Performance of virus isolation and Directigen® Flu A to detect influenza A virus in experimental human infection

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Abstract

Background: few data exist to assess the sensitivity of different specimen types for viral detection during the course of influenza virus infection. Objectives: this study assessed the relationships between quantitative influenza A virus replication and antigen detectability by the enzyme immunosorbent assay (EIA) Directigen® Flu A in different type of samples during experimental human infection. Study design: fourteen volunteers were inoculated with influenza A virus A/Texas/36/91 (H1N1). Four specimens types were collected in sequence for quantitative isolation in cell culture and antigen testing from days 1 to 8 after inoculation. Results: seventy-one (63%) of nasopharyngeal wash specimens were culture positive, compared to 51 (46%) of throat gargles, 51 (46%) of nasal swabs, and 27 (24%) of throat swabs. All subjects shed virus in their nasopharyngeal wash at least one day and 86% of subjects had a positive nasopharyngeal wash culture on day 2 after inoculation. The mean viral titers were highest on day 2 post inoculation for all specimen types and averaged 3.6 log10 TCID50/ml for nasal washes, 1.2 log10 TCID50/ml for throat gargles, 1.8 log10 TCID50/ml for the nasopharyngeal swabs, and 0.6 log10 TCID50/ml for the throat swabs. Mean viral titers in the nasal washes were significantly different (P<0.05) compared to other specimen types. The peak of sensitivity of EIA (compared to culture) was the second day after inoculation. Nasopharyngeal and throat swab results were combined for this analysis and considered positive by culture if positive in either or both samples. Thus, on day 2 the number of EIA positive samples relative to the number culture positive was 9/12 (75%) for nasopharyngeal wash specimens, 2/9 (22%) for throat gargles, and 7/11 (64%) for the combined throat and nasal swabs specimens. Conclusions: nasopharyngeal washes are the most sensitive sample type detecting influenza A virus in adults. For rapid diagnosis the Directigen® Flu A is a an alternative with a sensitivity compared to culture ranging between 64 and 78% if performed on nasopharyngeal specimens on day two or three after experimental infection in adults. However, if performed on other specimens or later in the course of infection the sensitivity is lower.

Introduction

The ability to detect influenza viruses in clinical specimens depends on the assay used, the type and site of sampling, and the time during the course of infection. The current reference method to detect influenza A in clinical samples is isolation in cell culture (Frank et al., 1979, Monto et al., 1981). Virus replication is detected usually by hemadsorption with erythrocytes or when cytopathic effect is observed. Hemadsorbing activity is present in over 90% of positive cultures by 3 days and cytopathic effect is observed in 90% of cases by 5 days (Johnston and Siegel, 1991, Johnston et al., 1992). Many observations suggest that influenza virus is more readily cultured from nasopharyngeal specimens than from throat specimens (Kaiser and Hayden, 1999). In addition, nasopharyngeal washes provide better yield than swab specimens. However, little comparative data exist to assess the sensitivity of different specimen types during the course of infection.

Rapid diagnosis of influenza enables appropriate antiviral treatment of patients with influenza like-illness. New anti-influenza agents, such as neuraminidase inhibitors (Calfee and Hayden, 1998) will be soon available, which emphasizes the need for appropriate diagnostic procedures. Rapid diagnosis may also improve surveillance and allow implementing earlier effective control measures against influenza epidemics and localized outbreaks in closed populations. The combination of cell culture in shell vial or plate format, combined with type or sub-type antigen detection (Harmon and Pawlik, 1982, Espy et al., 1986, Mills et al., 1989) allows detection of influenza virus in 80% of cases within 1 or 2 days (Espy et al., 1986, Stokes et al., 1986, Ray and Minnich, 1987, Waner et al., 1991, Ryan-Poirier et al., 1992, Johnston and Bloy, 1993, Leonardi et al., 1994). Direct immunofluorescence for detection of viral antigens on respiratory cells is also possible but these assays have a widely variable sensitivity (Schmidt et al., 1982, McQuillin et al., 1985). Molecular techniques such as RT-PCR have a sensitivity ranging between 92 and 95% compared to culture (Claas and Van Sprenger, 1993, Wright et al., 1995, Atmar et al., 1996, Kaiser and Hayden, 1999). Although, RT-PCR can be performed in less than 24 h this method is not a rapid diagnostic technique that is applicable at the point-of-care in out patient settings. One currently available assays is the Directigen® Flu A, an enzyme immunosorbent assay that detects influenza A type specific nucleoprotein antigen. Qualitative detection of viral antigen occurs in less than fifteen minutes. Studies comparing Directigen® Flu A to viral culture reported sensitivity values ranging from 39 to 100% for varying sample types and patient populations (Espy et al., 1986, Ryan-Poirier et al., 1992, Johnston and Bloy, 1993, Leonardi et al., 1994, Kaiser and Hayden, 1999).

The purpose of the current study was to assess the relationships between quantitative viral replication/viral dynamics, and antigen detectability by the Directigen® Flu A assay, in different type of clinically relevant samples during the course of experimentally induced influenza in healthy volunteers.

Section snippets

Participants and design

Fourteen healthy volunteers (median age 21 years, range 19–40) were experimentally infected with influenza A virus A/Texas/36/91 (H1N1), at the University of Virginia Health Sciences Center. The volunteers were confirmed to be susceptible to the challenge virus based on serum hemagglutination inhibition antibody titers of<1:8 performed 60 days and 1 day before the inoculation. Nasopharyngeal washings were collected before inoculation to exclude a wild type virus infection.

Participants were

Viral cultures

Fourteen subjects were inoculated with influenza A A/Texas/36/91 virus. One specimen in each sampling site was obtained for each subject during the 8 days of the study. On the last day samples were not obtained from one participant. All subjects inoculated shed influenza A (H1N1) virus in nasopharyngeal washes on at least one day post-inoculation. The mean duration of shedding was 5.1 days (range 1–8 days). Seventy-one (64%) of nasopharyngeal wash specimens were culture positive, 51 (46%) of

Discussion

Our results show that nasopharyngeal washes are the most sensitive sample type detecting influenza A virus in adults experimentally infected. During the 8 days following influenza A virus inoculation 63% of daily specimens were culture positive compared to 24 to 46% with the other types of specimens. Viral titers in nasopharyngeal specimens were also significantly higher than in any other specimen, both at peak and overtime after inoculation. As expected the mean viral titer were higher on day

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