Search

A method was developed for the detection and quantitation of HAdV (human adenovirus) and HBoV (human bocavirus) based on a duplex real-time PCR, the AB PCR, using a Smartcycler instrument. A control real-time PCR was carried out on albumin DNA to standardise the non-homogenous respiratory samples. No cross-reactivity was observed with viruses or bacteria that could be found in the respiratory tract. The diagnosis rate using the AB PCR on clinical samples was 10.7%: 3.4% for HBoV detection, 6.9% for HAdV detection and 0.3% double detection HBoV–HAdV. The clinical and epidemiological characteristics of the HAdV- and HBoV-infected patients were evaluated. In the HAdV-positive group and the HBoV-positive group the samples were classified according to the severity of the disease. The HAdV viral load did not appear to be linked to the severity of the disease. Conversely, the difference between the two HBoV groups, severe and non-severe, was significant statistically when the comparison was based on the viral load (P = 0.006) or after adjustment of the viral load to the number of cells in the samples (P = 0.02).

Aim: This study has two objectives: to study the clinical symptoms associated with the detection of the four human coronaviruses (HCoVs), 229E, OC43, NL63 and HKU1 types, in the respiratory specimens sampled from hospitalised children in France between September 2004 and May 2005; and to develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay allowing for the simultaneous detection of the four HCoVs. Methods: 1002 respiratory specimens were tested for HCoVs. The clinical and epidemiological data were compared on the basis of the type HCoV infection. Results: A hundred coronaviruses, 33 NL63, 2229E, 27 OC43 and 38 HKU1, were detected in 97 (9.8%) of 1002 samples negative in routine tests. The clinical and epidemiological characteristics of the study children were compared in three groups, 24 OC43-, 27 NL63- and 34 HKU1-infected children. HCoVs were identified mainly in children with upper and lower respiratory tract infections (50.5% vs. 29.4%). The significant difference in clinical presentation between the three coronavirus groups was the very low association between lower respiratory tract illness and HKU1 detection. Conclusions: HCoV detection in hospitalised children without any other respiratory virus detection was associated with upper and a significant rate of lower respiratory tract illness. The four types of HCoVs were detected, and new types NL63 and HKU1 represented a substantial portion of detection. The multiplex RT-PCR enabled a sensitive one-time detection and the characterisation of all of the known HCoV types with the exception severe acute respiratory syndrome-coronavirus.

From November 2004 to April 2007, specimens were obtained from 2,281 patients with acute respiratory tract illness in Normandy, France. Eighteen strains of influenza C virus were detected in these samples using a combined tissue culture/RT‐PCR diagnostic method. Most patients with influenza C virus infection (13/18) were infants or young children (

Background Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. Objective To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. Study design After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. Results Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the “gold standard” were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k = 0.80). Conclusions The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.

Coronavirus NL63 was found in hospitalized children with upper and lower respiratory infections., The human coronavirus NL63 (HCoV-NL63) was first identified in the Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory specimens for HCoV-NL63. Of the 300 samples, 28 (9.3%) were positive for HCoV-NL63. The highest prevalence was found in February (18%). The main symptoms were fever (61%), rhinitis (39%), bronchiolitis (39%), digestive problems (33%), otitis (28%), pharyngitis (22%), and conjunctivitis (17%). A fragment of the spike protein gene was sequenced to determine the variety of circulating HCoV-NL63. Phylogenetic analysis indicated that strains with different genetic markers cocirculate in France.

