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The University of Louisville Journal of Respiratory Infections

Abstract

Background: Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn), and Legionella pneumophila (Lpn) can cause both epidemic and endemic occurrences of acute respiratory disease and are responsible for up to 22% of cases of community acquired pneumonia. Due to the limited availability of FDA-approved molecular diagnostic assays, we developed and evaluated a multiplexed Real-time PCR assay for the detection of these agents in two respiratory specimen types on the Luminex ARIES® instrument. The instrument provides for nucleic acid extraction plus PCR amplification and target detection in the same cassette. The ARIES® instrument generates a cycle threshold value and a confirmatory melt curve value for each reaction, including results for an internal sample processing control. The limit of detection for Mpn, Cpn and Lpn, was 100 CFU/mL, 1000 CFU/mL and 100 CFU/mL, respectively. In addition, accuracy, precision, specificity and stability studies were conducted to validate the assay for diagnostic use. Between November 2016 and June 2017, a total of 836 patient specimens were processed in our reference laboratory, with six positive Mpn and two positive Lpn. No specimens were positive for Cpn during this time period. The availability of a robust multiplex PCR assay greatly enhances the ability to rapidly diagnose infections caused by these three agents causing atypical pneumonia.

Funder

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

DOI

10.18297/jri/vol2/iss1/3

table 1 v2.pdf (36 kB)
Table 1. Amplification and Melt Curve Settings for ARIES®

Table 2.pdf (29 kB)
Table 2. Assay Sensitivity for Mpn, Cpn and Lpn in BAL Specimens

Table 3.pdf (28 kB)
Table 3. Accuracy Results for the Mpn, Cpn and Lpn PCR Assay

Table 4.pdf (26 kB)
Table 4. Precision Studies for the Mpn, Cpn and Lpn PCR Assay

table 5.pdf (43 kB)
Table 5. Specificity Studies for the Mpn, Lpn and Cpn PCR assay

figure 1 a Negative control-exported from ARIES.pdf (89 kB)
Figure 1A. Negative control result. Sample data from ARIES® instrument with a negative control illustrating the amplification curve and melt temperature for the internal control (SPC).

figure 1b Positive control exported from ARIES.pdf (106 kB)
Figure 1B. Positive control result. Sample data from ARIES® instrument with a positive control illustrating the amplification curve and melt temperature for Mpn, Cpn, and Lpn (SPC).

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