The ProPneumo1 Assay: Detection of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and an Internal Control Using Multiplex PCR on Multiple Real Time PCR Systems S. Dollhopf,, W. Majewski,, P. Douglass, M. Khanna and S. Visuri Poster ID Association for Molecular Pathology Scottsdale, Arizona November 1112, 0
INTRODUCTION Chlamydophila pneumoniae and Mycoplasma pneumoniae cause communityacquired pneumonia, accounting for an estimated 600,000 pneumoniarelated hospitalizations per year in the U.S. These atypical organisms are not susceptible to betalactam antibiotics and lack of diagnosis impedes directed prevention and therapy. Current diagnostic techniques (culture and serology) are not efficient and lack both sensitivity and specificity, resulting in misdiagnosis and mistreatment. Development of a rapid, accurate, and costeffective diagnostic test would be helpful to facilitate the diagnosis of atypical pneumonias. Molecular assays could fill the need for rapid, sensitive, and specific tests for diagnosis and management of atypical communityacquired pneumonia. MATERIALS & METHODS A Real Time multiplex PCR assay (ProPneumo1 ) was developed that simultaneously detects Chlamydophila pneumoniae (FAM), Mycoplasma pneumoniae (CalO) and an Internal Control (Q670) in a single test. Primers and probes were designed to highly conserved regions of specific genes and were combined together in a single µl PCR reaction. Amplification and detection were performed using a similar protocol on four different Real Time instruments: Smart Cycler II (Cepheid), ABI 700 (Applied Biosystems), RotorGene 00 (Corbett Research) and the LightCycler 2.0 (Roche). Clinical validation was performed on the Cepheid Smart Cycler II and Corbett Research RotorGene 00. Clinical specimens (24 sputum, 24 bronchoalveolar lavages, and 24 nasopharyngeal swabs) were spiked with C. pneumoniae (ATCC #2822) and M. pneumoniae (ATCC #1293) at concentrations of 10 1, 10 0, and 10 1 /ml and 10, 10 4, and 10 3 dilutions of ATCC stock culture, respectively. Clinical specimens were extracted with the Roche High Pure Viral Nucleic Acid kit. µl of the resulting extract were added to µl of multiplex supermix. Each specimen was tested independently with three separate lots of reagents on the Cepheid Smart Cycler II and RotorGene 00. This resulted in 216 tests: 90 spiked with C. pneumoniae and internal control, 90 spiked with M. pneumoniae and internal control, and 36 spiked with only internal control. RESULTS Reaction : Cloned DNA fragments of known size and quantity were used to determine amplification efficiency using the following equation: E = 10 (1/slope) 1 (Figure 1).
Figure 1: Amplification efficiency of the ProPneumo1 assay on different real time PCR systems Chlamydophila pneumoniae Cepheid Smart Cycler II RotorGene 00 R 2 = 0.9966 E = 97% 0 1 2 3 R 2 = 0.9727 E = 97% 0 1 2 3 ABI 700 PCR System R 2 = 0.9997 E = 100% 0 1 2 3 4 LightCycler 2.0 R 2 = 0.99 E = 114% 0 1 2 3 4 Mycoplasma pneumoniae Cepheid Smart Cycler II R 2 = 0.9838 E = 91% 0 1 2 3 RotorGene 00 34 32 28 26 24 22 R 2 = 0.9843 E = 97% 0 1 2 3 ABI 700 PCR System R 2 = 0.99 E = 90% 0 1 2 3 4 LightCycler 2.0 R 2 = 0.996 E = 97% 0 1 2 3 4
