C. Gazin

RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

Point of Care Strategy for Rapid Diagnosis of Novel A/H1N1 Influenza Virus

Background Within months of the emergence of the novel A/H1N1 pandemic influenza virus (nA/H1N1v), systematic screening for the surveillance of the pandemic was abandoned in France and in some other countries. At the end of June 2009, we implemented, for the public hospitals of Marseille, a Point Of Care (POC) strategy for rapid diagnosis of the novel A/H1N1 influenza virus, in order to maintain local surveillance and to evaluate locally the kinetics of the pandemic. Methodology/Principal Findings Two POC laboratories, located in strategic places, were organized to receive and test samples 24 h/24. POC strategy consisted of receiving and processing naso-pharyngeal specimens in preparation for the rapid influenza diagnostic test (RIDT) and real-time RT-PCR assay (rtRT-PCR). This strategy had the theoretical capacity of processing up to 36 samples per 24 h. When the flow of samples was too high, the rtRT-PCR test was abandoned in the POC laboratories and transferred to the core virology laboratory. Confirmatory diagnosis was performed in the core virology laboratory twice a day using two distinct rtRT-PCR techniques that detect either influenza A virus or nA/N1N1v. Over a period of three months, 1974 samples were received in the POC laboratories, of which 111 were positive for nA/H1N1v. Specificity and sensitivity of RIDT were 100%, and 57.7% respectively. Positive results obtained using RIDT were transmitted to clinical practitioners in less than 2 hours. POC processed rtRT-PCR results were available within 7 hours, and rtRT-PCR confirmation within 24 hours. Conclusions/Significance The POC strategy is of benefit, in all cases (with or without rtRT-PCR assay), because it provides continuous reception/processing of samples and reduction of the time to provide consolidated results to the clinical practitioners. We believe that implementation of the POC strategy for the largest number of suspect cases may improve the quality of patient care and our knowledge of the epidemiology of the pandemic.

Novel virus influenza A (H1N1sw) in South-Eastern France, April-August 2009.

Background In April 2009, the first cases of pandemic (H1N1)-2009 influenza [H1N1sw] virus were detected in France. Virological surveillance was undertaken in reference laboratories of the seven French Defence Zones. Methodology/Principal Findings We report results of virological analyses performed in the Public Hospitals of Marseille during the first months of the outbreak. (i) Nasal swabs were tested using rapid influenza diagnostic test (RIDT) and two RT-PCR assays. Epidemiological characteristics of the 99 first suspected cases were analyzed, including detection of influenza virus and 18 other respiratory viruses. During three months, a total of 1,815 patients were tested (including 236 patients infected H1N1sw virus) and distribution in age groups and results of RIDT were analyzed. (ii) 600 sera received before April 2009 and randomly selected from in-patients were tested by a standard hemagglutination inhibition assay for antibody to the novel H1N1sw virus. (iii) One early (May 2009) and one late (July 2009) viral isolates were characterized by sequencing the complete hemagglutinine and neuraminidase genes. (iiii) Epidemiological characteristics of a cluster of cases that occurred in July 2009 in a summer camp were analyzed. Conclusions/Significance This study presents new virological and epidemiological data regarding infection by the pandemic A/H1N1 virus in Europe. Distribution in age groups was found to be similar to that previously reported for seasonal H1N1. The first seroprevalence data made available for a European population suggest a previous exposure of individuals over 40 years old to influenza viruses antigenically related to the pandemic (H1N1)-2009 virus. Genomic analysis indicates that strains harbouring a new amino-acid pattern in the neuraminidase gene appeared secondarily and tended to supplant the first strains. Finally, in contrast with previous reports, our data support the use of RIDT for the detection of infection in children, especially in the context of the investigation of grouped cases.

The viral etiology of an influenza-like illness during the 2009 pandemic.

Many viruses are known to cause influenza‐like illness (ILI); however, in nearly 50% of patients, the etiologic agent remains unknown. The distribution of viruses in patients with ILI was investigated during the 2009 A/H1N1 influenza pandemic (A/H1N1p). From June 2009 to January 2010, 660 patients with suspected influenza were questioned and examined, and nasal swabs were collected. All patient samples were tested for influenza virus, and 286 negative nasal swabs were tested further for 18 other respiratory viruses using real‐time RT‐PCR. Two waves of ILI were observed in the epidemic curve (weeks 35–42 and 42–49). At least eight viruses co‐circulated during this period: human rhinovirus (HRV) (58), parainfluenza 1–4 viruses (PIV) (9), human Coronavirus (hCoV) OC43 (9), enterovirus (5), adenovirus (AdV) (4), and human metapneumovirus (hMPV) (2); however, 204 samples remained negative for all viruses tested. ILI symptoms, according to the Centers for Disease Control and Prevention criteria for ILI definition, were reported in 75% of cases. These patients had positive swabs for A/H1N1p, HRV, hCoV‐OC43, PIV, AdV, and hMPV without significant difference with non‐ILI patients. This study found that many respiratory viruses circulated during this period and that the A/H1N1p did not impact on the kinetics of other respiratory viruses. The proportion of non‐documented cases remains high. ILI could not distinguish A/H1N1p infection from that due to other respiratory viruses. However, in multivariate anlaysis, cough, chills, hyperemia, and dyspnea were associated significantly with influenza virus versus other respiratory viruses. J. Med. Virol. 84: 1071–1079, 2012.

