Papers Multiplex Polymerase Chain Reaction for the Simultaneous

J Clin Pathol 1999;52:257-263 257 Papers Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples C Y W Tong, C Donnelly, G Harvey, M Sillis Abstract Mycoplasma and chlamydia are major causes Aims-To develop a multiplex polymerase of community acquired atypical pneumonia.1-3 chain reaction (PCR) for the simultaneous Mycoplasma pneumoniae activity occurs in the detection of Mycoplasma pneumoniae, United Kingdom in predictable four yearly Chlamydia pneumoniae, and Chlamydia cycle. So far, outbreaks have been documented psittaci in respiratory samples. during 1983, 1987, 1991, and 1995.4 Methods-Oligonucleotide primers for Chlamydia pneumoniae is rare in children the amplification of the DNA of these under five but becomes more common with three organisms were optimised for use in increasing age,6 with an adult seroprevalence combination in the same reaction. PCR of up to 40-70%.] Chlamydia psittaci, a zoono- products were detected by hybridisation sis acquired from avian exposure, is less com- with pooled internal probes using an mon than Mycoplasma pneumoniae and enzyme linked immunosorbent assay. Chlamydia pneumoniae, but is still an impor- Those with positive signals were further tant pathogen in the United Kingdom, with differentiated using species specific reported outbreaks involving contact with probes. Quality of DNA extraction and psittacine birds' and poultry,9 causing severe PCR inhibition were controlled by ampli- systemic illness in addition to pneumonia. fication ofa human mitochondrial gene. A Culture of these organisms is difficult and time panel of 53 respiratory samples with consuming.1' " Differentiation between M p- known results was evaluated blindly. This neumoniae and M genitalium, both of which was followed by a retrospective study on can be isolated from respiratory samples, is not sputa collected from 244 patients with easy,'2 '1 and the culture of C psittaci could be suspected community acquired pneumo- hazardous.'4 Frequently, insensitive and cross nia. reacting serological techniques, 15-17 such as the Department of complement fixation test, are being used as the Medical Microbiology Results-The multiplex assay had a lower and Genitourinary sensitivity than PCR with individual prim- main diagnostic test. As a result of these Medicine, University ers by about one log. The resultant sensitiv- factors, and also of the timing of the research, of Liverpool, 81F ity was considered acceptable for conflicting prevalences of these atypical infec- Duncan Building, diagnostic use. Of the panel of 53 samples, tions have been reported, ranging from 1 % to Royal Liverpool nine of 11 M pneumoniae, 11 of 11 C pneu- 40%.1-3 18 19 Many recent studies have explored University Hospital, the use of the polymerase chain reaction Liverpool L69 3GA, moniae, six ofseven C psittaci, and 24 of24 UK negative samples were correctly identified. (PCR) to detect either mycoplasma or chlamy- C Y W Tong Of the 244 patients with pneumonia, seven dia in atypical pneumonia.20.26 Here, we report C Donnelly (2.9%) had detectable M pneumoniae, six on the development and evaluation of a multi- (2.5%) had C pneumoniae, and one (0.4%) plex PCR that could simultaneously detect Department of had C psittaci. The case notes from 11 and differentiate these three organisms in res- Medical Microbiology, piratory samples. Royal Liverpool patients were studied. The PCR finding was University Hospital of possible significance in at least eight of G Harvey these patients. Methods Conclusions-This multiplex PCR assay PANEL OF KNOWN POSITIVE AND NEGATIVE Norwich Public Health has the potential to be used as a diagnostic SAMPLES Laboratory, Norwich, A panel of 53 respiratory samples, thoroughly Norfolk, UK and epidemiological tool. Further pro- M Sillis spective studies are needed to establish its investigated previously using direct antigen clinical value. detection, culture, serology, and PCR25 27 28 for Correspondence to: (3 Clin Pathol 1999;52:257-263) mycoplasma and chlamydia, was used for the Dr Tong. initial evaluation of the multiplex PCR assay. email: [email protected] Keywords: atypical pneumonia; polymerase chain The samples were collected over many years Accepted for publication reaction; Mycoplasma pneumoniae; Chlamydia by the Norwich Public Health Laboratory and 15 December 1998 pneumoniae; Chlamydia psittaci the University of Liverpool and were stored at 258 Tong, Donnelly, Harvey, et al Table 1 Test results of the positive members of the blood kit (Qiagen) according to the blood and evaluation panel body fluid protocol recommended by the Direct manufacturer. Briefly, the sample was digested antigen Previous Multiplex with the supplied protease at 70°C for 