Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens

Abstract

The ResPlex I assay (Qiagen) was designed to amplify and detect DNA of six bacterial respiratory pathogens. This assay was compared with real-time PCR assays based upon the same target sequences for the ability detect the target bacteria by use of both stock strains and specimens from respiratory disease patients. The ResPlex I assay is somewhat less sensitive than real-time PCR assays but offers the advantage of multiple assays in a single reaction.

Differential diagnosis of bacterial respiratory diseases such as pneumonia is a public health priority because of high morbidity and mortality rates. Each year, an estimated 500,000 bacterial pneumonia-related hospitalizations and 40,000 deaths occur in the United States (4, 5). Acute respiratory infections are caused by a wide range of microorganisms (1, 2). Clinically, it is difficult to make a distinction between causative infectious agents, and conventional diagnosis is usually based on microbial culture. Culture techniques are time-consuming, and some pathogens like Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydia pneumoniae are very difficult to culture. Also, Streptococcus pneumoniae can be cultured from the upper respiratory tract for as many as 68% of children and 15% of adults in the absence of respiratory disease (8). In addition, individuals with pneumococcal infections who are receiving antibiotics can have negative culture results (10). Thus, a variety of other diagnostic methods including antigen testing and PCR applied to different clinical specimens such as urine, blood, bronchoalveolar lavage fluid, transthoracic needle aspirates, and nasopharyngeal (NP) and oropharyngeal swabs have been used to define both invasive and noninvasive respiratory infections (12, 15, 16).

For fatal cases, formalin-fixed, paraffin-embedded (FFPE) tissues are often the only specimens available. Nucleic acid extracts from such specimens are limited in volume and therefore the number of assays that can be performed for various pathogens are limited. Real-time PCR assays are generally rapid, sensitive, and specific. Multiplex PCR assays could be useful for the rapid and simultaneous detection of coinfection of respiratory bacteria in a specimen without additional cost or processing. However, multiplex PCR is still challenging because amplification conditions for multiple targets are often incompatible and the high concentration of primers typically yields elevated background readings and reduced amplification efficiency. Target-enriched multiplex PCR addresses these issues via application of liquid-phase bead-based array (Luminex xMAP) technology.

The multiplex PCR ResPlex I assay (Qiagen, Valencia, CA) was developed as part of a cooperative research and development agreement between the Centers for Disease Control and Prevention (CDC) and Genaco Biomedical Products, Inc. It is not intended for clinical diagnostic use. The assay amplifies and detects gene-specific DNA sequences of six respiratory bacterial pathogens: S. pneumoniae (lytA), Neisseria meningitidis (ctrA), encapsulated or nonencapsulated Haemophilus influenzae (bexA, ompP2), L. pneumophila (mip), Mycoplasma pneumoniae (ATPase), and C. pneumoniae (ompA) (4, 6, 9, 11, 13, 17, 18). The objective of this study was to design the ResPlex I assay using modifications of CDC real-time PCR primers and probes to C. pneumoniae, L. pneumophila, M. pneumoniae, N. meningitidis, and S. pneumoniae and examine the application of this assay for the detection of bacteria associated with respiratory infections. Analytical specificity and the limit of detection of the ResPlex I assay were determined and the assay was compared with immunohistochemistry (IHC) and real-time PCR for its ability to detect these specimens in NP swabs and FFPE tissues.

The following 98 bacterial isolates were used to determine the analytical sensitivity of the ResPlex I assay: S. pneumoniae types 1, 3, 4, 6A, 7F, 8, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 21, 22F, 23F, 33F, 35A, and NT (n = 28); L. pneumophila strains representing serogroups 1 to 15 (n = 16); M. pneumoniae types I and II (strains M129 and FH, respectively) (n = 2); C. pneumoniae strains CM-1, TW-183, CW-011, A-03, K66, IOL 1515, FML-19, BAL 16, IOL 207, and T2023 (n = 10); H. influenzae serotypes a, b, c, d, e, f, and nontypeable (NT) (n = 35); and N. meningitidis serogroups A, B, C, W135, X, Y, and nongroupable (NG), (n = 7). The analytical specificity of the ResPlex I assay was determined using a panel of DNA from 111 microorganisms normally found in the respiratory tract or other species of phylogenetically related organisms (Table 1). The Qiagen QIAamp DNA mini kit (Qiagen, Valencia, CA) was used for the extraction of nucleic acid from all of the bacterial isolates. DNA concentration was determined by use of an ND 1000 spectrophotometer (Nano Drop Technology, DE), and the extracts were stored at −20°C. For comparison of the limits of detection between the ResPlex I assay and real-time PCR, the DNA templates were serially diluted. Equal amounts of DNA were used in both assays.

TABLE 1.

