis or central nervous system symptoms in children aged <5 years. HPeV can be considered to be the second cause of viral sepsis and meningitis in young children, and rapid identification of HPeV by PCR could contribute to shorter duration of both antibiotic use and hospital stay.

High Prevalence of Human Parechovirus (HPeV) Genotypes in the Amsterdam Region and Identification of Specific HPeV Variants by Direct Genotyping of Stool Samples

ABSTRACT Human parechoviruses (HPeV) are widespread pathogens belonging to the Picornavirus family. Six genotypes, which have predominantly been isolated from children, are known. Data on prevalence of HPeV genotypes are generally based on cell culture, which may underestimate the prevalence of certain HPeV strains that are difficult to grow. We studied 1,824 stool samples from 1,379 children (<5 years old) sent to the Academic Medical Center, Amsterdam, The Netherlands, between 2004 and 2006. Samples were screened using specific human enterovirus (HEV) and HPeV real-time PCRs based on the 5′ untranslated region. A high percentage of HPeV infections (16.3%), comparable to the percentage of HEV infections (18.4%), were found by PCR in stool samples. HPeV-positive stool samples were directly genotyped based on the VP1 region for the first time to avoid a culture bias. HPeV1 was found to be the most prevalent type. The majority of the HPeV1 strains clustered separately from the prototype strain, Harris, which has not been reported to circulate lately. However, we could identify three strains as HPeV1 Harris. HPeV3 was identified as the second most predominant type during 2004 and 2006 but was not found in 2005. HPeV4 to -6 were found in smaller numbers. One strain could not be associated with a known HPeV type (VP1 gene nucleotide similarity: 71%), possibly indicating a new genotype. Also, we report the first identification of three HPeV5 strains and one HPeV1 strain with a different motif at the C-terminal end of VP1, where the arginine-glycine-aspartic acid (RGD) motif is normally located.

Human Bocavirus Can Be Cultured in Differentiated Human Airway Epithelial Cells

ABSTRACT In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.

The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription

Equine arteritis virus (EAV) (ARTERIVIRIDAE:) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

Alarming incidence of hepatitis C virus re-infection after treatment of sexually acquired acute hepatitis C virus infection in HIV-infected MSM

Background:Recent data indicate that seroprevalence of sexually transmitted hepatitis C virus (HCV) infection among MSM is stabilizing in Amsterdam. However, little is known about the incidence of HCV re-infection in MSM who have cleared their HCV infection. We, therefore, studied the incidence of re-infection in HIV-infected MSM who were HCV RNA-negative following HCV treatment of acute primary infection. Methods:Our study population comprised HIV-infected MSM at two large HIV outpatient clinics in Amsterdam, who were previously diagnosed with a sexually transmitted acute HCV infection and tested HCV RNA-negative at the end of treatment. We defined HCV re-infection as detectable HCV RNA in individuals with an undetectable HCV RNA at the end of treatment accompanied by a switch in HCV genotype or clade. Person–time methods were used to calculate the incidence of re-infection. Results:Fifty-six persons who became HCV RNA-negative during primary acute HCV treatment were included. Five of the 56 cases relapsed and were not analysed. Eleven persons were re-infected. The incidence of HCV re-infection in this group was 15.2 per 100 person-years (95% confidence interval 8.0–26.5). The cumulative incidence was 33% within 2 years. Discussion:An alarmingly high incidence of HCV re-infection was found in this group. This high re-infection rate indicates that current prevention measures should be discussed, frequent HCV RNA testing should be continued after successful treatment and, in case of possible relapse, clade typing should be performed to exclude re-infection.