10 min- Samples detectiona Culture Serology PCR6 PCR utes, precipitated in ethanol, and centrifuged M pneumoniae through a spin column. The spin column with 1 NA Neg Pos Pos Neg the bound DNA was washed and eluted in 200 2 NA Pos Pos Pos Pos 3 NA Neg Pos NT Pos jtl of the supplied AE buffer. These were tested 4 NA Pos Pos Pos Pos either immediately or stored at -20°C. 5 NA Pos Pos Pos Pos 6 NA Pos Pos Pos Neg 7 NA Pos Pos Pos Pos' POLYMERASE CHAIN REACTION AND PRODUCT 8 NA Neg Pos Neg Pos DETECTION 9 NA Pos Pos Pos Pos The PCR primers for mycoplasma (P4A, P4B) 10 NA Neg Pos Pos Pos 11 NA Neg Pos Neg Pos and chlamydia (CP1, CP2) were described previously; they respectively amplify a 345 bp C pneumoniae 1 Pos Neg Pos Pos Pos region of the P1 adhesin gene of 2 Pos Neg Pos Pos Pos Mpneumoniae22 and a 333 bp region of the 3 Pos Neg Pos Pos Pos major outer membrane protein gene of both 4 Pos Neg Pos Pos Pos 5 Pos Neg Pos Pos Pos C pneumoniae and C psittaci.25 The reverse 6 Pos Neg Pos Pos Pos primers P4B and CP2 were both biotinylated 7 Pos Neg Pos Pos Pos at the 5' end to facilitate PCR product 8 Pos Neg NA Pos Pos 9 Pos Neg Pos Pos Pos' detection (Life Technologies). PCR reactions 10 NT NT NA Pos Pos for mycoplasma and chlamydia were initially 11 NT NT NA Pos Pos evaluated separately using serial dilutions of C psittaci culture materials. The reactions were subse- 1 Pos Neg Neg Pos Pos quently combined. Optimisation of the multi- 2 Pos Neg Neg Pos Pos plex assay was performed by testing different 3 Pos NT Pos Pos Pos 4 Pos NT Pos Neg Negd combinations of primer concentration and 5 Pos NT Pos Pos Pos' magnesium concentration, using a fixed dilu- 6 Pos Neg Pos Pos Pos tion of the culture materials at about 2 log 7 Pos NT Pos NT Pos above the detection threshold of the individual NA, test or sample not available; NT, not tested. assays, to establish a combination with the 'Direct antigen detection for chlamydia included ELISA and direct immunofluorescence, not applicable fpr mycoplasma. most comparable result. The annealing tem- bPrevious PCR performed according to refs 25 and 28. perature was set at 60°C as a compromise cPositive after dilution. between the original individual PCRs (Touch- dBoth multiplex PCR and human DNA PCR remained negative down PCR from 65°C to 55°C for chlamydia after dilution. and 65°C for mycoplasma). Apart from the annealing temperature, the -20°C. The expected results included: M condition of the optimised multiplex PCR pneumoniae (n = 1 1), C pneumoniae (n = 1 1), assay was not significantly different from the C psittaci (n = 7), and negative (n = 24). All individual assays and consisted of 10 jl of the positive samples of the panel contained a extracted DNA per reaction with 20 mM Tris single organism with no double infection. The HC1, pH 8.4,50 mM KC1, 1.5 mM MgCl2, 200 details of these positive samples, some of FM of dNTP, 0.5 jM of each primer, and 0.6 which had previously been reported,25 are units of Taq polymerase (Life Technologies) in listed in table 1. The negative samples were a final volume of 50 jil. random specimens with little known clinical Thermocyclers with heated lids (Perkin information but were tested negative for Elmer 2400 or 9600) were used and the mycoplasma and chlamydia previously. To test programme consisted of an initial denaturation the specificity of the assay, three of the known at 94°C for five minutes, followed by 40 cycles negative samples were also spiked with M sali- of 94°C for 30 seconds, 60°C for 30 seconds, varium, M genitalium, and M orale. and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. Internal probes were HOSPITAL BASED STUDY designed for PCR product detection as follows: Sputum samples (n = 279) from 244 patients (1) an M pneumoniae specific probe (P4P: 5' (mean age 60 years, range 5 to 95; 122 females GACTlITCTGAAAGCAACGCCGCAAAG- and 122 males) with suspected community ATGA); (2) a C pneumoniae specific probe acquired pneumonia, received and stored by (CPDP: 5' AAACTATACTACTGCCGTA the department of medical microbiology of the GAT), and (3) a common probe for both Royal Liverpool University Hospital during the C pneumoniae and C psittaci (CPCP: 5' TTA- winter of 1996-1997, were studied retrospec- TTAA'TTGATGGYACWATRTGGGARGG). tively using the multiplex assay. Each probe was labelled at the 3' end with dig- oxigenin using a commercial oligonucleotide 3' SPECIMEN PREPARATION AND DNA EXTRACTION end labelling kit (Boehringer Mannheim). An aliquot of each sputum sample was diluted The PCR products were denatured with an 1 in 5 with phosphate buffered saline (PBS) equal volume of 1.6% NaOH and 1 mM and centrifuged for 10 minutes in a microfuge EDTA at room temperature for 20 minutes.

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