Comparison of PCR, Culture, and Serological Tests for Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infection in Children

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.     

Diagnosis of Mycoplasma pneumoniae Pneumonia in Children

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of Mycoplasma pneumoniae infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for M. pneumoniae-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of M. pneumoniae DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of M. pneumoniae pneumonia in children of any age.     

Evaluation of PCR, Culture, and Serology for Diagnosis of Chlamydia pneumoniae Respiratory Infections

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of Chlamydia pneumoniae by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a C. pneumoniae infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute C. pneumoniae infection; nine (39%) of these subjects were C. pneumoniae PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute C. pneumoniae infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several “gold standards” were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive C. pneumoniae PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory Chlamydia infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of C. pneumoniae infections in a given community.     

Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia

In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by Streptococcus pneumoniae. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy.     

Viruses and Bacteria in the Etiology of the Common Cold

Two hundred young adults with common colds were studied during a 10-month period. Virus culture, antigen detection, PCR, and serology with paired samples were used to identify the infection. Viral etiology was established for 138 of the 200 patients (69%). Rhinoviruses were detected in 105 patients, coronavirus OC43 or 229E infection was detected in 17, influenza A or B virus was detected in 12, and single infections with parainfluenza virus, respiratory syncytial virus, adenovirus, and enterovirus were found in 14 patients. Evidence for bacterial infection was found in seven patients. Four patients had a rise in antibodies against Chlamydia pneumoniae, one had a rise in antibodies against Haemophilus influenzae, one had a rise in antibodies against Streptococcus pneumoniae, and one had immunoglobulin M antibodies against Mycoplasma pneumoniae. The results show that although approximately 50% of episodes of the common cold were caused by rhinoviruses, the etiology can vary depending on the epidemiological situation with regard to circulating viruses. Bacterial infections were rare, supporting the concept that the common cold is almost exclusively a viral disease.     

Rapid detection of blaKPC carbapenemase genes by real-time PCR

Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla_KPC) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla_KPC genes using TaqMan chemistry. The q-PCR amplification of bla_KPC DNA was linear over 7 log dilutions (r² = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla_KPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla_KPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla_KPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla_KPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla_KPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.     

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.     

Streptococcus pneumoniae DNA Load in Blood as a Marker of Infection in Patients with Community-Acquired Pneumonia

Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.Streptococcus pneumoniae is the most prevalent cause of community-acquired pneumonia (CAP) in adults (1, 10). Identification of S. pneumoniae as a cause of CAP can be achieved with Gram stain microscopy, culture of respiratory tract samples, blood culture, PCR, and antigen detection testing (26). A positive blood culture result provides a definite diagnosis. However, blood cultures have a low sensitivity for the confirmation of the pneumococcal etiology of CAP. This is due to the low prevalence of bacteremia in CAP, especially in uncomplicated cases, and the use of antibiotics prior to drawing blood samples for culture (11, 18, 32). In addition, blood cultures usually require 1 or 2 days before results are available and thus may have limited impact on the initial choice of empirical antimicrobial treatment (6, 29). PCR and antigen detection provide non-culture-based tools for the rapid identification of S. pneumoniae in respiratory tract specimens, pleural aspirate samples, and blood samples (7, 8, 13). These tests can add to culture methods in the etiological diagnosis of patients with CAP, particularly for patients who received antimicrobial treatment prior to sampling for culture (13, 26, 33).Several studies have evaluated the use of real-time PCR assays for the detection of S. pneumoniae DNA in blood samples from patients suspected of infection with invasive pneumococcal disease, with variable results (5, 14, 24, 31). These studies all used blood culture as the reference standard. However, positive PCRs relate not only to bacteremia but also to detection of DNA from nonviable bacteria, i.e., within phagocytes or due to the use of antibiotics. As such, it is understandable that most studies found cases with positive S. pneumoniae DNAemia and negative blood cultures. In this study, we used microbiologically documented S. pneumoniae infection as the gold standard for evaluation of PCR results and hypothesized that the bacterial DNA load (BDL) can be used to discriminate between patients with invasive infection and those with localized infection.The magnitude of bacteremia, as based on quantitative blood cultures, relates to the severity of S. pneumoniae infection (2). A high bacterial count in blood samples from children with bacteremia is associated with an increased risk of development of more-serious disease (27). Also, a shorter amount of time needed to detect positivity for blood cultures, supposedly reflecting a higher initial bacterial load, is associated with higher severity of disease (20). This relation between bacterial concentration and severity of disease may also hold true for the level of S. pneumoniae DNA in blood samples: the S. pneumoniae BDL was higher in human immunodeficiency virus-infected children who died from invasive pneumococcal disease than in survivors (5).In this study, we evaluated the S. pneumoniae BDL as a putative diagnostic tool and clinical marker of infection in adult patients with CAP.     

Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae

A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ≤88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted.     

