Anny Dewilde

Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT‐PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT‐PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT‐PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 ± 2.07 vs. 5.04 ± 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants. J. Med. Virol. 61:341–346, 2000.

Detection of enteroviral RNA by polymerase chain reaction in endomyocardial tissue of patients with chronic cardiac diseases

Enteroviruses are suspected to be etiologic agents in myocarditis and cardiomyopathy. The prevalence of enteroviral (EV) heart infection in patients with chronic cardiomyopathy was determined through detection of specific EV genomic sequences using reverse transcription and polymerase chain reaction (RT‐PCR) followed by slot blotting. Endomyocardial biopsies from the explanted hearts of 19 patients with dilated cardiomyopathy (DCM) and 14 patients with chronic coronary disease (CCD) were examined. EV genome was detected in 11 of 19 patients with DCM and in 8 of 14 patients with CDD. Ventricular biopsies from the control group, which included 35 healthy heart patients and 33 patients with myocardial infarction, were negative by EV RT‐PCR. The percentage of patients showing presence of EV‐RNA was almost similar in the DCM (57.9%) and CCD (57.1%) groups. The present study demonstrates that enterovirus RNA sequences persist in the myocardium in a significant proportion of patients suffering from end‐stage ischaemic and dilated cardiac diseases and supports the hypothesis of a possible direct link between EV infection and the pathogenesis of chronic heart disease.

Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay.

A rapid detection method of enteroviral RNA in clinical samples using PCR and a microwell capture hybridization assay is described. PCR products were labelled directly by digoxigenin-dUTP during the amplification step. The labelled amplicons were hybridized with a biotinylated oligo-probe and captured on commercially available test microwells coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and colorimetric reaction automatically measured. This method detected as few as 0.01 PFU/100 microl of biological sample with a result obtained within 8 h. Using this method, we were able to detect enteroviral RNA in 23 of 35 clinical specimens from 16 of 17 patients with suspected acute or chronic enteroviral infection. The samples included cerebrospinal fluid, broncho-pulmonary lavage, pericardial effusion, throat swabs, stools, sera, muscular and myocardial biopsies. In contrast, virus was isolated in cell culture in only 8 of 28 clinical specimens from 6 of the 17 patients. This easy-to-perform assay has useful potential in the rapid detection of enterovirus in acute or chronic infection. This methodology could be used for a rapid qualitative detection of other RNA viruses.

A clinical and epidemiological study of human parvovirus B19 infection in fetal hydrops using PCR southern blot hybridization and chemiluminescence detection

Ninety‐eight samples from 80 cases of spontaneous abortions after fetal death or hydrops fetalis from 12,000 pregnant women were examined using PCR. DNA was extracted from amniotic fluid, fetal blood, ascitic fluid and fetal biopsies or placenta specimens using QIA amp kits (QIAGEN). A 270‐bp length fragment located within the B19 gene NS1 was amplified using PCR followed by electrophoresis and southern‐blot hybridization assay using a horseradish peroxidase‐labelled probe and chemiluminescence detection. This assay was able to detect 1 to 10 DNA copies in a 10 |gml sample. Parvovirus B19 was identified in 11 cases (14% of fetal hydrops; 1 case for 1,100 pregnancies). Amniotic fluid was the most common and reliable sample to assess the diagnosis. Gestational age ranged from 17 to 28 weeks (mean 23 weeks). IgM antibodies were detected in 3 maternal sera, 2 patients of which reported an exposure to B19 infection during pregnancy. In 2 cases, intrauterine blood transfusions led to the cessation of symptoms and to birth of normal babies. J. Med. Virol. 54:140–144, 1998.

Virucidal activity of pure singlet oxygen generated by thermolysis of a water-soluble naphthalene endoperoxide

Using the water-soluble naphthalene carrier of singlet oxygen NDPO2, we have shown that pure singlet oxygen is able to inactivate enveloped viruses (human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus, vesicular stomatitis virus), but has no effect on non-enveloped viruses (adenovirus and poliovirus 1). These results are related to the experiments on photoinactivation of viruses by hydrophobic photosensitizers (merocyanine 540, hypericin, phthalocyanines, hematoporphyrin and benzoporphyrin derivatives) and they strengthen the hypothesis that singlet oxygen plays a predominant role in this process.

Virus and cystic fibrosis: Rhinoviruses are associated with exacerbations in adult patients

Abstract Background Few studies have suggested the potential role of respiratory viruses in cystic fibrosis (CF) exacerbation, but their real impact is probably underestimated. Method Sixty-four sputum samples collected from 46 adult patients were included in the study: 33 samples were collected during exacerbation of CF, and 31 during the stable phase. After extraction, nucleic acids were tested for the presence of respiratory viruses. When rhinovirus (HRV) was detected, the 5′UTR and VP4/2 regions were sequenced, and phylogenetically analyzed. The characteristics of patients in exacerbation and stable phase were compared. Results Viruses were found in 25% of samples. The HRV viruses were the most frequently detected followed by coronaviruses. Only the HRV detection was significantly associated with the occurrence of CF pulmonary exacerbation (p <0.027). Characterization of 5′UTR and VP4/2 regions of the HRV genome specified that HRV-A, -B, -C were detected. All HRV-C were recombinant HRV-Ca. Conclusions HRV were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. The reality of viral recombination between HRV was demonstrated in CF patients for the first time, raising the role of viruses in lung microbiota. Further studies are now warranted to decipher virus impact in CF.