Microorganisms used to determine the analytical specificity of the ResPlex I assay

Genus	Species	No. tested (total no., 111)
Streptococcus	pseudopneumoniae, pyogenes, agalactiae, oralis, mitis, crista, gordonii, sanguinis, parasanguinis, vestibularis, salivarius, peroris, australis, oligofermentans, infantis, cristatus, sinensis, “viridans”	41
Chlamydia	trachomatis (serovars A, B, C, D, E, F, G, H, J, K, L2), psittaci (serovars A, B, D, E)	16
Mycoplasma	salivarium, fermentans, orale, genitalium	4
Legionella	lansingensis, erythra, oakridgensis, jordanis, wadsworthii, maceachernii, birminghamensis, sainthelensi, tucsonensis, dumoffii, longbeachae, bozemanae, gormanii, micdadei, feeleii, anisa, hackeliae, parisiensis, rubrilucens, nautarum, cincinnatiensis, worsleiensis, shakespearei	23
Haemophilus	haemolyticus, parainfluenzae, aegyptius	5
Neisseria	gonorrhoeae, subflava, lactamica	3
Bordetella	pertussis, bronchiseptica	2
Enterococcus	faecalis	1
Dolosigranulum	pigrum	1
Branhamella	catarrhalis	1
Staphylococcus	aureus, epidermidis, warneri	5
Mycobacterium	tuberculosis, fortuitum	2
Escherichia	coli	1
Corynebacterium	diphtheriae, pseudotuberculosis	2
Nocardia	farcinica, asteroides	2
Klebsiella	pneumoniae	1
Pseudomonas	aeruginosa	1

Twenty-six FFPE samples from fatal cases of pneumonia or meningitis/meningococcemia with bacterial etiologies (S. pneumoniae [n = 15], H. influenzae [n = 3], N. meningitidis [n = 4], L. pneumophila [n = 4], and multiple-agent infection of S. pneumoniae, H. influenzae, L. pneumophila, and N. meningitidis [n = 3]) confirmed by IHC or culture were tested with the ResPlex I assay and organism-specific real-time PCR assays. DNA was extracted from the specimens with the QIAamp DNA mini kit (Qiagen, Valencia, CA) following the tissue extraction protocol. Forty-nine NP swab samples were obtained from patients hospitalized for pneumonia (16). The NP samples had been tested previously using real-time PCR assays for M. pneumoniae and C. pneumoniae. These samples were also analyzed for S. pneumoniae by real-time PCR while being tested by the ResPlex I assay. All clinical samples were extracted using the QIAamp virus BioRobot MDx extractor (Qiagen, Valencia, CA).

The ResPlex I assay protocol follows. A 5-μl sample of bacterial DNA (5 ng/μl) was added to a mixture containing 25 μl of HotStart Taq master mix (Qiagen Inc., CA), 6 μl of ResPlex I SuperPrimers, and 14 μl of sterile water. A no-template control sample was included by adding 5 μl of water in place of the DNA sample volume. Samples were amplified in a thermocycler under the following conditions: initial PCR activation, 95°C for 15 min; enrichment cycling, 94°C for 30 s, 52°C for 1 min, and 72°C for 1 min (15 cycles); two-step cycling, 94°C for 15 s and 70°C for 1.5 min (6 cycles); three-step cycling, 94°C for 15 s, 52°C for 15 s, and 72°C for 15 s (30 cycles); and final extension, 72°C for 3 min. PCR products were hybridized with probe-coupled beads in a 96-well flat-bottom plate by use of a Luminex XY platform heater prewarmed to 52°C. The ResPlex I bead mix was resuspended by vortexing for at least 20 seconds. Forty-five microliters of the hybridization master mix was prepared by mixing 35 μl of hybridization buffer with 10 μl of ResPlex I bead mix and dispensed into each well. Five microliters of PCR product was added and mixed thoroughly. Samples were hybridized at 52°C for 10 min. After hybridization, 10 μl of streptavidin-phycoerythrin diluted in detection buffer was added to each sample. Samples were mixed briefly and incubated at 52°C for 5 min. The reactions were stopped by adding 120 μl of stopping buffer prewarmed at 52°C. Samples were analyzed at 52°C on a Luminex 100 following the manufacturer's instructions. Collected data were exported to Excel (Microsoft Corporation, Redmond, WA).

For real-time PCR assays, 2- to 5-μl samples of 10-fold serial dilutions of extracted DNA from each organism were added to 20 μl of a PCR master mixture consisting of 1× TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA), 100 to 900 nM of each primer, and 100 to 200 nM of fluorescently labeled probe, depending on each PCR protocol. The 10-fold serial dilutions contained 5 × 10⁵ to 1 × 10⁶ copies of DNA per PCR. Primers and probes were previously titrated to check for amplification efficiency. Amplification and detection were performed with an ABI Prism 7500 sequence detection system.

The ResPlex I assay detected all DNA samples from the 98 strains except for 1 H. influenzae serotype a isolate that was not detected by the ompP2 probe but was positive for the bexA probe. A comparative analysis of the limits of detection of the multiplex PCR assay and real-time PCR assays demonstrated that the ResPlex I assay has a similar sensitivity for N. meningitidis and is 10-fold less sensitive in detecting S. pneumoniae, L. pneumophila, and M. pneumoniae. The ResPlex I assay is 100-fold less sensitive for the detection of C. pneumoniae than the respective real-time PCR assays. The limits of detection for H. influenzae were not compared to those of real-time PCR assays because real-time PCR assays for both targets (bexA, ompP2) are not available at CDC. The ResPlex I assay did not react with DNA from any of the 111 organisms representing other respiratory pathogens and normal respiratory flora (Table 1).