Analytical Specificity: The ProPneumo1 assay was tested for cross reactivity with several respiratory viral and bacterial pathogens (Table 1). No cross reactivity was observed, yielding 100% analytical specificity. Table 1. Specificity of the ProPneumo1 assay tested against common respiratory pathogens Microorganism Quantity CP () MP () C. pneumoniae ATCC #2822 (TW183) 10 1 /ml 28.1 C. pneumoniae ATCC #1310 (CDC029) 10 1 /ml.6 C. pneumoniae ATCC #1360 (CM1) 10 1 /ml 29.8 C. pneumoniae ATCC #1 (TWAR43) 10 1 /ml 32.7 M. pneumoniae ATCC #1293 (M2) 10 dilution 27.4 L. pneumophila 10 3 dilution L. micdadei 10 3 dilution L. birminghamensis 10 3 dilution L. maceacherii 10 3 dilution M. fermentans 10 3 dilution M. hominis 10 3 dilution B. pertussis 10 3 dilution B. parapertussis 10 3 dilution H. influenzae 10 6 dilution Adenovirus type A Adenovirus type D Parainfluenza type 2 Parainfluenza type 3 Influenza A Influenza B RSV
Analytical Sensitivity: The analytical sensitivity of ProPneumo1 for Chlamydophila pneumoniae and Mycoplasma pneumoniae whole organisms and quantified DNA controls was determined on all four PCR systems (Table 2). The limit of detection did not differ by more than one log across all instruments. The limit of detection for recombinant DNA controls for each organism was copies/reaction (Table 2). Table 2. Limit of detection for whole organisms and recombinant DNA controls on four real time PCR systems Smart Cycler RotorGene 00 ABI 700 Light Cycler Chlamydophila pneumoniae Whole Organism ( /ml) 10 2 10 2 10 2 10 2 DNA Control (Copies/reaction) Mycoplasma pneumoniae Whole Organism (dilution of culture) 10 6 10 6 10 6 10 7 DNA Control (Copies/reaction) 0. a The limit of detection is defined as the lowest concentration which is detected > 0 % of the time (n = 6).
Clinical Validation: ProPneumo1 detected >90% of the 90 Chlamydophila pneumoniae positive samples tested and >93% of the 90 Mycoplasma pneumoniae positive samples tested (Table 3). Specificity was 100% for C. pneumoniae on both the Cepheid SmartCycler II and the RotorGene 00. For M. pneumoniae, specificity was 100% on the Cepheid SmartCycler II and 9.2% on the RotorGene 00. Table 3: Clinical validation of ProPneumo1 assay on the Cepheid SmartCycler II and RotorGene 00. Chlamydophila pneumoniae Positives (detected/tested) Negatives (negative/tested) Sensitivity Specificity SmartCycler 81/90 126/126 90 % 100 % RG00 86/90 126/126 9.6 % 100 % Mycoplasma pneumoniae Positives (detected/tested) Negatives (negative/tested) Sensitivity Specificity SmartCycler 89/90 126/126 98.9 % 100 % RG00 84/90 1/126 93.3% 9.2 %
Internal Control Performance: The performance of the Internal Control in clinical samples is shown in Figure 2. The coefficient of variation (CV) was less than 3% on all four PCR systems. The Internal Control was detected between.4 and.9 cycles on all PCR systems. Detection of the Internal Control was the most variable on the Cepheid SmartCycler II, with a range of 3.23 cycles and a CV of 2.6%. Figure 2. Internal Control performance in clinical samples Cepheid Smart Cycler II RotorGene 00 Average = 28.7 CV = 2.6 % Range = 3.23 Average = 26.7 CV = 2.1 % Range = 2.2 ABI 700 PCR System Average = 26.8 CV = 1.0 % Range = 0.94 LightCycler 2.0 Average = 26. CV = 1. % Range = 1.67
CONCLUSIONS ProPneumo1 is a rapid, sensitive, and specific assay that is beneficial to clinicians in diagnosis of atypical pneumonias. The assay demonstrated high sensitivity, specificity, and reproducibility. The assay demonstrated no cross reactivity between closely related pathogens that manifest similar symptoms. Most significantly its ease of use, competitive performance, and reliability on different real time machines makes it an excellent tool for detection of C. pneumoniae or M. pneumoniae in a single test. Contact Information: Prodesse, Inc. W229 N1870 Westwood Drive Waukesha, WI 3186 12624460700 sdollhopf@prodesse.com www.prodesse.com