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Utility of real time PCR in the rapid diagnosis of pyogenic meningitis.

Introduction

Bacterial meningitis is a serious and sometimes fatal infection affecting the central nervous system (1-3). Detection of bacteria in these body sites reassures clinicians about the chosen empirical antimicrobial therapy, may help to streamline antibiotic treatment once the antibiotic sensitivity of the isolate has been assessed, and allows for prognostic information. Potential benefits are reductions in side effects of antimicrobial therapy, in treatment costs, and in selection of resistant bacterial strains (4). The current standard for the diagnosis of invasive bacterial infections is microscopic examination and culture of body fluids considered to be sterile in healthy subjects. Nevertheless, this approach is neither very fast nor optimally sensitive. Microscopy, although rapid, requires a relatively large concentration of bacteria ([10.sup.4] CFU/ml) to become positive (5-6), and identification based on morphology is often not possible. Culture results may be available only after 24 h to 72 h. Moreover, culture results may be false negative when fastidious or culture-resistant bacteria are involved or when patient samples are obtained after antimicrobial therapy has started. Public Health Laboratory Service (PHLS) and Communicable Disease Surveillance Centre (CDSC) data show a growing discrepancy between the numbers of clinically suspected and culture-confirmed cases of bacterial meningitis, with particular reference to Streptococcal infection and N. meningitidis in India.

To address this problem, non-culture methods like PCR have been employed (7) and shown to confirm additional cases of Streptococcal disease (8-10). Broad-range PCR could offer an important benefit, as it can detect any kind of bacterial DNA present in a sample through targeting conserved bacterial sequences. The identification of all bacterial pathogens would be desirable, and to this end, amplification of conserved ribosomal nucleotide sequences has provided a strategy for universal detection of bacteria. Unfortunately, this approach is compromised by the presence of residual bacterial DNA contaminating the manufacture of commercially available reagents (11), which has frustrated attempts to exploit the 16S rRNA gene to develop a highly sensitive PCR assay for the universal detection of bacterial causes of meningitis (12-15).

TaqMan (Sequence Detection System) enables amplification and detection to be carried out at the same time in a closed-tube system. Continuous real-time PCR monitoring permits the rapid throughput of large numbers of specimens in a highly standardized format (16-18). Multiplex PCR is particularly economical for small-volume samples such as cerebrospinal fluid (19-20). Recent developments in molecular technique have exploited the TaqMan platform to enable the introduction of sensitive and specific assays for the non-culture detection of Streptococci pneumoniae and Neisseria meningitidis (21). Advances in real-time PCR technology have now made possible the selective amplification of multiple genes in one reaction vessel by utilizing spectrally distinct phosphoramidite dye-labeled probes. Thus, multiple targets can be specifically identified in a single assay, obviating the need for repeated analysis.

The three major pathogens causing bacterial meningitis which are difficult to culture are N. meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae type b. These three organisms accounted for 88.9% of all bacterial meningitis in India.

This study outlines the evaluation of a single-tube multiplex real-time PCR for the simultaneous detection of N. meningitidis, H. influenzae, and S. pneumoniae in clinical samples using the TaqMan system. The sensitivity and specificity for the detection of the three major meningitis-causing pathogens are assessed. The application of the multiplex PCR as an epidemiological tool for improved non-culture diagnosis was evaluated.

Materials And Methods

Bacterial strains and culture methods

(i) Sensitivity. The sensitivity of each of the primer sets was evaluated using samples from culture-confirmed cases of meningococcal disease (20 Blood, 16 CSF, and 8 throat swab samples), H. influenzae disease (6 CSF samples, and 1 whole-blood-heparin sample), and pneumococcal disease (14 CSF samples and 07 Blood) that had been obtained from diverse sources. For determination of comparative sensitivities of the primer sets in multiplex and individual PCR formats, serial dilutions (undiluted to [10.sup.-4]) of quantitated DNA preparations for N. meningitidis, H. influenzae, and S. pneumoniae were tested. Different concentrations of template DNA from each organism were tested in combination in order to ascertain the ability of the assay to co-amplify multiple gene targets.