Background: Antigen detection assays and viral isolation techniques are routinely used to detect adenoviruses (Ad) associated with respiratory infections, and the value of the polymerase chain reaction (PCR) has recently been assessed. Objectives: This paper describes a PCR-hybridization-immunoenzymatic assay (PCR Adenovirus consensus ® ) used to detect Ad and identify Ad species in respiratory specimens. Results: On seven representative serotypes Ad 12, Ad 3, Ad 7, Ad 11, Ad 1, Ad 8, Ad 4, the mean genome equivalents per ml and the mean 50% infectious doses per ml were 10 6.3 and 10 4 , respectively. Using 362 nasal aspirates from children, Ad were detected by immunofluorescence (IF) and culture in 97 cases (27%), by the PCR-Ad hexon method in 107 cases (29.5%) and by the PCR Adenovirus Consensus ® method in 113 cases (31.2%); 13 samples were found positive by both PCR and negative by the IF and culture methods; five samples were only positive according to the PCR Adenovirus Consensus ® method. The sensitivity, specificity, predictive positive value and predictive negative value of the PCR Adenovirus Consensus ® method were 97.9%, 93.2%, 84%, 99.1%, respectively. The method identified the species (sp) from 91 positive amplicons: 1 Ad sp A, 44 Ad sp B, 42 Ad sp C, 3 Ad sp E, and 1 Ad sp F; 85 isolates were identified by IF or the neutralisation in culture, and 86 by a PCR-RE digestion method. The PCR Adenovirus Consensus ® detected six positive samples that were negative according to the IF and culture methods, and it identified the precise species of nine IF-positive and culture-negative nasal aspirates. Conclusion: The PCR Adenovirus Consensus ® technique is more efficient than the classical IF or culture techniques for the detection of Ad in respiratory samples. An internal control is included to validate the screening results, and specific probes are used to identify the Ad species.

Background: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients . It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. Objective: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. Study design: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. Results: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r =0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r =0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200 000 polymorphonuclear leukocytes (PMNLs) is log 4.095 copies per ml of blood. Conclusions: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.

The 2 groups of human coronaviruses (HCoVs) represented by the prototype strains HCoV 229E and HCoV OC43 are mostly known as viruses responsible for common cold syndrome. HCoVs are difficult to detect, and epidemiological data are rare. From October 2000 through April 2001, we tested 1803 respiratory samples for HCoV by reverse-transcriptase polymerase chain reaction. From 8 February through 27 March 2001, HCoV OC43 was detected in samples obtained from 30 (6%) of 501 patients. The other viruses detected were respiratory syncytial virus (6.1%), parainfluenza virus 3 (1%), influenza virus A (7.8%), influenza virus B (7.2%), rhinovirus (6.4%), enterovirus (1%), and adenovirus (2%). Infection with HCoV OC43 was detected in patients of all age groups. The following clinical symptoms were noted: fever (in 59.8% of patients), general symptoms (in 30%), digestive problems (in 56.8%), rhinitis (in 36.6%), pharyngitis (in 30%), laryngitis (in 3.3%), otitis (in 13.3%), bronchitis (in 16.6%), bronchiolitis (in 10%), and pneumonia (in 6.6%). This study shows that an outbreak of HCoV OC43 respiratory infection was responsible for the lower respiratory tract symptoms observed in nearly one-third of patients identified by active surveillance for coronavirus infection.

A method was developed for the quantitation of respiratory syncytial virus (RSV) based on real-time RT-PCR using a LightCycler instrument. A control real-time RT-PCR was undertaken on GAPDH mRNA (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. The real-time RT-PCR method was one log more sensitive for the detection of RSV according to the endpoint dilution technique than the culture method or a conventional qualitative RT-PCR-hybridization-EIA. No cross-reactivity was observed with any of the viruses that could be found in the respiratory tract. RSV and GAPDH were quantified in nasal aspirates from 75 children hospitalised for acute respiratory tract disease: 31 (41.3%) were positive according to the immunofluorescence assay (IFA), 34 (45.3%) were culture-positive and 42 (56%) were positive according to our real-time RT-PCR method. The sensitivity, specificity, positive and negative predictive values of the real-time RT-PCR were 100, 90, 92, 100%, respectively. The samples found to be positive for RSV were classified according to the severity of the disease. The mean number of RSV RNA copies was higher in the severe disease group than in the non-severe group 4.05×107 vs 9.1×106 (P=0.055). However, the mean ratio of RSV RNA copies to GAPDH mRNA copies was 42.8 in the severe group, and 22.2 in non-severe group (P=NS).