A simple method for molecular detection of swine-origin and human-origin influenza A virus

Here we present a real-time one-step reverse transcriptase-polymerase chain reaction SYBR Green assay derived from the method reported by van Elden and collaborators ( 2001 ) that ensures the rapid, sensitive, and cost-effective detection of both seasonal influenza A virus and emerging (H1N1) swine-origin influenza A virus (S-OIV). In addition to this screening test, which successfully detected both seasonal influenza A virus and S-OIV in human clinical samples, we showed that the probe initially designed by van Elden and collaborators could detect seasonal influenza A virus, but not S-OIV; a new probe was designed and tested that specifically detects S-OIV, but not seasonal influenza A. Both probe-based assays were validated by testing human clinical samples and specifically detected either seasonal influenza virus or S-OIV. Finally, in silico analysis of databases predicted that minor modifications of the van Elden primers would facilitate the use of this assay for the broad spectrum detection of all currently characterized variants of influenza A virus, including avian strains.

Evaluation of the Xpert Flu test and comparison with in‐house real‐time RT‐PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France

Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/H1N1-2009) and 52 were positive for influenza B. The Xpert(®) Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/H1N1-2009, and 80.7% and 100% for Flu B.

Interim report on the A/H1N1 influenza virus pandemic in Marseille, France, April-November 2009

Abstract We report here the results of a 7-month survey of the influenza A/H1N1 pandemic in the Virology laboratory of the public hospitals of Marseille (April–November 2009). In total, 8 587 samples were analysed during this period, of which 1 974 (23%) were positive for the novel influenza variant. The analysis of results obtained using rapid influenza diagnostic tests (RIDTs) revealed a global sensitivity of 49.4% (vs. molecular qRT-PCR detection), strongly correlated with age groups (varying from 30% to 58% for patients >40 age and <10, respectively), indicating that RIDTs can be helpful in accelerating the management of suspected cases. Epidemiological analysis showed that the winter influenza wave began in October in Marseille (i.e. 2 to 3 months earlier than usual seasonal influenza outbreaks) and that the majority of autochthonous cases were observed in patients younger than 20 years old, with a low number of cases in patients over 60 years old. In November 2009, 22.2% (167/754) of patients with a laboratory diagnosis of influenza A/H1N1 infection were hospitalized, of which 9% (15/167) were admitted to an intensive care unit (ICU). Patients in the extreme age groups (>40 years old and <1) were significantly more often hospitalized than others, and 2.4% of hospitalized patients died. During the last 3 weeks of the period, the average number of bed-days attributable to H1N1sw-positive patients was 31.4, of which 5.9 were in ICUs.

Molecular detection of human rhinoviruses in respiratory samples: a comparison of Taqman probe-, SYBR green I- and BOXTO-based real-time PCR assays

BackgroundHuman Rhinoviruses (HRV) are major causative agents of acute respiratory tract infections in all age group and important contributing factors of childhood morbidity and mortality. Clinical presentation is poorly specific and the great antigenic and genetic variability of HRVs renders the biological diagnosis complex. Here, we have evaluated several molecular diagnostic protocols, including Taqman probe-based and intercalating agent-based RT-PCR assays.Methods5,627 respiratory samples sent to the laboratory of Virology of the University Hospitals of Marseille, France, from March 2011 to February 2012, were tested using a real-time RT-PCR assay in the 5’NCR of the rhinoviral genome that associated a Taqman probe and the detection of DNA-BOXTO-dye complexes. A sample of 500 BOXTO-positive samples were further tested using the same probe assay (without BOXTO), and a SYBR Green assay (using the same amplification primers). The specific amplification of HRV sequences was assessed by NGS amplicon sequencing.ResultsThe Taqman probe RT-PCR assay identified 696/5,627 samples (12,4%) as HRV-positive. BOXTO-positive samples included all probe-positive samples and 1,913 additional samples, of which only 24.3% were confirmed by sequencing. The SYBR Green assay was more specific (16/550 samples were probe-negative/SYBR Green-positive, all confirmed by 5′NCR sequencing), but 3/500 samples were probe-positive/SYBR Green-negative.ConclusionsOur results highlight the difficulty in detecting HRVs in clinical samples using a single molecular detection system. Amongst the 3 systems tested, the best compromise was obtained with the SYBR Green assay, which, by comparison with our probe-based assay provided an improved sensitivity without altering the detection specificity. Interestingly, a majority of probe-negative/BOXTO- or SYBR Green-positive samples were not associated with mutations in the sequence targeted by the probe. Sequence-based modifications of the secondary structure of the HRV 5′NCR may be associated with a limited access to the probe hybridisation region. Further investigations may identify a test combining a probe based- and an intercalating agent-based detection, which will significantly improve the diagnosis of HRV infections.

Improved sensitivity of the novel Xpert® Flu test for detection of influenza B virus

Fully automated easy-to-use integrated molecular systems have demonstrated their capability for rapid documentation of respiratory specimens, especially in point-of-care (POC) laboratories. In our previous study ([1][1]), the Xpert Flu test exhibited respective sensitivity (Se) and specificity (Sp)

SYBR Green Real-Time PCR for the Detection of All Enterovirus-A71 Genogroups

Enterovirus A71 (EV-A71) has recently become an important public health threat, especially in South-East Asia, where it has caused massive outbreaks of Hand, Foot and Mouth disease every year, resulting in significant mortality. Rapid detection of EV-A71 early in outbreaks would facilitate implementation of prevention and control measures to limit spread. Real-time RT-PCR is the technique of choice for the rapid diagnosis of EV-A71 infection and several systems have been developed to detect circulating strains. Although eight genogroups have been described globally, none of these PCR techniques detect all eight. We describe, for the first time, a SYBR Green real-time RT-PCR system validated to detect all 8 EV-A71 genogroups. This tool could permit the early detection and shift in genogroup circulation and the standardization of HFMD virological diagnosis, facilitating networking of laboratories working on EV-A71 in different regions.