(ii) Specificity. The specificities of the three primer sets were determined using genomic DNAs from bacteria and most likely to be present in CSF and blood samples and from other Neisseria species. The bacterial strains obtained from the Pune, reference centre, India had been isolated from blood or CSF samples and stored--80[degrees]C. The different bacterial strains obtained are S. pneumonia NCTC 11887, NCTC 11899, NCTC 11903, NCTC 11904, and NCTC 11908, H. infuenzae strains NCTC 8466, NCTC 8467, NCTC 8469, NCTC 8470, NCTC 8455, and NCTC 8473. S. pneumonia strains were cultured overnight on 5% (vol/vol) blood agar (Hi-media, India) and H. influenzae cultured on heated blood agar (Hi-media) at 37[degrees]C in 5% C[O.sub.2].

Quantitation of DNA in bacterial cultures. A sweep of colonies from a pure culture obtained using a sterile cotton swab was emulsified in 2 ml of sterile injectable water in a microbiological class 2 safety cabinet. Using a spectrophotometer set at 650 nm, the bacterial suspension was standardized to an optical density of 0.1 and adjusted to a concentration of approximately 20,000 bacteria / ml, which represents 40 bacteria per 2 [micro]l of inoculum.

DNA extraction. For the clinical isolates and samples, a 100-[micro]l aliquot of standardized suspension or sample was added to 1 ml of DNAzol (Gene Lab, India), vortexed, and incubated for 5 min at 20[degrees]C. A 500-[micro]l volume of 100% ethanol (SD Chemicals, India) was added, and the tube was vortexed and incubated for a further 10 min at 20[degrees]C. Following centrifugation at 12,000 x g for 10 min, the supernatant was aspirated and a further 1 ml of 75% (vol / vol) ethanol was added to the tube, vortexed, and centrifuged at 12,000 x g for 5 min. The supernatant was aspirated (with care being taken to remove any residual ethanol), resuspended in 50 [micro]l of sterile water added to the tube, and incubated for a minimum of 10 min in a water bath at 50[degrees]C.

PCR design. Oligonucleotide primers and dye-labeled probes (Table-1) were designed using the ABI Primer Express Software Package based on previously published ctrA (13, 14)), bexA (24), and ply (25) gene sequences. The ctrA sequence-specific probe was 6-carboxyfluorescein labeled, the bexA probe was tetrachloro-6-carboxyfluorescein labeled, and the ply probe was VIC (chemical name not disclosed by ABI at present) labeled.

PCR components and amplification profile. Based on a 25-[micro]l reaction volume, the master mixture was prepared from the TaqMan Universal Master Mix kit (ABI). Briefly, this comprises a 300 nM concentration of each oligonucleotide primer; 25 nM 6-carboxyfluorescein-labeled probe; 100 nM (each) VIC and tetrachloro-6-carboxyfluorescein fluorescently labeled probes; 5.5 mM Mg[Cl.sub.2]; 200 [micro]M (each) deoxynucleoside triphosphates dATP, dCTP, dGTP, and dUTP; and 0.125 U of Taq DNA polymerase. A negative (no-template) control and control DNA preparations (2 [micro]l) for each of the bacterial pathogens were included in every run. DNA was amplified with the TaqMan system using the following cycling parameters: heating at 95[degrees]C for 10 min followed by 45 cycles of a two-stage temperature profile of 95[degrees]C for 15 s and 60[degrees]C for 1 min. Real-time PCR results were based on the fluorescence readings taken by the TaqMan machine, which are used to calculate a baseline reading for each reaction. The cycle threshold ([C.sub.T]) value is the PCR cycle number (out of 45) at which the measured fluorescent signal exceeds a calculated background threshold identifying amplification of the target sequence. If no increase in fluorescent signal is observed after 45 cycles, the sample is assumed to be negative.