An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID50 of HCoV-229E and 0.01 TCID50 of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.

Background: A high frequency of virus infections has been recently pointed out in the exacerbations of asthma in children. Objectives: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children. Study design: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae. Results: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8- and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9.8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively. Conclusion: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma.

Depuis quelques annees, le VHE est clairement defini comme responsable de cas sporadiques d’hepatites aigues autochtones (genotypes 3 et 4), de formes graves chez les sujets porteurs d’une hepatopathie chronique et de formes prolongees, voire chroniques chez les patients immunodeprimes. Nous rapportons le cas d’un patient de 42 ans (Hogdkin en 2006, autogreffe en 2011) admis en reanimation le 8 juillet 2015, a 21 jours de son allogreffe pour cytolyse hepatique et defaillance multiviscerale, compatibles avec un synfrome d’activation macrophagique (SAM), d’evolution fatale, chez qui ont ete mises en evidence une viremie VHE (6,7log), BK virus (5log) et une parasitemie (toxoplasmose). Des analyses serologiques (IgG/IgM VHE) et genomiques (ARN VHE) retrospectives de 5 serums preleves entre mai 2015 et octobre 2012 ont permis de dater la seroconversion fin decembre 2012 et la viremie en octobre 2012. Le sequencage des souches 2012 et 2015 revele le meme genotype G3i, objectivant l’existence d’une hepatite chronique depuis 2012 avec viremie fluctuante et reactivation. Parmi les produits sanguins labiles recus par le patient, seuls 2 concentres de plaquettes d’apherese, administres en aout et septembre 2012, etaient suspects dans la mesure ou ce sont les 2 seules transfusions du patient au cours des 14 mois precedents la viremie. Si l’enquete ascendante a conduit a identifier un donneur ARN VHE positif (2,2log) lors de son don, la faible viremie n’a pas permis le genotypage et la comparaison phylogenique des souches donneurs–receveurs. Ainsi, le resultat de ces analyses plaide en faveur d’une contamination transfusionnelle, sans qu’une autre origine ne puisse pas etre formellement exclue. En conclusion, ce cas d’hepatite E, probablement post-transfusionnel, meconnu mais evolutif sous forme chronique a bas bruiit pendant 3 ans, incite a appliquer uen vigilance accrue, tout particulierement chez les sujets transfuses en contexte d’hemopathie et d’immunosuppression.

Les bronchiolites a VRS sont frequentes, epidemiques et necessitent parfois l'hospitalisation des nourrissons, voire une reanimation.Le determinisme des lesions pulmonaires est complexe, lie au virus et a la reaction immunitaire. Le traitement est symptomatique, et l'utilisation de la ribavirine est limitee aux formes severes. Le diagnostic virologique doit etre rapide, et la recherche immunologique directe des proteines virales est la technique la plus utilisee.

Human metapneumovirus (hMPV) is responsible for respiratory tract disease, particularly in the young and elderly population. An epidemiological and phylogenic study was performed on children admitted to hospital with an acute lower respiratory tract infection (LRI). Data were obtained and analyzed over three consecutive winters, from 2002–2003 to 2004–2005. Each year during the winter period, from November to March, 2,415 nasal swabs were tested by a direct immunofluorescence assay (DFA) for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses, and adenoviruses. Rhinoviruses, enteroviruses, and coronaviruses OC43 and 229E were detected by RT‐PCR. A RT‐PCR designed for the M gene was performed on negative samples for hMPV detection and phylogenic analyses. For the three consecutive winters, hMPV represented 10%, 22.6%, and 8.8% of virus‐negative samples, respectively. In most cases, clinical symptoms indicated a LRI with a final diagnosis of bronchiolitis. During the winter of 2003–2004, all viral clusters (A1, A2, B1, and B2) that circulated in France shifted progressively from the A group to the B group. This study determined the prevalence of hMPV in Normandy, its clinical impact and permitted the analysis of the molecular evolution during the successive outbreaks. J. Med. Virol. 83:517–524, 2011. © 2011 Wiley‐Liss, Inc.

