Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study

Abstract

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR-enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

FIG. 1

Separation of m-RT-PCR products on an agarose gel. A total of 10 μl of each of the m-RT-PCR products was separated on a 2% agarose gel. The m-RT-PCR was performed with 1 μl of viral or bacterial nucleic acid as the template, as described in Materials and Methods. The expected product lengths are given in the text. The sizes of the DNA fragments of the marker (0.7 μg of MspI-digested pUC19) are as follows: 1, 501 and 489 bp; 2, 404 bp; 3, 331 bp; 4, 242 bp; 5, 190 bp; 6, 147 bp; 7, 111 and 110 bp). Inf A and Inf B, influenza A and B viruses, respectively.

FIG. 2

Proportion of positive m-RT-PCR results. The number of samples with positive m-RT-PCR results and the total number of samples are given on the y axis. The time scale on the x axis is from November 1995 to April 1998.

FIG. 3

Frequency of clinical specimens with positive m-RT-PCR–ELISA results. The number of samples with positive m-RT-PCR results for each of the nine organisms is given on the y axis. The time scale on the x axis is from November 1995 to April 1998. InfA and InfB, influenza A and B viruses, respectively.

FIG. 4

Percentage of organisms causing infections. The proportions of organisms causing a respiratory disease are given as a percentage of the total number of patients infected with organisms causing the disease. Organisms not included in the test are not indicated in the figure. InfA and InfB, influenza A and B viruses, respectively.