Simultaneous detection of 6 human herpesviruses in cerebrospinal fluid and aqueous fluid by a single PCR using stair primers

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), human cytomegalovirus (HCMV), varicella‐zoster virus (VZV), Epstein‐Barr virus (EBV), and human herpes virus 6 (HHV‐6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at −80°C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an “in‐house” PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts. J. Med. Virol. 62:349–353, 2000.

Diagnosis of Viral Infections Using Myxovirus Resistance Protein A (MxA)

BACKGROUND: Myxoma resistance protein 1 (MxA) is induced during viral infections. MxA testing could be helpful to differentiate between viral and bacterial infections. METHODS: A prospective multicenter cohort study was performed in pediatric emergency departments. MxA blood values were measured in children with confirmed viral or bacterial infections, uninfected controls, and infections of unknown origin. First patients were used to determine MxA threshold for viral infection. The diagnostic performance of MxA was determined by using receiver operating characteristic (ROC) analysis. Sensitivities (Se), specificities (Sp), and positive and negative likelihood ratios (LR+, LR–) were calculated. RESULTS: The study included 553 children; 44 uninfected controls and 77 confirmed viral infections (mainly respiratory syncytial virus and rotavirus) were used to determine an MxA threshold at 200 ng/mL. In the 193 other patients with confirmed infections and uninfected controls (validation group), MxA was significantly higher in patients with viral than in those with bacterial infections and uninfected controls (P < .0001). The area under the ROC curve (AUC) were 0.98, with 96.4% Se and 85.4% Sp, for differentiating uninfected from virus-infected patients and 0.89, with 96.4% Se and 66.7% Sp, for differentiating bacterial and viral infections. MxA levels were significantly higher in patients with clinically diagnosed viral versus clinically diagnosed bacterial infections (P < .001). Some patients with Streptococcus pneumonia infections had high MxA levels. Additional studies are required to elucidate whether this was due to undiagnosed viral coinfections. CONCLUSIONS: MxA is viral infection marker in children, at least with RSV and rotavirus. MxA could improve the management of children with signs of infection.

Ultrasound diagnosis, management and prognosis in a consecutive series of 27 cases of fetal hydrops following maternal parvovirus B19 infection.

Introduction: Fetal hydrops caused by anemia from parvovirus B19 infection (FH-B19) is rare. Doppler measurement of the middle cerebral artery peak systolic velocity (PSV-MCA) improves its prenatal diagnosis, but its frequency and prognosis are still poorly known. Despite improved survival due to in utero transfusions, the possibility of late neurological sequelae makes prognosis uncertain. Objectives: To assess the frequency, management and prognosis of a consecutive series of FH-B19 observed over a 15-year period. Methods: Retrospective study of 27 cases of FH-B19, that is, 3/100,000 births, 24 of them discovered during routine second-trimester ultrasound. All but 1 case (96.2%) had at least four of the six ultrasound signs that Saltzman et al. [Obstet Gynecol 1989;74:106–111] suggested as indicators of anemia. Of the fetuses tested, 80% had a PSV-MCA >1.5 MoM, also indicative of anemia. Of the 19 fetuses treated by exchange transfusions, 11 were liveborn compared with 2 of the 6 not so treated (57.8 vs. 33.3%, NS). The survival rate was higher during the second half of the study period (23.1 vs. 71.4%, p < 0.02) for less severe anemia (p < 0.03) and for repeated transfusions (p = 0.03). In our series, 1 case of prenatal cerebral atrophy was identified on screening. All 13 liveborn children appeared healthy at the age of 1 year. Conclusion: In cases of fetal hydrops, Saltzman et al.’s ultrasound criteria and PSV-MCA measurement made it possible to determine the likelihood that anemia is the cause of the hydrops and to measure its intensity. Use of these techniques allowed us to choose the most appropriate treatment (transfusion or not, depending on the degree of anemia), and survival improved notably in our series.

Supramolecular effects on the antifungal activity of cyclodextrin/di-n-decyldimethylammonium chloride mixtures

Candidiasis infections are growing problem worldwide especially for the immunocompromised individuals. Di-n-decyldimethylammonium chloride is one of the most common antifungal agents used to clean medical devices. The current study examines the antifungal mechanism of di-n-decyldimethylammonium cation and its cyclodextrin inclusion complexes. Depending on the type of cyclodextrin (α-, β- or γ-CD), inclusion complexes can be as active as ammonium alone in terms of microorganism death (fungicidal activity). Moreover, with β-CD inclusion complexes, synergism is observed against fungus growth (fungistatic activity). Based on molecular dynamics, we propose a mechanism supported by cell number, selective electrode and ζ-potential measurements as a function of time. The mechanism involves four steps: (i) the positively-charged complex diffuses through the solution, (ii) it adsorbs onto the fungus membrane surface by electrostatic interaction, (iii) then it dissociates and the ammonium inserts in the microorganism membrane, and (iv) the change of the cell surface charge induces cell lysis.