Techniques used for the diagnostic of upper and lower respiratory tract viral infections Techniques used for the diagnostic of upper and lower respiratory tract viral infections include conventional procedures and the new molecular tools. Serology has no interest for diagnosis. Antigen detection can be carried out by simple, easy and cheap tools. They are available at hospital and in community practice as well for the detection of influenza virus, RSV, hMPV, parainfluenza virus and adenovirus. To look for viruses which are non detectable by antigen detection assays (rhinovirus, coronavirus, bocavirus, polyomavirus...) or in severe or complicated respiratory lower infections, the use of molecular tools is highly indicated.

Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene, Argene, France), a pp65 antigenemia assay and four different "in-house" real-time PCR assays were compared to the CMV R-gene for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r=0.63, p0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200000 polymorphonuclear leukocytes was 5.26log(10)copies/mL of whole blood. When the CMV R-gene kit was compared to the four other "in-house" real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good (r=0.82 for laboratory 1, r=0.66 for laboratory 3) to excellent (r=0.99 for laboratory 2, r=0.94 for laboratory 4) correlation between CMV R-gene and the four real-time "in-house" PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene test were constantly higher than those generated by three out of four "in-house" PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene and "in-house" PCRs was of 0.77log(10), 0.04log(10), 0.77log(10) and 0.97log(10), for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an "inter-laboratory" comparison of results. Our study showed that CMV R-gene is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.

Human coronavirus OC43 (HCoV-OC43) causes acute, self-limited respiratory infections. A close relationship between bovine coronaviruses (BCoVs) and HCoV-OC43 has recently been demonstrated. This study includes seven clinical, non-cell culture-adapted, contemporary HCoV-OC43 strains detected in France in 2003. By using RT-PCR and clonal sequencing of the S1 gene of HCoV-OC43, the inter-variant heterogeneity of the HCoV-OC43 circulating strains was studied and the intra-variant diversity was assessed by investigation of a quasispecies cloud. This paper brings to the forefront a high genetic diversity of circulating HCoV-OC43 variants. Genetically different groups are defined among the variants described in this study. One of these variants holds characteristics of an outlier and presents a deletion of 12 nt, also found in BCoV strains. Moreover, the presence of HCoV-OC43 as a quasispecies cloud in vivo during an acute respiratory-tract illness was discovered. It has also been revealed that quasispecies-cloud sizes are similar for the two viral populations tested.

The performances of four multiplex PCR (m‐PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA‐positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR‐positive. The m‐PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m‐PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m‐PCR, the prevalence of each virus was compared between in‐patient and out‐patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in‐patient group than in the out‐patient group, and influenza viruses were detected more frequently in the out‐patient group than in the in‐patient group. Moreover, the use of m‐PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m‐PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children. J. Med. Virol. 78:1498–1504, 2006. © 2006 Wiley‐Liss, Inc.

Background. Human coronavirus HKU1 (HCoV-HKU1), a new group 2 coronavirus, was first characterized in 2005 from 2 adults with pneumonia in Hong Kong, China. To the best of our knowledge, there is no other report to date about the detection of this new virus. We report a molecular method allowing for the detection of HCoV-HKU1 and also report the clinical presentation of 6 infected patients. Methods. We screened 141 specimens (135 nasal samples and 6 stool samples) received in February and March 2005 in our laboratory and obtained from 135 hospitalized patients (61.5% of whom were 20 years old) for HCoV-HKU1. Results. HCoV-HKU1 was detected in 6 (4.4%) of the 135 nasal specimens and in 2 (33.3%) of the 6 stool samples; the positive samples were obtained from 6 patients (5 children and 1 adult). The clinical presentation of these 6 patients was as follows: 3 were admitted to the hospital for acute enteric disease resulting in severe dehydration associated with upper respiratory symptoms; 1 had fever, otitis, and febrile seizure; 1 had a sample obtained to investigate failure to thrive; and 1 had a sample obtained for exploration of X-linked agammaglobulinemia and hyperleucocytosis. Conclusion. HCoV-HKU1 can be detected in respiratory and stool samples from children and adults in a part of the world other than Hong Kong. Our results suggest that HCoV-HKU1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions.

