Eric C. J. Claas

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

Rapid and Sensitive Method Using Multiplex Real-Time PCR for Diagnosis of Infections by Influenza A and Influenza B Viruses, Respiratory Syncytial Virus, and Parainfluenza Viruses 1, 2, 3, and 4

ABSTRACT Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.

Improved Diagnosis of the Etiology of Community-Acquired Pneumonia with Real-Time Polymerase Chain Reaction

Abstract Background. Community-acquired pneumonia (CAP) remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods and, therefore, may improve the accuracy of microbiological diagnosis for patients with CAP. Methods. Conventional detection techniques and multiplex real-time PCR for atypical bacteria and respiratory viruses were performed on samples collected from 105 adults enrolled in a prospective study. An infiltrate was visible on each patients chest radiograph, and a pneumonia severity index score was determined for each patient. Results. Microbiological diagnoses were determined for 52 (49.5%) of 105 patients by conventional techniques and for 80 (76%) of 105 patients by real-time PCR. The time to obtain the result of real-time PCR could be reduced to 6 h. PCR methodology was significantly more sensitive for the detection of atypical pathogens and viruses (P ⩽ .001). Respiratory viral infections and mixed infections were detected in 15 (14.2%) and 3 (2.8%) of 105 patients, respectively, by conventional methods, but were detected in 59 (56.2%) and 28 (26.5%) of 105, respectively, by real-time PCR. Presence of a mixed infection was significantly associated with severe pneumonia (P = .002). Human rhinoviruses and coronaviruses, OC43 and 229E, were frequently identified pathogens. Conclusions. The combined real-time PCR assay is more sensitive for diagnosis of the main viruses and atypical bacteria that cause CAP compared with conventional methods, and obtains results in a clinically relevant time period.

Evaluation of 12 Commercial Tests and the Complement Fixation Test for Mycoplasma pneumoniae-Specific Immunoglobulin G (IgG) and IgM Antibodies, with PCR Used as the “Gold Standard”

ABSTRACT Serology and nucleic acid amplification are the main diagnostic tools for the diagnosis of Mycoplasma pneumoniae infection. Since no reference standard is generally accepted, serologic assays for M. pneumoniae have not been evaluated on a broad scale. In this study, 12 commercially available serologic assays (for immunoglobulin G [IgG] and IgM) and the complement fixation test (CFT) were evaluated by using M. pneumoniae DNA detection by real-time PCR as the “gold standard.” The assays tested were Platelia EIA (Bio-Rad), SeroMP EIA (Savyon), Serion classic EIA (Virion/Serion), Biotest EIA (Biotest), Ridascreen EIA (r-Biopharm), AniLabsystems EIA (Labsystems), Novum EIA (Novum Diagnostica), Diagnosys EIA (MP products), Genzyme/Virotech EIA, ImmunoWell EIA (Genbio), ImmunoCard EIA (Meridian), and SerodiaMycoII microparticle agglutination (Fujirebio). Serum samples (n = 46) from 27 PCR-positive patients with a known first day of disease and sera (n = 33) from PCR-negative controls were obtained from prospective studies of acute lower respiratory tract infections. Additionally, control sera (n = 63) from patients with acute viral or bacterial respiratory infections other than those caused by M. pneumoniae were tested. The results showed low specificities for both the Novum and the ImmunoCard IgM assays. The IgM assays with the best performances in terms of sensitivity and specificity were AniLabsystems (77% and 92%, respectively), SeroMP (71% and 88%, respectively), and CFT (65% and 97%, respectively). Good receiver operating characteristic areas under the curve were found for CFT (0.94), the Platelia assay (0.87), and the AniLabsystems assay (0.85). We conclude that there are few commercial serologic assays for the detection of M. pneumoniae infections with appropriate performances in terms of sensitivity and specificity and that PCR has become increasingly important for the diagnosis of M. pneumoniae infections in defined groups of patients.

Internally Controlled Real-Time PCR Monitoring of Adenovirus DNA Load in Serum or Plasma of Transplant Recipients

ABSTRACT Adenoviruses have been recognized as important pathogens in immunocompromised hosts. Particularly in pediatric allogeneic stem cell transplant recipients, the morbidity of the patients and mortality in those patients with disseminated infections have been found to increase over the last few years. Severe infections are predominantly but not exclusively caused by subgroup C adenoviruses. A multiplex real-time PCR assay using molecular beacons as probes was developed to enable monitoring of adenovirus DNA in those patients with simultaneous identification of subgroups. An internal control was coamplified in the multiplex PCR to check for the DNA isolation procedure as well as the presence of inhibitors in the clinical samples. The assay has been applied retrospectively in patient groups with different clinical outcomes of infection. In fatal cases, significantly higher adenovirus loads developed, exceeding even 1011 copies/ml of serum or plasma. Patients with viral loads over 106 copies/ml appear to have an increased risk for fatal complications. This quantitative real-time PCR assay has been prospectively used clinically since 2002 to study the course of adenovirus infection. In addition, the assay provides objective start and end points of therapeutic interventions, including the clinically important evaluation of antiviral drugs.