Comparative results of IHC staining, real-time PCR, and the ResPlex I assay for the 26 FFPE specimens are shown in Table 2. There was fairly good agreement among the three assays, although the ResPlex I assay detected slightly fewer agents in specimens known to contain nucleic acids of multiple pathogens.

TABLE 2.

Number of FFPE specimens positive for bacterial respiratory pathogens by three assays

Organism (no. of specimens)	% Positive (no. positive/no. tested) by:
	IHC	Real-time PCR	ResPlex I assay
S. pneumoniae (n = 15)	100 (15/15)	100 (15/15)	87 (13/15)
H. influenzae (n = 3)	67 (2/3)	NDb	100 (3/3)
N. meningitidis (n = 4)	75 (3/4)	100 (4/4)	100 (4/4)
L. pneumophila (n = 4)	100 (4/4)	75 (3/4)	75 (3/4)
Multiple agentsa (n = 3)	0c (0/3)	100 (3/3)	33 (1/3)

^aMultiple pathogens detected in a single specimen included combinations of L. pneumophila, N. meningitidis, H. influenzae, and S. pneumoniae.

^bND, not determined.

^cIHC assays detected at least one pathogen. The percentage reflects the number of specimens where a particular assay detected more than one of the potential pathogens.

The ResPlex I assay was performed on 49 NP swab specimens known to be positive by real-time PCR for three pathogens (C. pneumoniae, M. pneumoniae, and S. pneumoniae) (Table 3). The ResPlex I assay detected 50, 59, and 81% of the C. pneumoniae-, M. pneumoniae-, and S. pneumoniae-positive samples, respectively. In 16 specimens, where two of these organisms were detected by real-time PCR, the ResPlex I assay detected 43%.

TABLE 3.

Results of ResPlex I assay testing of 49 NP swabs positive for three bacterial pathogens by real-time PCR

Organism (no. of patients)	% Positive (no. positive/no. tested) by ResPlex I assay
S. pneumoniae (n = 16)	81 (13/16)
C. pneumoniae (n = 10)	50 (5/10)
M. pneumoniae (n = 39)	59 (23/39)
Multiple agentsa (n = 16)	44 (7/16)

^aMultiple pathogens detected in a single specimen included combinations of C. pneumoniae, M. pneumoniae, and S. pneumoniae.

There is a clear need for multiplexed PCR assays, especially for the diagnosis of respiratory disease. Real-time PCR assays for respiratory pathogens can be sensitive and specific, but finite volumes of clinical specimens can limit the number of these assays that may be performed for a single patient. The need for rapid assays is highlighted by events such as the 2003 severe acute respiratory syndrome outbreak and the threat of pandemic influenza. A variety of strategies have been advanced to combine nucleic acid amplification assays. Many of these suffer from the consequences of incompatible primer sets and nonspecific reactions (3). The ResPlex I assay attempts to overcome these problems by use of nested primers for each target along with “super primers” which are used at higher concentrations later in the PCR. Amplicon detection utilizes the Luminex platform to allow the recognition of multiple targets in a single assay.

The ResPlex I assay was designed, in part, through a Cooperative Research and Development Agreement with CDC (CRADA no. CID04-186-00) with primer and probe sequence information from CDC real-time PCR assays for M. pneumoniae, C. pneumoniae, L. pneumophila, N. meningitidis, and S. pneumoniae. This study compares these real-time PCR assays and the ResPlex I assay using a number of stock strains to establish analytical sensitivity (limit of detection) and analytical specificity. In addition, a number of previously tested clinical specimens were simultaneously tested by both assays. IHC testing of available tissue specimens is important, since it can define if an organism is causing the pathology by demonstrating the organism in areas of inflammation. This is in contrast to organisms identified with other methods (culture or PCR) from contaminated specimens (such as NP swabs), where the clinical significance cannot be clearly defined.

The ResPlex I assay did not detect any DNA from the other 111 strains of bacteria associated with the respiratory tract, suggesting a high analytical specificity. The limit of detection for the ResPlex I assay is somewhat lower for some of the pathogens, especially C. pneumoniae. The assay may lack sensitivity to detect some NT H. influenzae strains because of the high diversity of the ompP2 gene and considering that NT H. influenzae will not react with the bexA probes designed to detect encapsulated H. influenzae. In addition, some encapsulated H. influenzae strains may not react with both probes. The ResPlex I assay was less sensitive in detecting respiratory pathogens on NP swabs, as shown in Table 3, detecting 50% of the samples positive for C. pneumoniae, 59% of those positive for M. pneumoniae, and 81% of those positive for S. pneumoniae. This study is biased in favor of the real-time PCR assays, since many of the specimens were selected based upon the fact that they were positive by these assays. The somewhat lower sensitivity of the ResPlex I assay may be compensated for by its overall efficiency as a multiplexed assay that can detect multiple pathogens from a single specimen. The ResPlex I assay should be further evaluated by prospective surveillance studies of respiratory disease in comparison with validated assays.