Assessment of poly-L-lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept.

AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = -0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.

Rapid quantification of viable fungi in hospital environments: analysis of air and surface samples using solid-phase cytometry.

BACKGROUND: Environmental surveillance is important in high-risk areas of hospitals to prevent fungal infections in immunosuppressed patients. Conventional culture methods for enumerating environmental fungi are time-consuming. AIM: In this field study, a solid-phase cytometry technique (SPC) and a more conventional culture-based method to quantify fungal contamination of hospital air and surface samples were compared. METHODS: For the air sampling, a liquid cyclone air sampler was used with a flow rate of 300 L/min for 10 min in each of four hospital locations. Surface swabbing was done in two locations, with two different swab types. Samples from all areas were processed by SPC and by culture on malt extract agar. FINDINGS: The mean airborne concentrations of viable fungi determined by SPC were about 1.5-fold higher than the mean concentrations obtained with the culture-based method. These differences for air samples were significant in three hospital environments. No significant difference was observed for surface samples between the two swab types and between the two analytical methods. One of the prominent advantages of SPC was its rapidity in comparison with the culture-based method (5 h versus 5 days). CONCLUSION: In this study, we showed that SPC allows for rapid monitoring of viable fungi in hospital environments. SPC can thus be used to provide an early warning and a rapid implementation of corrective measures. Viable fungi detection may be an important tool to assess infectious risk in wards with immunosuppressed patients.

High-throughput typing method to identify a non-outbreak-involved Legionella pneumophila strain colonizing the entire water supply system in the town of Rennes, France.

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.

A large multi-pathogen waterborne community outbreak linked to faecal contamination of a groundwater system, France, 2000.

A large waterborne outbreak of infection that occurred during August 2000 in a local community in France was investigated initially via a rapid survey of visits to local physicians. A retrospective cohort study was then conducted on a random cluster sample of residents. Of 709 residents interviewed, 202 (28.5%) were definite cases (at least three liquid stools/day or vomiting) and 62 (8.7%) were probable cases (less than three liquid stools/day or abdominal pain). Those who had drunk tap water had a three-fold increased risk for illness (95% CI 2.4-4.0). The risk increased with the amount of water consumed (chi-square trend: p < 0.0001). Bacteriological analyses of stools were performed for 35 patients and virological analyses for 24 patients. Campylobacter coli, group A rotavirus and norovirus were detected in 31.5%, 71.0% and 21% of samples, respectively. An extensive environmental investigation concluded that a groundwater source to this community had probably been contaminated by agricultural run-off, and a failure in the chlorination system was identified. This is the first documented waterborne outbreak of infection involving human C. coli infections. A better understanding of the factors influencing campylobacter transmission between hosts is required.

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

An improved method for the detection of Norwalk-like caliciviruses in environmental samples.

An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.

Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes.

Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles.

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-gamma and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8+ T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.

Detection of antibodies to hepatitis E virus in patients with autoimmune chronic active hepatitis and primary biliary cirrhosis.

BACKGROUND: Viral hepatitis E (HIV) frequently causes epidemic outbreaks in many developing countries. It is also present in developed countries as imported cases. The role of hepatitis A virus as trigger for autoimmune chronic hepatitis has been demonstrated, and it has been suggested that this may also apply to HEV. METHODS: The presence of anti-HEV antibodies in serum samples from patients with autoimmune chronic active hepatitis (n = 52) and with primary biliary cirrhosis (n = 25) was investigated using an Abbott assay and a peptide-based test. RESULTS: Anti-HEV antibodies were detected with the Abbott test in 13% (7 of 52) of the patients with autoimmune chronic active hepatitis, but not of these were positive in the synthetic peptide-based test. CONCLUSION: These findings indicate the HEV is not associated with primary biliary cirrhosis but may be implicated in some cases of autoimmune chronic active hepatitis.

Detection by PCR of human papillomavirus genotypes in cervical lesions of Senegalese women.

In order to analyse human papillomavirus (HPV) infection in the Senegalese population, HPV DNA was sought in 65 women with evidence of cervical cytological abnormality and in 72 pregnant women. Ninety-four percent of the patients were positive for HPV DNA as compared to 24% of pregnant women. HPV 16 was detected in cervical smears in 42% of cases, HPV 18 in 39%, HPV 6 in 26%, HPV 11 in 15%, HPV 45 in 10%, HPV 52 in 3%, and HPV 31, HPV 33 and HPV 68 in 1.5%. HPV 16 and HPV 18 were detected in 16% and 7% respectively of pregnant women. HPV DNA of unknown type was detected in 6% of cases, and multiple HPV infections were observed in 28% of cases. Low risk genital HPVs (6/11) were detected in smaller proportions (17%) among high grade squamous intraepithelial lesions (SILs) than the low grade SILs (43%). High risk HPVs (16/18) were detected in high proportions both in low and high grade SIL lesions, though the highest frequency (70%) was observed among patients with high grade lesions. In conclusion, the results confirm that HPV infections are frequent in Senegal and that HPV 18 and 45 are detected in a high proportion of patients in Africa.

