M. Van Ranst

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Phase II Double-Blind, Placebo-Controlled Study of the Safety and Efficacy of Cidofovir Topical Gel for the Treatment of Patients with Human Papillomavirus Infection

Genital condylomata acuminata are nonmalignant human papillomavirus (HPV)-induced tumors in which HPV types 6 and 11 are most commonly found. Usual treatments for condylomata acuminata are nonspecific and are based on the destruction or removal of infected tissue. These procedures are often painful and are characterized by a high relapse rate. We report here what is to our knowledge the first double-blind, placebo-controlled study of the use of cidofovir, a nucleotide analogue, for the treatment of genital papillomavirus infections. Thirty patients were enrolled in the study; 19 received cidofovir, and 11 received placebo. The median number of warts and the median baseline wart area were comparable for both groups. Nine (47%) of 19 patients in the cidofovir group had a complete response (total healing), compared with 0 of the patients in the placebo group (P=.006). None of the patients in the cidofovir group experienced progression of the disease, compared with 5 (45%) of 11 patients in the placebo group. The side effects recorded for both groups were comparable.

Rotavirus Surveillance in Europe, 2005–2008: Web-Enabled Reporting and Real-Time Analysis of Genotyping and Epidemiological Data

BACKGROUND The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations.

The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate types being detected.

A unifying hypothesis and a single name for a complex globally emerging infection: hantavirus disease.

In the January 2011 issue of this journal, Swedish authors described three severe cases of predominantly pulmonary infection with the European hantavirus Puumala virus (PUUV), leading to death due to refractory shock in two of these patients [1]. Of note, the kidneys in these two fatal cases had, on autopsy, no prominent inflammatory infiltrates or haemorrhages, in contrast with the lungs which showed extensive interstitial oedema and mononuclear cell infiltrates, mainly CD8 T lymphocytes. As the authors justly point out, even in the title of their communication, it is now, perhaps, time to revise the sacro-saint paradigm existing since 1994 of two “different” infectious diseases caused by the same genus of rodent-borne hantaviruses of the Old and the New World, respectively. The former would target mainly the human kidney, and the latter mainly the human lung, resulting in the so-called “haemorrhagic fever with renal syndrome” (HFRS) [2] or the “hantavirus pulmonary syndrome” (HPS) [3]. When it appeared that the failing heart was, in fact, the most direct cause of death in refractory shock in HPS, yet another name, “hantavirus cardio-pulmonary syndrome” (HCPS), was added to the already bewildering list of over 60 mostly exotic synonyms for a disease described already in 1913 by Russian doctors in Vladivostok. On the other side of the Eurasian landmass and in 1934, Swedish doctors described an epidemic renal affection which they called “nephropathia epidemica” (NE), which was proven in 1979 to be, in fact, a milder variant of HFRS, caused by PUUV [4]. To date, 23 distinct species of hantaviruses have been recognized, each carried by their more or less specific rodent or insectivore reservoir. The most important pathogens are Hantaan (HTNV) and Seoul virus (SEOV) in the Far-East (where >90% of worldwide hantavirus infections occur), PUUV and Dobrava-Belgrade virus (DOBV) in Europe and Russia, and Sin Nombre (SNV) and Andes virus (ANDV) in the Americas [5]. The first HPS report that appeared in 1994 [3] described “a newly recognized disease” which was of paramount importance for a better understanding of an emerging viral infection in the New World, and generated an explosive surge in hantavirus research. However, H(C)PS was, and remains, a rare disease, with only 560 confirmed cases in the USA during the 1993–2010 period [6] (meaning 30 cases/year) and about 2,500 cases in the entire Americas, albeit with a much higher case fatality rate (CFR) of 35% [7]. This is in stark contrast with the current 0.1–0.2% CFR for PUUV infections with high morbidity in the Old World, with now over 50,000 registered NE cases in Europe, and even over 175,000 in Western Russia. In some peak years, Russia witnessed more than 10,000 cases/year (e.g. 11,413 in 1985), whereas registered NE numbers in Western Europe recently took epidemic proportions (e.g. 3,259 in Finland, 2008, and 1,874 in Germany, 2010), probably as a result of global warming [8]. Thus, it should not come as a surprise that Russian or European clinicians had or have now identified clinical characteristics claimed as being specific for “American HPS”. The Centers for Disease J. Clement (*) : P. Maes :K. Lagrou :M. Van Ranst National Reference Laboratory for Hantavirus Infections, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium e-mail: jan.clement@uzleuven.be

Mannan-binding lectin (MBL) gene polymorphisms in ulcerative colitis and Crohn's disease.