Clinical evaluation of culture-negative samples. After establishing the specificity and sensitivity of the multiplex assay, it was used to assess the incidences of the three pathogens in 242 samples that had been submitted to the microbiology laboratory for PCR testing (24). Clinical findings were suggestive of meningitis being part of the differential diagnosis. These samples had been DNAzol extracted and stored at -20[degrees]C. Before PCR screening for the three pathogens in the multiplex format, the samples were vortexed to resuspend any bacterial DNA. Any PCR-positive result was confirmed by testing with single primer sets for each gene target (ctrA, bexA, and ply). In addition, newly identified N. meningitidis ctrA-positive samples were tested with the serogrouping siaD PCR assay as a means of confirmation (10). Where it was not possible to confirm a multiplex result, the original specimen was re-extracted and retested using the single primer in an attempt to confirm the initial result.

Result

Assay evaluation

(i) N. meningitidis ctrA PCR. The sensitivity of the meningococcal ctrA PCR was 88.4% when tested against samples from culture-confirmed cases of meningococcal disease (Table-2). There was no difference in the sensitivity of the ctrA primer set when compared in multiplex and single-primer-set formats using serially diluted N. meningitidis DNA (Table-3). The ctrA primer set amplified DNAs from meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z and diverse serotypes and sero-subtypes. The primers did not amplify DNA from any of the other bacterial DNA extracts tested (100% specificity) (Table-4). There was no cross-reaction with human genomic DNA.

(ii) H. influenzae bexA PCR. The sensitivity of the bexA PCR when tested against nine samples from culture-confirmed cases of Hib disease was 100%.There was no difference in the sensitivity of the bexA primer sets when compared in multiplex and single-primer-set formats using serially diluted H. influenzae DNA (Table-3). The bexA primer set amplified DNAs from H. influenzae Pittman types b and c. The primers did not amplify Pittman type a, d, e, or f. There was no cross-reactivity with any of the other bacterial and viral DNA extracts tested (100% specificity) (Table-4) and no cross-reaction with human genomic DNA.

(iii) S. pneumoniae ply PCR. The S. pneumoniae ply PCR sensitivity was assessed using 16 samples from culture-confirmed cases of S. pneumoniae disease and was determined to be 91.3% for CSF and 92.3% for serum or plasma, giving an overall sensitivity of 91.8%. There was no difference in the sensitivity of the ply primer set when compared in multiplex and single-primer-set formats using serially diluted S. pneumoniae DNA (Table-3). The primer set amplified the 23 pneumococcal serotypes tested (serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31). There was no cross-reactivity with any of the other bacterial and viral DNA extracts tested (100% specificity) (Table-4) and no cross-reaction with human genomic DNA.

Multiple target amplification. Two different gene targets were tested using culture extracts and were shown to co-amplify without a loss in sensitivity (Table-5). Three samples from which one organism had been cultured and one sample previously PCR positive were simultaneously positive with two of the three gene targets. These results were reproducible with the respective individual PCR assay. Of the three samples PCR positive for both N. meningitidis and S. pneumoniae, two had been confirmed as N. meningitidis infections by culture. Hib was cultured from the H. influenzae and S. pneumoniae PCR-positive sample (Table-6). These results were reproducible upon re-extraction of the original sample.

Culture-negative samples. By testing a large number of culture-negative samples, the N. meningitidis primers developed for use in the multiplex assay were found to be significantly more sensitive (P < 0.0001) than the previously reported ctrA primer set (14). This represented an improvement in the meningococcal detection rate of 2.9% (13.0 versus 15.9% of total ctrA PCR-positive samples), or 87 additional cases identified by PCR alone. Of these samples, 53.8% were confirmed as serogroup B or C by siaD PCR. Conversely, 62 previously ctrA PCR-positive results were not detected using the multiplex ctrA primer set on repeat testing. Of these, 24.2% had been confirmed by siaD PCR. The total number of specimens ply PCR reactive by the multiplex assay was 73, of which 48 (65.7%) were confirmed using the ply primer set only. This represents an additional 46 cases confirmed by PCR alone, as two patients were ply positive for CSF and plasma samples. One sample not previously Hib culture positive was identified with the bexA primers in the multiplex assay.

