Development of a multiplex RT-PCR for simultaneous diagnosis of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) from clinical specimens

Abstract

Human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) are ubiquitous respiratory viral pathogens. They belong to the family Paramyxoviridae (subfamily Pneumovirinae) and is responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. We designed and tested a multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) as a cost-effective alternative to real-time PCR and cell culture based detection for HMPV and HRSV. The newly developed PCR was used to screen nasal/throat swab samples from 356 patients with suspected acute respiratory infection attending the Government Medical College, Thiruvananthapuram, Kerala, India. The method was compared with a commercially available kit employing real time PCR, for its sensitivity and specificity. 53 (14.9 %) samples were positive for at least one tested pathogen by mRT-PCR. All except one among the positive samples showed similar pathogen profile when tested using real time PCR. 8 (15.1 %) among these 53 were positive for HRSVA, 33 (62.3 %) positive for HRSVB and 12 (22.6 %) were positive for HMPV. 17 (32.7 %) samples showed co-infections in them. Sensitivity and specificity of the mRT-PCR was comparable to that of the commercial kit. Our findings indicate that this newly developed mRT-PCR can be used as a cost-effective alternative for laboratory diagnosis of HMPV/HRSV infection and will significantly reduce diagnostic costs for these viruses in clinical settings.

Keywords: Acute respiratory infection; Human metapneumovirus; Human respiratory syncytial virus; Multiplex reverse transcriptase PCR; Real-time reverse transcriptase PCR.

Fig. 1

Agarose gel analysis of PCR products from mRT-PCR of HRSV and HMPV from clinical samples. Two H1N1 positive clinical samples were used as controls for testing primer specificity

Fig. 2

Representative amplification curves obtained with the HRSV (a) and HMPV (b) rRT-PCR. Each reaction also had an internal control (not shown)

Fig. 3

Neighbor joining tree representing the phylogenetic relationship of the sequences from RGCB RSV, India isolates with partial genome sequences of HRSVA and B isolates in the NCBI database

Fig. 4

Neighbor joining tree representing the phylogenetic relationship of the sequences from RGCB HMPV, India isolates with partial genome sequences of HMPV isolates in the NCBI database