The inflammatory bowel diseases (IBD), Crohn’s disease (CD), and ulcerative colitis (UC), are complex multifactorial traits involving both environmental and genetic factors. Mannan-binding lectin (MBL) plays an important role in non-specific immunity and complement activation. Point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene are associated with decreased MBL plasma concentrations and increased susceptibility to various infectious diseases. If these MBL mutations could lead to susceptibility to putative IBD-etiological microbial agents, or could temper the complement-mediated mucosal damage in IBD, MBL could function as the link between certain microbial, immunological and genetic factors in IBD. In this study, we investigated the presence of the codon 52, 54 and 57 mutations of the MBL gene in 431 unrelated IBD patients, 112 affected and 141 unaffected first-degree relatives, and 308 healthy control individuals. In the group of sporadic IBD patients (n = 340), the frequency of the investigated MBL variants was significantly lower in UC patients when compared with CD patients (P = 0.01) and with controls (P = 0.02). These results suggest that MBL mutations which decrease the formation of functional MBL could protect against the clinical development of sporadic UC, but not of CD. This could be explained by the differential T-helper response in both diseases.

JC viral loads in patients with Crohn’s disease treated with immunosuppression: can we screen for elevated risk of progressive multifocal leukoencephalopathy?

Background and aims: Anti-α4 integrin therapy with natalizumab is efficacious in refractory Crohn’s disease and in multiple sclerosis, but carries an estimated 1/1000 risk of progressive multifocal leukoencephalopathy (PML) caused by reactivation of latent JC virus infection. Athough anti-α4 integrin therapies are likely to be introduced in the clinic, screening for the risk of PML has not been developed. Methods: We prospectively collected urine, serum, plasma and buffy coats from 125 patients with Crohn’s disease, 100 control subjects with gastrointestinal (GI) disease, and 106 healthy volunteers. Four to eight weeks after this first sample collection, we additionally collected a set of urine, serum, plasma and buffy coat samples from the 125 patients with Crohn’s disease, and a next set of samples was collected 12–16 weeks after the first collection. JC viral loads were determined with quantitative real-time polymerase chain reaction (PCR), and JC virus seroprevalence with a specific enzyme-linked immunosorbant assay (ELISA). Results: The overall JC virus seroprevalence was 65%. JC virus DNA copies were detected in the urine from 29–44% of subjects, both those with Crohn’s disease and controls. Median viral loads were significantly higher in patients with Crohn’s disease who were immunosuppressed (7.36×106 copies/ml) compared to healthy volunteers (2.77×105 copies/ml) and compared to GI controls (1.8×106 copies/ml). Clearance at any time point occurred in 4/107 (3.7%) subjects only. JC viraemia was found in two patients with Crohn’s disease. Conclusions: The natural history of JC virus in patients with Crohn’s disease is still unknown. Our study results show that JC virus latency and urine viral shedding is frequent in immunosuppressed patients with Crohn’s disease. More prospective studies are needed in order to agree on possible recommendations concerning the exclusion of patients with JCV viraemia from anti-α4 integrin treatment.

Clinical Predictive Value of Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples

The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.

Evaluation of the New Architect Cytomegalovirus Immunoglobulin M (IgM), IgG, and IgG Avidity Assays

ABSTRACT A panel of new cytomegalovirus (CMV) assays for use on the Architect instrument has been developed, including a CMV avidity assay based on a new technology. The purpose of this study was to compare the performance characteristics of the fully automated CMV immunoglobulin M (IgM), IgG, and IgG avidity tests on the Architect instrument with those of other available assays. A total of 503 consecutive fresh patient serum specimens (routine serum specimens) and 96 serum specimens from 33 pregnant women with a recent CMV primary infection (seroconversion serum specimens) were tested for CMV IgM and IgG by the Architect (Abbott), Vidas (BioMérieux), and Enzygnost (Siemens) assays. The seroconversion sera and 100 preselected serum specimens IgM negative and IgG positive by the AxSYM assay were also tested by the IgG avidity tests on the Architect and Vidas instruments. The relative agreements for CMV IgM determination with routine sera between the Architect assay and the Vidas, Enzygnost, and AxSYM assays were 97%, 94%, and 93%, respectively, for the CMV IgM tests and 99%, 98%, and 98%, respectively, for the CMV IgG tests. The specificities of the CMV IgG avidity test were 98% for the Architect assay and 76% for the Vidas assay. No high CMV IgG avidity test results were found within the first 3 months after seroconversion by either of those assays. The correlation between the results of the newly developed CMV IgM and IgG tests on the Architect instrument with the Vidas and Enzygnost assays was excellent (≥94%). The CMV IgG avidity test reliably excluded patients with recent infections and showed an excellent specificity (98%).

Human Papillomavirus Genotype Spectrum in Czech Women: Correlation of HPV DNA Presence With Antibodies Against HPV-16, 18, and 33 Virus-Like Particles

Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV‐genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus‐primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot‐blot hybridisation with type‐specific oligonucleotide probes and thermocycle DNA sequencing. Twenty‐two different HPV types were detected, HPV‐16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV‐DNA‐positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low‐grade lesions, 58.0% in high‐grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV‐specific IgG antibodies was tested by an enzyme‐linked immunosorbent assay (ELISA) using virus‐like particles (VLPs) of HPV‐16, ‐18, and ‐33. Antibodies were significantly more prevalent in HPV‐DNA‐positive than in HPV‐DNA‐negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV‐16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV‐16 or HPV‐33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV‐18‐DNA‐positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV‐specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV‐16, ‐18, and ‐33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age. J. Med. Virol. 58:378–386, 1999.