Evaluation of a Rapid Immunochromatographic ODK0501 Assay for Detecting Streptococcus pneumoniae Antigen in Sputum Samples from Patients with Lower Respiratory Tract Infection

A novel, rapid, and noninvasive test (ODK0501) to detect Streptococcus pneumoniae antigen was evaluated in a Japanese multicenter study. ODK0501 uses polyclonal antibodies to detect C polysaccharide of S. pneumoniae from sputum samples by an immunochromatographic assay. The utility of ODK0501 was evaluated for 161 adult patients with lower respiratory tract infection between March 2006 and March 2007. Bacterial culture and identification, real-time PCR, and ODK0501 assays were performed on sputum samples, and the Binax Now Streptococcus pneumoniae antigen test was performed using urine samples obtained from the same patients. The performances of all tests were compared based on the results of bacterial culture and identification. The sensitivity and specificity of ODK0501 were 89.1% (49/55 samples) and 95.3% (101/106 samples), respectively. We then compared the Binax Now Streptococcus pneumoniae antigen test with ODK0501 using samples from 142 patients. The sensitivities of ODK0501 and the Binax Now S. pneumoniae antigen test were 90.0% (45/50 samples) and 62.0% (31/50 samples), respectively (P = 0.002). The relative quantity of S. pneumoniae in expectorated sputum was calculated using real-time PCR and indicated that the possibility of false-positive results for ODK0501 due to indigenous S. pneumoniae was low. The positive and negative concordance rates of ODK0501 and Binax Now were 96.8% (30/31 samples) and 21.1% (4/19 samples), respectively. Binax Now was less capable of detecting S. pneumoniae antigen among patients with underlying chronic obstructive pulmonary disease. In conclusion, ODK0501 is noninvasive, rapid, and an accurate tool for diagnosing respiratory infection caused by S. pneumoniae.Streptococcus pneumoniae is a frequent cause of community-acquired bacterial pneumonia and lower respiratory tract infection (10, 11, 19, 21) and a major cause of significant morbidity and mortality in Japan and the rest of the world (5).Since pneumococcal infections are common and can be severe, appropriate initial selection of antimicrobial agents is crucial to an optimal outcome. Rapid and precise identification of the causative agents of infectious diseases is critical but challenging. Pathogen-oriented, prompt selection and application of antimicrobial agents improve prognosis, reduce medical costs, and prevent the development of drug-resistant bacteria due to their inappropriate use. The “gold standard,” bacterial culture to identify causative microorganisms, requires several days to yield a result and is thus unhelpful for the initial selection of appropriate antibacterial drugs. Although Gleckman et al. previously reported that classic Gram staining of sputum, which is simple and inexpensive, is useful for the diagnosis of bacterial infections (7), Garcia-Vazquez et al. and Reed et al. found that it is ineffective for rapid diagnoses (6, 18). In addition, staining results rely on several factors such as the quality of the sputum samples and the skill of laboratory personnel in processing samples (7). Thus, Gram staining is not recommended for all patients with community-acquired pneumonia, according to the most recent “Infectious Diseases Society of America/American Thoracic Society Consensus Guidelines on the Management of Community-Acquired Pneumonia in Adults” (12).Urinary antigen detection is an alternative rapid diagnostic technique for detecting antigens of S. pneumoniae and Legionella pneumophila as respiratory pathogens. The Binax Now Streptococcus pneumoniae urinary antigen test (Binax, Inc., Portland, ME) detects cell wall antigens secreted in urine using an immunochromatographic method to separate capsular polysaccharide (C-ps) of S. pneumoniae. The test is noninvasive and rapid (the entire assay can be completed in about 15 min), and the specificity and sensitivity of detecting pneumococcal infections in adult patients are >90% and 50% to 80%, respectively, which allows early diagnosis (3, 8, 16). Moreover, even when S. pneumoniae cannot be identified by bacterial culture tests after the start of antibiotic therapy, the assay can still detect this organism. However, Binax Now has significant problems, including false-positive results due to indigenous S. pneumoniae in children (17) and antigens that can be detected even 1 to 3 months after treatment (13, 15).ODK0501 (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) is a diagnostic kit that uses polyclonal antibodies to detect antipneumococcal C-ps, a cell wall antigen of S. pneumoniae (4). This immunochromatographic method is rapid and noninvasive and analyzes sputum derived directly from a local infection site. Here, we evaluated the ability ODK0501 to detect S. pneumoniae among patients with lower respiratory tract infection at several Japanese medical institutions and compared its performance with that of the Binax Now assay. The goal of this study is to prove the usefulness of ODK0501 and its performance in clinical settings.     

Pneumolysin PCR-Based Diagnosis of Invasive Pneumococcal Infection in Children

Blood-based pneumolysin PCR was compared to blood culture and detection of pneumolysin immune complexes, as well as to detection of antibodies to pneumolysin and to C polysaccharide, in the diagnosis of pneumococcal infection in 75 febrile children. Invasive pneumococcal infection was suspected on clinical grounds in 67 of the febrile children, and viral infection was suspected on clinical grounds in 8 of the febrile children. In addition, 15 healthy persons were examined to test the specificity of the PCR assay. Plasma, serum, and leukocyte fractions were analyzed by PCR. The combination of all test results led to the diagnosis of pneumococcal infection in 25 patients. Pneumolysin PCR was positive in 44% of these children, an increase occurred in the pneumolysin antibodies in 39% and in the C polysaccharide antibodies in 30% of the patients; pneumolysin immune complexes were found in convalescent serum in 30%, pneumolysin immune complexes occurred in acute-phase serum samples in 16%, and a positive blood culture was found in 20% of the patients. None of the healthy controls had positive results by PCR. The results suggest that the diagnosis of Streptococcus pneumoniae infection from blood samples necessitates the use of several different assays. Pneumolysin PCR was the most sensitive assay, but its clinical value is reduced by the fact that three blood fractions are needed.     