Frequent HCV reinfection and superinfection in a cohort of injecting drug users in Amsterdam

BACKGROUND/AIMS This study investigates the occurrence of HCV reinfection and superinfection among HCV seroconverters participating in the Amsterdam Cohort Studies among drug users from 1985 through 2005. METHODS HCV seroconverters (n=59) were tested for HCV RNA at five different time points: the last visit before seroconversion (t=-1), the first visit after seroconversion (t=1), six months after (t=2) and one year after (t=3) seroconversion, and the last visit prior to November 2005 (t=4). If HCV RNA was present, part of the NS5B region was amplified and sequenced. Additional phylogenetic analysis and cloning was performed to establish HCV reinfection and superinfection. RESULTS Multiple HCV infections were detected in 23/59 (39%) seroconverters; 7 had HCV reinfections, 14 were superinfected, and 2 had reinfection followed by superinfection. At the moment of HCV reinfection, 7/9 seroconverters were HIV-negative: persistent HCV reinfection developed in both HIV-positive cases but also in 4/7 HIV-negative cases. In total, we identified 93 different HCV infections, varying from 1 to 4 infections per seroconverter. Multiple HCV infections were observed in 10/24 seroconverters with spontaneous HCV clearance (11 reinfections, 3 superinfections) and in 13/35 seroconverters without viral clearance (20 superinfections). CONCLUSIONS HCV reinfection and superinfection are common among actively injecting drug users. This might further complicate the development of an effective HCV vaccine.

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.

Female sex and IL28B, a synergism for spontaneous viral clearance in hepatitis C virus (HCV) seroconverters from a community-based cohort.

Background & Aims Since acute hepatitis C virus (HCV) infection is often asymptomatic, it is difficult to examine the rate and determinants of spontaneous clearance. Consequently, these studies are subject to bias, which can potentially lead to biased rates of viral clearance and risk estimates. We evaluated determinants of spontaneous HCV clearance among HCV seroconverters identified in a unique community-based cohort. Methods Subjects were 106 drug users with documented dates of HCV seroconversion from the Amsterdam Cohort Study. Logistic regression was used to examine sociodemographic, behavioral, clinical, viral and host determinants, measured around acute infection, of HCV clearance. Results The spontaneous viral clearance rate was 33.0% (95% confidence interval (CI) 24.2–42.8). In univariate analyses female sex and fever were significantly associated with spontaneous clearance. The favorable genotypes for rs12979860 (CC) and rs8099917 (TT) were associated with spontaneous clearance, although borderline significant. In multivariate analysis, females with the favorable genotype for rs12979860 (CC) had an increased odds to spontaneously clear HCV infection (adjustedOR 6.62, 95% 2.69–26.13), whereas females with the unfavorable genotype were as likely as men with the favorable and unfavorable genotype to clear HCV. Chronic Hepatitis B infection and absence of HIV coinfection around HCV seroconversion also favor HCV clearance. Conclusions This study shows that co-infection with HIV and HBV and genetic variation in the IL28B region play an important role in spontaneous clearance of HCV. Our findings suggest a possible synergistic interaction between female sex and IL28B in spontaneous clearance of HCV.

Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles

ABSTRACT Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countries, including The Netherlands. Using a molecular epidemiological approach, the present study investigates the genetic diversity and evolutionary origin of HCV-4 in Amsterdam, The Netherlands. Phylogenetic analysis of the NS5B sequences (668 bp) obtained from 133 patients newly diagnosed with HCV-4 infection over the period from 1999 to 2008 revealed eight distinct HCV-4 subtypes; the majority of HCV-4 isolates were of subtypes 4d (57%) and 4a (37%). Three distinct monophyletic clusters were identified, with each one having a specific epidemiological profile: (i) Egyptian immigrants infected with HCV-4a (n = 46), (ii) Dutch patients with a history of injecting drug use infected with HCV-4d (n = 44), and (iii) Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) infected with HCV-4d (n = 26). Subsequent molecular clock analyses confirmed that the emergence of HCV-4 within these three risk groups coincided with (i) the parenteral antischistosomal therapy campaigns in Egypt (1920 to 1960), (ii) the popularity of injecting drug use in The Netherlands (1960 to 1990), and (iii) the rise in high-risk sexual behavior among MSM after the introduction of highly active antiretroviral therapy (1996 onwards). Our data show that in addition to the influx of HCV-4 strains from countries where HCV-4 is endemic, the local spread of HCV-4d affecting injecting drug users and, in recent years, especially HIV-positive MSM will further increase the relative proportion of HCV-4-infected patients in The Netherlands. HCV-4-specific agents are drastically needed to improve treatment response rates and decrease the future burden of HCV-4-related disease.