Detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofluorescent-antibody test

Abstract

Human metapneumovirus (hMPV) is a recently discovered pathogen associated with respiratory tract infections, primarily in young children, immunocompromised individuals, and elderly individuals. Reverse transcription-PCR (RT-PCR) has been reported to be a more sensitive method for the diagnosis of hMPV infections than virus isolation by culture and serological study. However, there has been no report on rapid methods, such as an immunofluorescent-antibody test or an enzyme-linked immunosorbent assay, for the detection of hMPV antigens in nasopharyngeal secretions. In this study, we compared an indirect immunofluorescent-antibody test (IFA) with a monoclonal antibody with RT-PCR for detection of hMPV in nasal secretions from 48 hospitalized children with respiratory tract infections. Fifteen of the 48 children were positive for hMPV by RT-PCR. IFA results were positive for 11 of the 15 RT-PCR-positive children (sensitivity, 73.3%) and 1 of the 33 RT-PCR-negative children (specificity, 97.0%). Although the sensitivity of IFA is lower than that of RT-PCR, IFA is a rapid and useful test for the diagnosis of hMPV infections in children.

FIG. 1.

Typical IFA staining of hMPV-infected LLC-MK2 cells (A, granular staining [white arrow]; B, diffuse staining) and nasopharyngeal epithelial cells (C, IFA-positive staining; D, IFA-negative staining) with the anti-hMPV monoclonal antibody (monoclonal antibody 1B7).

FIG. 2.

Phylogenetic analysis of hMPV fusion nucleotide sequences. A tree was constructed by the neighbor-joining method with 100 bootstraps with random sequence addition. The length of the horizontal line represents the number of substitutions per site. Bootstrap values over 70% are shown. Avian pneumovirus subgroup C (APV; GenBank accession number AF187152) was the outgroup and was used to root this tree. *, samples negative for hMPV by IFA. The hMPV sequence analysis included the following isolates (GenBank accession numbers are given in parentheses): isolates 94-1 (AF371342), 99-1 (AF371344), 00-1 (AF371337), and 93-1 (AF371341) from The Netherlands (23) and isolates JPS04-396, JPS04-407, JPS04-389, JPS04-372, JPS04-379, JPS04-394, JPS04-399, JPS04-402, JPS04-405, JPS04-395, JPS04-401, JPS04-409, JPS04-406, JPS04-411, and JPS04-417 (AY653162 to AY653176) from Sapporo.