Loop-mediated isothermal amplification method targeting the TTS1 gene cluster for detection of Burkholderia pseudomallei and diagnosis of melioidosis

Melioidosis is a severe infection caused by Burkholderia pseudomallei. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of B. pseudomallei. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and LAMP was positive for 10 clinical B. pseudomallei isolates but negative for 5 B. thailandensis and 5 B. mallei isolates. A clinical evaluation was conducted in northeast Thailand to compare LAMP to an established real-time PCR assay targeting the same TTS1 gene cluster. A total of 846 samples were obtained from 383 patients with suspected melioidosis, 77 of whom were subsequently diagnosed with culture-confirmed melioidosis. Of these 77 patients, a positive result was obtained from one or more specimens by PCR in 26 cases (sensitivity, 34%; 95% confidence interval [CI], 23.4 to 45.4%) and by LAMP in 34 cases (sensitivity, 44%; 95% CI, 32.8 to 55.9%) (P = 0.02). All samples from 306 patients that were culture negative for B. pseudomallei were negative by PCR (specificity, 100%; 95% CI, 98.8 to 100%), but 5 of 306 patients (1.6%) were positive by LAMP (specificity, 98.4%; 95% CI, 96.2 to 99.5%) (P = 0.03). The diagnostic accuracies of PCR and LAMP were 86.7% (95% CI, 82.9 to 89.9%) and 87.5% (95% CI, 83.7 to 90.6%), respectively (P = 0.47). Both assays were very insensitive when applied to blood samples; PCR and LAMP were positive for 0 and 1 of 44 positive blood cultures, respectively. The PCR and LAMP assays evaluated here are not sufficiently sensitive to replace culture in our clinical setting.     

Diagnostic Value of PCR for Detection of Borrelia burgdorferi in Skin Biopsy and Urine Samples from Patients with Skin Borreliosis

Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology.     

Genus Level Identification of Mycobacteria from Clinical Specimens by Using an Easy-To-Handle Mycobacterium-Specific PCR Assay

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.     

Evaluation of PCR for Diagnosis of Bordetella pertussis and Bordetella parapertussis Infections

PCR, using primers PIp1 and PIp2, was evaluated for the detection of DNA from Bordetella pertussis in bacterial strains and in nasopharyngeal samples from patients with a cough lasting at least 7 days. The assay could detect DNA from 6 CFU of B. pertussis/10 μl of sample. Results of the PCR assay were compared with those of cultures, a determination of serum antibodies against pertussis toxin and filamentous hemagglutinin, and a clinical evaluation of 2,442 coughing episodes. The overall sensitivity of PCR was 65% (623 of 956), which was higher than the sensitivity of cultures (58%) (P < 0.001). Factors influencing the sensitivity of PCR were the interval between the onset of symptoms and sampling and the vaccination status of the patient. The specificity of PCR was 98% (1,451 of 1,486). The positive and negative predictive values were 95 and 81%, respectively. Parapertussis PCR, using primers BPPA and BPPZ, was positive in 11 of 18 culture-positive cases and was confirmed by serology in another 4 cases. In conclusion, PCR is a valuable complement to cultures and can probably replace cultures for diagnosis of B. pertussis and Bordetella parapertussis infections.     

Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella) catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ~370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) a Haemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive for Haemophilus, 3 (8.6%) were positive for Streptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.     

Validation of real-time PCR for laboratory diagnosis of Acanthamoeba keratitis

Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability to detect trophozoites and cysts, and (iii) by testing a battery of positive and negative samples. We also examined the inhibitory effects of a number of commonly used topical ophthalmic drugs on real-time PCR. The results of the real-time PCR limit of detection and amplification efficiency of the Rivière and Qvarnstrom assays were 11.3 DNA copies/10 μl and 94% and 43.8 DNA copies/10 μl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10 μl and 2.3 Acanthamoeba trophozoites/10 μl by both real-time PCR assays. The overall agreement between the assays was 97.0%. The clinical sensitivity and specificity of both real-time PCR assays based on culture were 100% (7 of 7) and 100% (37 of 37), respectively. Polyhexamethylene biguanide was the only topical drug that demonstrated PCR inhibition, with a minimal inhibitory dilution of 1/640 and an amplification efficiency of 72.7%. Four clinical samples were Acanthamoeba culture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoeba keratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results.     

Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples

Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n = 273) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80°C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.     

Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital Chlamydia trachomatis Infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.