In total, 38 samples of 536 (7.1%) which were positive by the multiplex assay were not confirmed by the appropriate single-primer-set PCR, of which 46 (93.9%) had a [C.sub.T] value of greater than 34 of 45 PCR cycles. Twenty-five PCR screen-positive samples could not be tested further due to insufficient specimen amount.

Discussion

Due to the problems associated with the development of a sensitive and specific universal PCR assay (10), a single-tube multiplex PCR was developed based on N. meningitidis, S. pneumoniae, and H. influenzae, which are responsible for upwards of 80% of cases of bacterial meningitis in developed and developing countries (18).

The ctrA gene is unique to N. meningitidis, and parts of the gene are highly conserved and common to all meningococcal serogroups (19). A previous study has demonstrated the rapid PCR amplification of the ctrA gene by continuous monitoring on the TaqMan system using samples from normally sterile sites (21). The limitation of this assay was the design of the forward primer near the 5' end of the gene, which contains sequence variation between different meningococcal serogroups, particularly those that contain sialic acid (B, C, Y, and W135) and those that do not (19). The ctrA primer set reported here amplified sialic acid-containing and non-sialic acid-containing meningococcal serogroups and was found to be significantly more sensitive than the previously described meningococcal ctrA PCR (21, 28). Of the discrepant results between the two ctrA assays, a higher proportion of the samples positive with the new primer set were confirmed by siaD PCR (53.8%) than were confirmed with the previous ctrA primer and probe set (24.2%). The high proportion being confirmed enhances the degree of confidence in the results obtained with the new ctrA primer set. Increased sensitivity is most likely due to improved primer design, a characteristic that has been noted by other scientists (18, 21). In addition, amplification of all meningococcal serogroups may account for some of the additional positive samples; for example, a case of serogroup A disease not detected with the previous primer set was identified using the new set. The new ctrA primers failed to amplify DNA from some samples that had been previously positive; however, almost all of these samples had been weak positives in earlier analyses. These samples remained negative upon repeat testing with the original ctrA primer set and also after the sample was reextracted and retested. This failure to repeatedly detect DNA in specimens indicates that degradation of DNA is likely to be occurring and is likely to be caused by long-term storage and/or repeated freezing and thawing of samples. In addition, sampling error is more likely to be associated with previously low-level-positive samples. The gene target is present in low numbers as indicated by the high [C.sup.T] value, and sample variation would lead to non reproducible amplification.

The bexA gene encodes the capsulation-associated bexA protein present in all capsulated H. influenzae strains. These strains express one of six capsular polysaccharides (types a to f) (22, 29, 30). The amplification of the bexA gene for the detection of H. influenzae in CSF samples has previously been reported, and the gene was shown to be amplified in all six H. influenzae types. Van Ketel et al. used a relatively insensitive, gel-based detection system and experienced problems with contamination (30). Optimal primer and probe sets for use in the TaqMan system were developed using the criteria in the Primer Express software (ABI) from the available Hib sequence. This primer and probe set amplified types b and c only, and the inability to detect other serotypes was assumed to be because of nucleotide sequence variation.

The S. pneumoniae pneumolysin gene encodes the hemolysin species-specific protein toxin produced intracellularly by all clinically relevant pneumococcal serotypes (21). PCR amplification from clinical material is indicative of invasive pneumococcal infection. There have been several reports of pneumococcal PCR utilizing amplification of the pneumolysin gene (16, 27, 29), with a report of PCR for the detection of S. pneumoniae DNA in culture-negative samples where meningitis was the diagnosis (5). This assay utilized the autolysin gene and was evaluated using only a small number of culture-negative clinical samples.

