Katja C. Wolthers

T cell telomere length in HIV-1 infection: No evidence for increased CD4(+) T cell turnover

Progression to acquired immunodeficiency syndrome (AIDS) has been related to exhaustion of the regenerative capacity of the immune system resulting from high T cell turnover. Analysis of telomeric terminal restriction fragment (TRF) length, a marker for cellular replicative history, showed that CD8+ T cell TRF length decreased but CD4+ T cell TRF length was stable during the course of human immunodeficiency virus type-1 (HIV-1) infection, which was not explained by differential telomerase activity. This observation provides evidence that turnover in the course of HIV-1 infection can be increased considerably in CD8+ T cells, but not in CD4+ T cells. These results are compatible with CD4+ T cell decline in HIV-1 infection caused by interference with cell renewal.

Increase in HCV incidence among men who have sex with men in Amsterdam most likely caused by sexual transmission

We retrospectively screened 1836 men who have sex with men (MSM) participating in the Amsterdam Cohort Studies (1984-2003) for hepatitis C virus (HCV) antibodies. HCV incidence was 0.18/100 person-years (PY) in human immunodeficiency virus (HIV)-positive MSM (8/4408 PY [95% confidence interval {CI}, 0.08-0.36]) but was 0/100 PY in MSM without HIV (0/7807 PY [95% CI, 0.00-0.05]). After 2000, HCV incidence among HIV-positive men increased 10-fold to 0.87/100 PY (5/572 PY [95% CI, 0.28-2.03]). Additional hospital cases (n=34) showed that MSM in Amsterdam who acquired HCV infection after 2000 reported high rates of ulcerative sexually transmitted infections (59%) and rough sexual techniques (56%), denied injection drug use, and were infected mainly with the difficult-to-treat HCV genotypes 1 (56%) and 4 (36%). Phylogenetic analysis showed 3 monophyletic clusters of MSM-specific HCV strains. The emergence of an MSM-specific transmission network suggests that HIV-positive MSM with high-risk sexual behaviors are at risk for sexually acquired HCV. Targeted prevention and routine HCV screening among HIV-positive MSM is needed to deter the spread of HCV.

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.

Epidemiology and Clinical Associations of Human Parechovirus Respiratory Infections

ABSTRACT Infections with human parechoviruses (HPeVs) are prevalent in young children and have been associated with mild gastroenteritis and, less frequently, with meningitis and neonatal sepsis. To investigate the involvement of these viruses in respiratory disease, a highly sensitive nested PCR was used to screen a large archive of respiratory specimens, collected between January and December 2007. Respiratory samples had previously been tested for eight respiratory viruses, including respiratory syncytial virus and adenovirus, by PCR. HPeV was detected in 34 of 3,844 specimens, representing 27 of 2,220 study subjects (1.2%). HPeV types were identified by sequencing the VP3/VP1 junction amplified by PCR directly from clinical specimens. The assay could amplify all HPeV types examined with high sensitivity (types 1 and 3 to 6) and also identified HPeV types in all but one of the screen-positive study specimens (25 HPeV1 and eight HPeV6 specimens). Infections with both HPeV1 and HPeV6 were seasonal, with highest frequencies in July and August, and restricted to children aged between 6 months and 5 years. Other respiratory viruses were frequently codetected in HPeV-positive specimens, with significant overrepresentation of adenovirus coinfections (37%). Most HPeV-positive specimens were referred from emergency departments, although no association with specific respiratory symptoms or disease was found. In summary, the low frequency of detection and lack of clear disease associations indicate that HPeV1 and -6 are not major pathogens in individuals presenting with respiratory disease. However, the screening and typing methods developed will be of value in further HPeV testing, including testing for meningitis cases and other suspected HPeV-associated disease presentations.

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.

Fourth Human Parechovirus Serotype

We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1–3, it should be classified as a fourth HPeV serotype.

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation

Epstein‐Barr virus lymphoproliferative disease (EBV‐LPD) following allogeneic stem cell transplantation (allo‐SCT) has a poor prognosis. We used a sensitive real‐time polymerase chain reaction (PCR) assay for quantitative detection of EBV‐DNA in plasma and serially measured EBV‐DNA levels to assess the response to treatment in allo‐SCT recipients with EBV‐LPD. Fourteen allo‐SCT recipients with EBV‐LPD who received a T cell‐depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV‐LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV‐LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV‐LPD diagnosis. Clinically responding patients showed a rapid decline of EBV‐DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non‐responders showed an increase of EBV‐DNA levels. Absolute EBV‐DNA levels at the time of EBV‐LPD diagnosis did not predict for response, but the pattern of EBV‐DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0·001). Quantitative monitoring of EBV‐DNA levels from the start of and during therapy for EBV‐LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV‐LPD following allo‐SCT.

Parechoviruses in children: understanding a new infection

Purpose of review Human parechoviruses (HPeVs) within the large and growing family of Picornaviridae are common human pathogens associated with a wide spectrum of disease presentations. Although 10 different HPeV types have been published to date, there is increasing evidence for a specific role of HPeV type 3 (HPeV3) in severe neonatal disease. In this review, we will describe both the disease associations and underlying epidemiological and/or biological basis for the often marked differences in disease outcomes between HPeV types. Recent findings Application of molecular-based diagnostic techniques has revealed an association between neonatal sepsis, encephalitis and hepatitis with HPeV3 but not with other parechovirus types. HPeV3 shows evidence for very recent emergence in human populations as well as inferred differences in cellular receptor usage. Summary The recently discovered HPeV3 has been shown to play an important role in severe neonatal infections, observations possibly linked to its very recent emergence or possibly different cellular tropism that underlie its targeting of the most susceptible individuals. HPeV infections are currently under-diagnosed and should be considered in the clinical and diagnostic evaluation of severe neonatal disease presentations.

Absolute Level of Epstein-Barr Virus DNA in Human Immunodeficiency Virus Type 1 Infection Is Not Predictive of AIDS-Related Non-Hodgkin Lymphoma

To study whether Epstein-Barr virus (EBV) load can be used to predict the occurrence of acquired immunodeficiency syndrome-related non-Hodgkin lymphoma (AIDS-NHL), we determined EBV load longitudinally for individuals infected with human immunodeficiency virus type 1. EBV load in peripheral blood mononuclear cells (PBMC) was high and displayed considerable fluctuations over time, indicating that absolute EBV load in PBMC is not predictive of the development of AIDS-NHL. EBV DNA was also detectable in serum at some time points but at a lower level.

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.