The significance of blood TcRγδ+ lymphocyte level was evaluated in the context of immunodeficiency and infections in 209 HIV-1-infected patients. Blood TcRγδ+ lymphocyte values were found higher in patients belonging to the CDC group II/III than those in the CDC groups IV C1 and IV D (P < 0.001) and P < 0.01, respectively). TcRγδ+ lymphocyte counts were lower in patients with oral candidiasis (P < 0.01), and in association with pneumocystosis or toxaplasmosis (P < 0.001). In 81 patients with a detectable HIV-1 p24 antigenemia, TcRγδ+ lymphocyte counts were lower than those in nonantigenemic patients (P < 0.001). In the, CDC II/III group, p24-antigenemic patients exhibited lower TcRγδ+ cell counts than those in patients without antigenemia (P = 0.06). Data suggest that depletion of the TcRγδ+ lymphocyte subset characterizes HIV-1-infected patients with oral candidiasis, pneumocystosis, toxoplasmosis, and/or HIV-1-antigenemia.

Background: The viral isolation technique (VIT) is largely used as a gold standard for the detection of influenza A and B viruses in respiratory samples. Some recent studies have pointed out that the polymerase chain reaction (PCR) assays allow sensitive and rapid detection of influenza viruses, also providing excellent correlation with traditional methods. Objectives and design study: The aim of this study was to evaluate the efficiency of three non-nested PCR, two PCR-hybridization assays using primers defined in M and NS genes, and one PCR which uses primers defined in NP, NS and HA genes and combines the detection of H3N2 and H1N1 hemagglutinin genes using defined primers in NP, NS and HA genes (PCR3), in comparison with an IF assay (IFA) and viral isolation technique (VIT). The study was carried out on 244 nasal samples collected mainly by practitioners of the GROG surveillance network during winter 1998–1999 for the detection of influenza A virus. Results: Overall influenza viruses were detected more frequently by PCR techniques in 157 (64.3%), 147 (60.2%), 110 (45%) cases for PCR1, PCR2, PCR3, respectively, than by VIT or IFA, in 100 (40.9%) and 74 (30.3%) cases, respectively. Taking the positive culture samples as a reference, 100 (41.8%) samples were found to be positive for influenza A, and the sensitivity of IFA, PCR 1, PCR 2 and PCR3 techniques were 70, 100, 99, and 90%, respectively as compared with viral isolation cultures. On the other hand, as 86.5% of positive samples were positive with at least two different techniques, the sensitivity, specificity, VPP and VPN of each technique were recalculated taking into account a further criterion defining a positive sample: positivity with two techniques. We observe that techniques PCR 2 and particularly PCR 1 have very good sensitivity, respectively 98.6 and 100%, far better than the traditional techniques, IFA and culture, whilst maintaining acceptable specificity: 94.1 and 86.1%, respectively. In both cases they enable 141 (57.7%) A-positive influenza samples to be detected instead of the 100 (40.9%) obtained when culture is the reference test. IFA, culture and PCR 3 are highly specific (VPP=100%), but in comparison with PCR 1 and 2 their sensitivity, respectively 51.7, 69.9, 77.6%, and negative predictive value are unsatisfactory. PCR 1 and 2 are superior to the other techniques to a statistically highly significant degree in terms of sensitivity, but the difference between the two is not significant.