The pneumococcal PCR developed here was specific for the 23 pneumococcal serotypes tested while simultaneously offering a high level of sensitivity. The 4,113 samples tested were from patients clinically suspected as having meningococcal disease; 48 samples from 38 cases were confirmed as pneumococcal PCR positive. These had not been identified by laboratory culture, emphasizing the beneficial impact of including pneumococcal PCR in the routine diagnostic testing strategy.

Data suggest that 1% of all cases of meningitis are due to more than one pathogen (9), and such cases have recently been reported in the literature (6, 23). However, traditional laboratory methods may not always identify multiple pathogens in a single clinical sample, as identification from culture is based on the predominating organism and may be influenced by the use of selective culture media. PCR assays have been shown to amplify multiple pathogens (15, 28), but these assays relied on a nested PCR approach for improved sensitivity. The multiplex PCR in this study co-amplified gene targets in a single-round PCR with a correlation between the organism identified by laboratory culture and the one with the lowest [C.sub.T] value (Table 6). In all cases, the cultured organism has the lower [C.sub.T] value, suggesting that this was the predominant organism in the specimen. The possibility of cross contamination of extracts was ruled out by reproducing the original results using another extract of the sample. The evidence confirms other observations that on some occasions, more than one organism may be present in a clinical sample and these may be underdetected by traditional laboratory methods.

In cases where it was impossible to confirm the multiplex PCR-positive results, the cycle number to reach the baseline threshold ([C.sub.T]) value was greater than 34 in 93.9% of specimens. Plasmid titration experiments for the generation of a standard curve have demonstrated that the detection of samples around cycle 35 represents a target input of fewer than 10 copies (14). This is therefore approaching the limits of detection of the PCR, and it would be expected that a positive result would not always be obtained on repeat testing due to sampling error.

By utilizing the available TaqMan technology, the introduction of a three-in-one multiplex PCR enables rapid identification and a high throughput of samples (130 min for 96 specimens), with a modest additional cost for primers and probes in each reaction. The multiplex PCR demonstrated that testing a large number of previously culture-negative specimens provides information on the incidence of meningococcal, H. influenzae, and pneumococcal infections in clinical specimens originally referred for meningococcal PCR testing. The inclusion of the multiplex PCR in the routine molecular diagnostic screening regimen would provide an adjunct in improved non-culture diagnosis and case ascertainment of meningitis and septicemia.

Acknowledgement

Authors would like to thank Department of Microbiology, Gulbarga University, Gulbarga for facilities and all the hospitals for the clinical samples and Gangagene for the facilities

Reference

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Shameem Sultlana (1), C. S. Vinod Kumar *, (2), Vandana Rathod (3), K. G. Basavarajappa (4) and K. N. Kalappanavar (5)

(1) Senior Research Scholar, Division of Research in Medical Sciences, Department of Microbiology, Gulbarga university, Gulbarga, Karnataka, Lecturer, KBNIMS, Gulbarga

(2) Assistant Professor, Department of Microbiology, S. S. Institute of Medical Sciences, Davangere-577005

(3) Reader, Division of Research in Medical Sciences, Department of Microbiology, Gulbarga university, Gulbarga, Karnataka

(4) Professor & Head, Department of Microbiology, S. S. Institute of Medical Sciences, Davangere-577005

(5) Professor & Head, Department of Pediatrics, S. S. Institute of Medical Sciences, Davangere-577005

* Correspondence Address
Table 1: Sequences and position of oligonucleotide primers and
probes.

