Evaluation of real-time PCR for detection of and discrimination between Bordetella pertussis,Bordetella parapertussis,and Bordetella holmesii for clinical diagnosis

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.     

Comparison of serological and real-time PCR assays to diagnose Bordetella pertussis infection in 2007

Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (>or=7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS481 and ptxA-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS481 PCR performance was superior to that of ptxA-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both ptxA-Pr and IS481 PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS481 or ptxA-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and ptxA-Pr PCR, and the most sensitive diagnostic tool was the combination of IS481 PCR with single serology.     

Rapid-cycle PCR method to detect Bordetella pertussis that fulfills all consensus recommendations for use of PCR in diagnosis of pertussis

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of Bordetella pertussis infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (Bordetella spp.). In specimens positive for Bordetella spp., B. pertussis was differentiated from Bordetella parapertussis and Bordetella bronchiseptica by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS481 and IS1001 PCR ( approximately 1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS481 and IS1001. Fifty-one specimens were positive for B. pertussis by POR and IS481 PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS481 PCR. A total of 652 specimens were negative by both methods. B. parapertussis was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of B. pertussis.     

Comparison of in-house PCR with conventional techniques and Cobas Amplicor M. tuberculosis kit for detection of Mycobacterium tuberculosis

PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis kit. MATERIALS AND METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor kit. CONCLUSION: In-house PCR and Cobas Amplicor kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis.     

Diagnosis of community-acquired pertussis infection: comparison of both culture and fluorescent-antibody assays with PCR detection using electrophoresis or dot blot hybridization

Diagnosis of Bordetella pertussis infection has been difficult due to the low sensitivity of culture. PCR tests have been shown to be more sensitive than culture, but the reported sensitivity of PCR is variable. We evaluated PCR product detection by using either agarose gel electrophoresis (PCR-gel) or dot blot hybridization with (32)P-labeled oligonucleotide probes, and we compared these methods to both culture and direct fluorescent-antibody (DFA) assays with microscopy for the detection of pertussis. This was done with 225 nasopharyngeal swab specimens collected in community clinic settings. The multiplexed PCR amplified the multiply repeated IS481 B. pertussis sequence and a sequence from the human globin gene as a positive control for specimen adequacy. Of 225 specimens, 179 were judged to be adequate for PCR analysis. Among the adequate specimens, 9, 4, and 10 were culture, DFA, and PCR-gel positive, respectively. The sensitivity of PCR-gel versus culture was 89% while the sensitivity of culture versus PCR-gel was 80%. DFA had the lowest sensitivity. Thirty specimens were positive by PCR with dot blot hybridization; no negative control specimens showed a signal above the background. Among the 79 (44%) adequate specimens with clinical data available, the rates of reported cough or persistent cough were similar for persons who were pertussis positive by each assay. The IS481 PCR, with either electrophoresis or dot blot hybridization, is a sensitive assay; however, at this time it cannot completely replace culture without an overall loss in sensitivity for the detection of pertussis. Further study is required to understand the clinical significance of B. pertussis PCR products detected by dot blot hybridization alone.     

Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii

Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported. Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.     

Multitarget PCR for diagnosis of pertussis and its clinical implications

PCR has greatly facilitated pertussis diagnosis due to the speed, sensitivity, and specificity of this assay compared to other detection methods. Various single-target PCR assays are currently utilized, but none is universally considered to be the "gold standard." Our aim was to assess the use of multitarget versus single-target PCR for the diagnosis of pertussis in clinical samples. PCR assays targeting insertion sequence IS481 (IS), pertussis toxin ptxA promoter region (PT), and outer membrane porin (PO), or recA (RA) were evaluated in respiratory specimens collected from 4,442 patients with suspected pertussis. The diagnosis of pertussis was confirmed in 309 (6.96%) patients by the 3-target IS-PT-PO/RA PCR versus 247 (5.56%) by the conventional single-target IS (P=0.007). Compared to single-target IS, the three-target combination increased the proportion of positive specimens by 1.25-fold, and two-target combinations increased the proportion of positive specimens by 1.10- to 1.24-fold. In addition, nine cases of B. parapertussis infection were also confirmed by using the discriminative features of this multitarget PCR. Of the 89 culture-proven pertussis cases, 17 (19.1%) and 5 of the 16 patients (31.3%) admitted to intensive care unit would have been missed had only the single-target IS PCR been applied. Patients with mild disease (P=0.004) and shorter hospitalization (P=0.006) were less likely to have positive cultures. This consensus generating real-time PCR approach permits a sensitive detection, as well as an accurate species identification of the causative Bordetella pathogens for the timely management of patients.     