Comparison and Evaluation of Real-Time PCR, Real-Time Nucleic Acid Sequence-Based Amplification, Conventional PCR, and Serology for Diagnosis of Mycoplasma pneumoniae

ABSTRACT Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.

High Levels of Adenovirus DNA in Serum Correlate with Fatal Outcome of Adenovirus Infection in Children after Allogeneic Stem-Cell Transplantation

An increase in the incidence of adenovirus (AdV) infection leading to death among children who have undergone allogeneic stem-cell transplantation has made it necessary to find new ways to monitor AdV infection. In this retrospective study, levels of AdV DNA in serum samples obtained from 36 transplant recipients with stool cultures positive for AdV were measured by polymerase chain reaction (PCR) semiquantitatively by analyzing serial dilutions of the DNA template. Six (86%) of 7 children who died of AdV infection, compared with only 2 (7%) of 29 other patients, had high serum levels of AdV DNA (detectable by PCR at a > or =100-fold dilution of the DNA template; P<.0001). High serum levels of AdV DNA were reached a mean of 18 days before death (range, 6-29 days). Quantification of adenoviral DNA in serum may prove to be a valuable tool to diagnose and monitor AdV infection and disease in immunocompromised children.

Real-Time PCR Assay Using Fine-Needle Aspirates and Tissue Biopsy Specimens for Rapid Diagnosis of Mycobacterial Lymphadenitis in Children

ABSTRACT A real-time PCR assay was developed to diagnose and identify the causative agents of suspected mycobacterial lymphadenitis. Primers and probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of the genus Mycobacterium and the species Mycobacterium avium and M. tuberculosis. The detection limit for the assay was established at 1,100 CFU/ml of pus, and the specificity tests showed no false-positive reaction with other mycobacterial species and other pathogens causing lymphadenitis. From 67 children with suspected mycobacterial lymphadenitis based on a positive mycobacterial skin test, 102 samples (58 fine-needle aspirates [FNA] and 44 tissue specimens) were obtained. The real-time PCR assay detected a mycobacterial infection in 48 patients (71.6%), whereas auramine staining and culturing were positive for 31 (46.3%) and 28 (41.8%) of the patients. The addition of the real-time PCR assay to conventional diagnostic tests resulted in the recognition of 13 more patients with mycobacterial disease. These results indicate that the real-time PCR is more sensitive than conventional staining and culturing techniques (P = 0.006). The M. avium-specific real-time PCR was positive for 38 patients, and the M. tuberculosis-specific real-time PCR was positive for 1 patient. Analysis of 27 patients from whom FNA and tissue biopsy specimens were collected revealed significantly more positive real-time PCR results for FNA than for tissue biopsy specimens (P = 0.003). Samples from an age-matched control group of 50 patients with PCR-proven cat scratch disease were all found to be negative by the real-time PCR. We conclude that this real-time PCR assay with a sensitivity of 72% for patients with lymphadenitis and a specificity of 100% for the detection of atypical mycobacteria can provide excellent support for clinical decision making in children with lymphadenitis.

Immune Reconstitution and Clearance of Human Adenovirus Viremia in Pediatric Stem-Cell Recipients

BACKGROUND Human adenovirus (HAdV) infections are increasingly frequent complications of allogeneic stem-cell transplantation (SCT), especially in children. Only a few data on the correlation between immune recovery and the course of HAdV infection are available, and data on HAdV-specific responses are lacking. METHODS In a prospective study, we determined the correlation between the HAdV DNA load in plasma and lymphocyte reconstitution in 48 children after allogeneic SCT. Additionally, HAdV-specific humoral and cellular immune responses were investigated. RESULTS HAdV infection occurred in 21 patients (44%), and, in 6 of these patients, the infection progressed to viremia, as demonstrated by the presence of HAdV DNA in plasma. Low lymphocyte counts at the onset of infection were predictive of HAdV viremia. Survival of patients with HAdV viremia was associated with an increase in lymphocyte counts during the first weeks after infection. In these patients, HAdV-specific CD4+ T cell responses, as well as increases in titers of neutralizing antibody, were detected after clearance of HAdV DNA from plasma. CONCLUSIONS Lymphocyte reconstitution appears to play a crucial role in clearance of HAdV viremia and survival of the host, warranting further development of therapeutic interventions aimed at improving immune recovery.

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

ABSTRACT Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A−/B+) strains. Thirty-nine clinical toxin A−/B+ isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A−/B+ isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A−/B+ isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A−/B+ isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A−/B+ strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.