Gene Sequence (59 to 39)
target
 Forward primer Reverse primer Dye-labeled probe (a)

ctrA 617-GCTGCGGTAG 727-TTGTCG CGG 6-FAM-680-
 GTGGTTCAA-635 ATTTGC AACTA-708 CATTGCCACG
 TGTCAGCTGCACAT-
 657

bexA 142-GGCGAAA 241-GGCCAA GA TET-189-
 TGGTGCTGG GATACTCA TAGA CACCACTCA
 TAA-160 ACGTT-217 TCAAACGAAT
 GAGCGTGG-163

Ply 894-TGCAGAGC 974-CTCTTACTC VIC-941-
 GTCCTTTGGT GTGGTTTCCAAC TGGCGCCCA
 CTAT-915 TTGA-950 TAAGCAACA
 CTCGAA-918

(a) 6-FAM, 6-carboxyfluorescein; TET,
tetrachloro-6-carboxyfluorescein.

Table 2: Sensitivity of meningococcal PCR with culture-confirmed

Sample type N. meningitidis ctrA result Sensitivity (%)

 Positive Negative

CSF 32 4 88.9
Whole blood-EDTA 28 6 82.4

Table 3: Comparative end point sensitivities of primer sets in
multiplex and single-set formats.

Target Assay CT value (a) at the following
 format dilution ofDNA

 Undiluted [10.sup-1] [10.sup.-2]

N. meningitidis Multiplex 26.94 30.39 32.91
 ctrA Single 26.85 30.07 34.25

H. influenzae Multiplex 28.54 31.58 34.62
 bexA Single 28.49 32.07 37.96

S. pneumoniae Multiplex 29.41 32.26 35.4
 ply Single 29.74 32.06 35.28

Target CT value (a) at the
 following dilution
 of DNA

 [10.sup.-3] [10.sup.-4]

N. meningitidis 37.25 >45.0
 ctrA 38.73 >45.0

H. influenzae >45.0 >45.0
 bexA >45.0 >45.0

S. pneumoniae 36.32 >45.0
 ply 36.5 >45.0

CT;-Cycle threshold

Table 4: Specificities of N.meningitidis ctrA, H. influenzae bexA
and S.pneumoniae ply primers in multiplex PCR.

 No. No. (%) reactive with
Organisms Tested following primer set

 ctrA bexA Ply

N. Meningitidis serogroup A 3 3(100)
N. Meningitidis serogroup B 24 24(100)
N. Meningitidis serogroup C 25 25(100)
N. Meningitidis serogroup X 3 3(100)
N. Meningitidis serogroup Y 3 3(100)
N. Meningitidis not groupable 8 8(100)
H. influenzae type b 9 9(100)
H. influenzae type c 1 1(100)
S.pneumoniae type
1,2,3,4,5,6,7,8,9,10A,11A,12, 15 15(100)
14,15B,17F, 18, 19, 20, 22,
23, 24, 31,

Total 90 66 10 15

Table 5: Co-amplification of the target genes.

 Cr Value for the
 following target gene *

Template ctrA bexA Ply

N.meningitidis 38.00
H.influenzae 31.37
S.pneumoniae 26.40
N.meningitidis plus H.influenzae 39.44 31.69
N. meningitides plus S.pneumoniae 36.29 27.07
H.influenzae plus S. pneumoniae 31.33 26.95

* Cr value is the cycle number at which the measured fluorescent
signal exceeds a calculated background threshold identifying
amplification of the target sequence

Table 6: Comparison of dual infection.

 Ct value for following
Multiplex Sample gene targets PCR and or culture
PCR result no.
 ctrA bexA Ply

 1 34.36 >45.0 36.34 N. meningitidis
 serogroup C by PCR
N.meningitidis 2 36.06 >45.0 41.85 N. meningitidis
plus serogroup b by culture
S.pneumoniae 3 35.09 >45.0 38.81 N. meningitidis
 serogroup C by culture
 & PCR

S.pneumoniae 1 >45.0 31.33 39.90 Hib by culture
plus
H.influenzae
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Author:Sultlana, Shameem; Kumar, C.S. Vinod; Rathod, Vandana; Basavarajappa, K.G.; Kalappanavar, K.N.
Publication:International Journal of Biotechnology & Biochemistry
Date:May 1, 2010
Words:4807
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