Human parvovirus B19 (PVB19) is a DNA virus ransmitted via the respiratory route, through parenteral dministration of products derived from blood, and vertically rom mother to fetus (Heegaard and Brown, 2002). PVB19 auses erythema infectiosum, arthropathy, transient aplasic crisis, and hydrops fetalis, but there are few reports of fatal outcome involving myocarditis (Murry et al., 2001; apadogiannakis et al., 2002; Zack et al., 2005). The patient, a 5-year-old girl, was admitted to the emerency unit after a malaise with loss of conscience for 3 min ithout convulsing. She complained of headache and abdomnal pain over 2 days, with five vomiting episodes and two iarrheic stools in a context of familial gastroenteritis. The hild was pale, with blood pressure of 92/59 mmHg, heart ate of 145 beats/min, and temperature 35 ◦C. The oxygen aturation level was 99%. A few hours later, a new episode of malaise occurred with drop in blood pressure to 46/25 mmHg and a heart rate f 54 beats/min, accompanied by loss of consciousness and lotching of the skin. The child appeared to recover following dministration of isotonic fluids to counteract hypovolemia. linical examination did not reveal other abnormal findings. ranial computer tomography and cardiac examination were ormal. There were no indications of hepatosplenomegaly, bdominal mass, meningeal syndrome, dehydration, or espiratory distress syndrome. The complementary blood xaminations showed no abnormal modifications. Tropoine, C-reactive protein, and hemoglobin were within

Background: Immunofluorescence assay (IFA) of viral antigens in nasal aspirates is largely used for the diagnosis of respiratory syncytial virus (RSV), parainfluenzavirus (PIV) type 3 and adenovirus (AdV) infections, whilst rhinovirus (RV) are detected by virus isolation technique (VIT) only. Using the two techniques, IFA and VIT, a significant number of specimens remain negative in spite of clinical and epidemiological presumptions of viral infection. Objectives and study design: The polymerase chain reaction (PCR) should improve the sensitivity of viral detection in clinical specimens. From October 1995 to March 1996, 277 nasal aspirates from hospitalized infants were tested simultaneously by IFA, VIT, polymerase chain reaction and hybridization with a DNA enzyme immunoassay (PCR-EIA) for RSV, PIV-3, AdV and RV. Results: RSV were detected in 177 (64%) samples, PIV-3 in 23 (8%), RV in 40 (14%), and AdV in 30 (10%). PCR-EIA detected RSV in more samples 173 (62%) than IFAVIT: 109 (39%) (P < 10−7). In most cases (79%), RSV-infected infants had lower respiratory tract disease, and routine and PCR techniques were positive. Out of the 23 PIV-3 infections, 12 were IFAVIT- and PCR-EIA-positive, and 11 IFAVIT-negative and PCR-EIA-positive. For RV, 35 (87%) specimens were PCR EIA-positive and 11 (27%) culture-positive; for AdV 30 samples were PCR-EIA-positive and four were culture-positive. Simultaneous viral infections were revealed in a significantly higher proportion than in conventional techniques: 18% (50277) versus 2.5% (7277); P < 10−7. One RSV infection in four was associated with the presence of another virus, mainly PIV-3 (16 cases) and AdV (13 cases). Conclusions: PCR-EIA detects more positive-specimens than IFAVIT, 1.5 times more for RSV, 1.9 for PIV-3, 4 for RV and 10 for AdV, respectively. This increased sensitivity of viral detection by PCR-EIA compared to the IFAVIT could suggest that samples containing low levels of virus are missed by routine methods IFAVIT, and consequently, RSV or PIV-3, and above all RV or AdV are overlooked as agents of respiratory diseases. However, apart from the fact that the economic and convenient aspects of virus diagnostic cannot be missed, it is difficult to answer the following questions: what is the meaning of the detection of a viral sequences in nasal aspirates of infants. or may PCR have detected virus in patients who would not developed disease?