Production of human papillomavirus type 45 virus-like particles in insect cells using a recombinant baculovirus.

The L1 major capsid protein of human papillomavirus type 45 was expressed in insect cells using recombinant baculovirus technology. Human papillomavirus type 45 L1 major capsid protein self-assembled into empty virus-like particles (VLPs). These 50-60 nm diameter particles were present in the nuclei of recombinant baculovirus-infected insect cells and had a density of 1.29 - 1.30 g/cm3 in cesium chloride. The expressed human papillomavirus type 45 L1 protein sequence is identical to the reference human papillomavirus type 45 strain except for one amino acid located at position 49 of the human papillomavirus type 45 L1 protein. It must be noted that the quantity of purified human papillomavirus type 45 virus-like particles is at a lower level than that previously observed with human papillomavirus type 16. Nevertheless, the ability to generate preparative amounts of human papillomavirus type 45 virus-like particles is of great importance for the production of an anti-human papillomavirus vaccine.

Detection of antibodies against human papillomavirus (HPV) type 16 virions by enzyme-linked immunosorbent assay using recombinant HPV 16 L1 capsids produced by recombinant baculovirus.

The L1 major capsid protein of human papillomavirus type 16 (HPV-16) was expressed in Sf-21 insect cells with a recombinant baculovirus. Virus-like particles obtained were purified and used to develop an enzyme-linked immunosorbent assay for detection of anti-HPV-16 antibodies in sera from 76 women with evidence of genital HPV infection and 79 controls. HPV-16-infected individuals developed antibodies directed at HPV-16 virions since reactivity against recombinant HPV-16L1 capsids was observed in 50% of them compared with only 6% in the general adult population. However, some cross-reactivities with sera from women infected with others HPV types were observed.

Detection of antibodies to L1, L2, and E4 proteins of human papillomavirus types 6, 11, and 16 by ELISA using synthetic peptides.

Antibodies against eight synthetic peptides spanning different epitopes located on L1, L2, and E4 proteins of human papillomavirus (HPV) types 16, 6, and 11 were examined in sera from 73 women infected by HPV and from 139 healthy controls. Only three of these peptides were reactive. Two located on proteins L2 and E4 of HPV 16 seem type specific since antibodies to these peptides were detected, respectively, in 21% and 15% of the HPV 16 infected patients and in 2.5% and none of women infected by other HPVs. The third peptide located on the L1 protein of HPV 6 bears a common epitope since antibodies to this peptide were detected not only in 85% of women infected by HPV 6 or 11, but also in 82% of women infected by other HPVs, and in 74% and 71% of the control groups (10-12-year-old children and adults, respectively). In conclusion, none of the peptides investigated seems useful to develop ELISAs for serological diagnosis of HPV infection.

Twelve-year follow-up study of hepatitis B immunization of Senegalese infants.

Numerous studies have documented the efficacy and safety of plasma-derived and recombinant hepatitis B vaccines. However, little is known about the long-term protection of hepatitis B vaccine, when anti-HBs declines to low or undetectable levels. This study reports results from a 9-12-year period follow up of infants immunized against hepatitis B in Senegal. At the end of the follow-up period anti-HBs were detected in 81% of children who received a booster dose at school age and in 68% of those who did not. HBsAg was detected in 19% of infants from the control group compared to only 2% of immunized infants, corresponding to a protective efficacy of 88%. The results show that long-term protection against HBsAg carriage of hepatitis B vaccination is very high and that a booster dose at school age does not significantly increase this protection.

Self-assembly of human papillomavirus type 16 capsids by expression of the L1 protein in insect cells.

The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty virions were identified by electron microscopy at densities of 1.29-1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.

Mapping of linear B cell epitopes on open reading frames 2- and 3-encoded proteins of hepatitis E virus using synthetic peptides.

Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84-88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.

Detection of hepatitis B virus DNA by polymerase chain reaction in vaccinated and non-vaccinated Senegalese children.

An hepatitis B immunization programme was initiated in Senegal in 1978, and infants included in this controlled study have been followed for a period of 2-12 years after immunization. During this period HBV infections have been observed both in vaccinated and non-vaccinated infants. The polymerase chain reaction was used to search for HBV DNA sequences in the sera of 153 children with evidence of serum markers of past or present HBV replication. Amplified HBV DNA sequences were detected in 93% of the HBsAg positive individuals, in 58% of those only positive for antiHBc antibodies and in 7.8% of antiHBs and antiHBc positive infants. The results confirm the high efficiency and long-lasting effectiveness of HB vaccine.

Mapping of linear B-cell epitopes of hepatitis B surface antigen.

Eight monoclonal antibodies directed against the surface protein of hepatitis B virus (HBV) were tested using an epitope-mapping system (Pepscan) for characterizing antigenic domains. Four different amino acid sequences corresponding to linear epitopes were identified: one in pre-S1 corresponding to the sequence 29-36, two in pre-S2 corresponding to overlapping sequences 134-141 and 137-144, and one in the S region of the protein corresponding to the amino acid sequence 117-126.