Diagnosis

Four principal methods are used for the diagnosis of respiratory virus infections: virus isolation by culture, antigen detection, RNA or DNA detection, and serological study. For a virus that is not easily detected by virus isolation in the laboratory, it is of great importance to develop rapid, sensitive and reproducible diagnostic tests. The identification of the two hMPV serotypes, A and B, with each serotype divided into genetic sublineages, 1 and 2, has implications for the development of RT-PCR assays and serological diagnostic tests. Because of the unavailability of rapid antigen detection tests and because of its fastidious growth in cell cultures, RT-PCR has become the method of choice. RT-PCR procedures have proved to be more sensitive than virus isolation, and can detect genetically distinct hMPV strains [32].

The cytopathic effect is variable, with RSV-like syncytia formation or focal rounding and cell destruction. The search by van den Hoogen et al. [63] of a cell line with similar susceptibility for the four hMPV lineages and with enhanced detection of the virus by cytopathic effects, resulted in the generation of a subclone of Vero cells (Vero cell clone 118). This cell line is now used routinely for virus isolation in the Netherlands. Commercially available antibodies are not yet available. Monoclonal antibodies (mAb) recognizing conserved epitopes will be useful for rapid viral diagnostics using immunofluorescence (IF) or direct IF techniques as currently used for diagnosing RSV. Confirmation of hMPV causing the cytopathic effect is achieved by RT-PCR testing of the viral culture.

Most RT-PCR protocols reported to date have relied on amplification of the L, N, or F gene with primer sequences mainly derived from the prototype strain 001 from the Netherlands. A comparative evaluation of RT-PCR assays performed in a LightCycler instrument for detection of hMPV in infected cell cultures showed positivity rates of 100%, 90%, 75%, 60%, and 55% using primers for the N, L, M, P, and F genes [64]. A second evaluation in the same study on nasopharyngeal aspirates positive for the hMPV N gene, the PCR positivity rate for the L, M, P, and F genes were 90%, 60%, 30% and 80%, respectively. From this study it can be concluded that RT-PCR assays aimed at amplifying the N and L genes, which code for two internal viral proteins and seemed to be more conserved regions of the genome, appear particularly suitable for detecting hMPV from both lineages [32, 64, 65]. Rapid and sensitive RT-PCR assays for the N gene (detection limit of 100 copies) have been developed allowing rapid amplification and detection of hMPV sequences directly from clinical samples in < 2 h [64, 65]. However, if inadequate primers are selected for PCR amplification, the hMPV detection might be underestimated.

Serological testing only permits a retrospective diagnosis. Because infection is almost universal in childhood, a seroconversion or a a fourfold increase in antibody titers must be demonstrated to confirm recent infection. The serological survey performed in the Netherlands was based on an indirect IF assay using hMPV-infected cells [1]. A homemade ELISA method has also been developed using cell lysates of hMPV [41]. To conduct large serological surveys, simpler ELISA tests using viral proteins possibly derived from the two serotypes will be needed.

mAbs used for diagnostic purposes can be directed against whole hMPV proteins or against individual proteins. Ishiguro et al. [66] used specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples, and these were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. Results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1% N protein; 40.8% M protein) when compared with immu-nofluorescence antibody (IFA) detecting whole proteins of hMPV. Western blot analysis using recombinant P protein was not successful due to nonspecific binding to human sera. The hMPV IFA-positive sera reacted with the F protein of hMPV by SDS-PAGE, but the signal was weak, suggesting that they were probably directed to conformational-type epitopes of the F protein [67]. Most of the antibodies detected by hMPV IFA were suspected to reaction with the F protein. These authors developed a baculovirus (Bac)-expressed hMPV protein IFA and showed that it was more sensitive than hMPV IFA. An ELISA using the N protein of hMPV has been developed recently [50] and was reported to detect in 58 (81.6%) of 71 adults antibodies against the N protein of hMPV. In previous studies, 20 (100%) of 20 adults aged > 20 years had antibodies detected by both hMPV IFA [1, 20], and Bac-F IFA [67]. In this Bac-F IFA study, 192 of 200 serum samples of Japanese subjects between 1 month and 41 years of age showed concordant results with conventional IFA based on hMPV-infected LLC-MK2 cells [67]. The titers obtained by Bac-F were equal or higher than those obtained by the conventional IFA. From the Bac-F IFA study it can be concluded that the availability of large quantities of Bac-expressed hMPV F protein offers an opportunity to use this recombinant protein as a diagnostic reagent (EIA, IFA, immunoblot) and to study antigenic and immunogenic characteristics of the F protein. Studies like these are important and urgently needed to be able to develop an hMPV vaccine in the near future.

Leung et al. [19] used vesicular stomatitis virus (which infects animals and seldom humans) to produce recombinant hMPV F protein in a seroepi-demiological study. The ELISA-based system has many advantages over the methods used in previous studies. The amount of hMPV-specific antigen can be standardized for each assay, antibody to genotype-specific viral glycoproteins can be measured, and the results are based on defined criteria rather than subjective determinations of a positive result in an IFA.

Two rapid antigen detection methods are available: an IFA test and an ELISA. This study compared the rate of virus detection in nasopharyngeal secretions by an indirect IFA with that by RT-PCR, and showed that the IFA with an anti-hMPV mouse mAb could detect hMPV in nasopharyngeal secretions with 73.3% sensitivity and 97.0% specificity compared with the results of RT-PCR [20]. ELISA is easier to perform in daily clinical practice and provides results that are more objective than IFA.

Immunofluorescence staining of clinical specimens and shell vial cen-trifugation cultures (SVCC) are methods commonly used in clinical virology laboratories for rapid diagnosis, but need sensitive and specific mAbs. Landry et al. [68] evaluated mAb-8 to hMPV M protein for its utility in the rapid diagnosis of hMPV by both IF and SVCC methods. Detection of hMPV was similar in A549, Hep-2, and LLC-MK2 SVCC, and mAb-8 staining was optimal on day 2 post inoculation. The ability to detect positive results by 1 or 2 days after inoculation is a great advantage over present conventional culture methods. The use of mAb-8 in IF staining of clinical specimens was, however, not successful due to nonspecific background staining. mAb-8 is commercially available (MAB8510, Chemicon International, Temecula, CA) and the results of its utility in the diagnosis of viral RTIs are awaited.

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