Detection and differentiation of Bordetella spp. by real-time PCR

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.     

Significant finding of Bordetella holmesii DNA in nasopharyngeal samples from French patients with suspected pertussis

Pertussis is routinely diagnosed with real-time PCR based on insertion sequence IS481, which is not specific for Bordetella pertussis. We conducted a retrospective study using real-time PCRs specific for Bordetella pertussis and for Bordetella holmesii on 177 samples positive for IS481 PCR. Bordetella holmesii DNA was detected in 20.3% samples collected from adolescents and adults.     

Multiplex LightCycler PCR Assay for Detection and Differentiation of Bordetella pertussis and Bordetella parapertussis in Nasopharyngeal Specimens

A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.     

Evaluation of four commercial real-time PCR assays for detection of Bordetella spp. in nasopharyngeal aspirates

We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical use.     

Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis

Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.     

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C_T of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C_T values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C_T values of 30 ≤ C_T <40.     

Evaluation of Amplicor MTB test as adjunct to smears and culture for direct detection of Mycobacterium tuberculosis in the French Caribbean.

A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.     

Prospective clinical evaluation of Amplicor Mycobacterium tuberculosis PCR test as a screening method in a low-prevalence population.

Of 656 respiratory samples analyzed for Mycobacterium tuberculosis by microscopy, culture, and the Amplicor PCR method, 25 were positive by culture, 12 were positive by microscopy, and 17 were positive by the Amplicor PCR method; 16 samples were Amplicor PCR positive and culture negative. No patient except one with culture-negative, Amplicor PCR-positive samples had clinical indications of tuberculosis. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 68 and 97.4%, respectively. For culture-positive, smear-negative samples, the sensitivity of the Amplicor PCR was 46%.     

Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of Bordetella pertussis

We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis.     

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.     

Comparison of in-house and commercial sample preparation and PCR amplification systems for detection of human immunodeficiency virus type 1 DNA in blood samples from Tanzanian adults.

This study compared the performance of several in-house nested PCR systems and the Amplicor human immunodeficiency virus type 1 (HIV-1) PCR kit in the detection of HIV-1 DNA in Tanzanian samples prepared by two different methods. All six of the in-house primer sets evaluated had a higher sensitivity for HIV DNA detection in samples prepared by the Amplicor PCR sample preparation method than in those prepared by the Ficoll-Isopaque (FIP) density gradient centrifugation method. A sensitivity of 100% was achieved by combining two in-house primer sets. The sensitivity of the standard Amplicor HIV-1 PCR kit was only 59%, whereas a modified Amplicor HIV-1 PCR test had a sensitivity of 98%. Our data show that Tanzanian samples prepared by the Amplicor preparation method are more suitable for HIV-1 PCR testing than samples prepared by the FIP method. The modified, but not the standard, Amplicor HIV-1 PCR kit provides an alternative to the nested in-house PCR technique for the diagnosis of HIV infection.     

Prevalence and sequence variants of IS481 in Bordetella bronchiseptica: implications for IS481-based detection of Bordetella pertussis

We report the prevalence in Bordetella bronchiseptica of IS481, a frequent target for diagnosis of Bordetella pertussis, as approximately 5%. However, PCR amplicons of the predicted size were detectable in 78% of IS481-negative strains. Our results suggest that PCR targeting IS481 may not be sufficiently specific for reliable identification of B. pertussis.     

Detection and serogroup determination of Neisseria meningitidis in CSF by polymerase chain reaction (PCR)

A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS1106 and the ctrA gene were used to detect meningococcal DNA, and a further two assays using the siaD gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The siaD B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture.