Franklin R. Cockerill

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

SUMMARY Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Infective Endocarditis Due to Unusual or Fastidious Microorganisms

Infective endocarditis due to fastidious microorganisms is commonly encountered in clinical practice. Some organisms such as fungi account for up to 15% of cases of prosthetic valve infective endocarditis, whereas organisms of the HACEK group (Haemophilus parainfluenzae, H. aphrophilus, and H. paraphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) cause 3% of community-acquired cases of infective endocarditis. Special techniques are necessary to identify these microorganisms. A history of contact with mammals or birds may suggest infection caused by Coxiella burnetii (Q fever), Brucella species, or Chlamydia psittaci. A nosocomial cluster of postsurgical infective endocarditis may be caused by Legionella species or Mycobacterium species. If risk factors that are commonly associated with fungal infections (cardiac surgical treatment, prolonged hospitalization, indwelling central venous catheters, and long-term antibiotic use) are present, fungal endocarditis is possible. Patients with endocarditis and a history of periodontal disease or dental work in whom routine blood cultures are negative might have infection due to nutritionally variant streptococci or bacteria of the HACEK group. Communication between the microbiologist and the clinician is of crucial importance for identification of these microorganisms early during the course of the infection before complications such as embolization or valvular failure occur. In this article, we review the microbiologic and clinical features of these organisms and provide recommendations for diagnosis and treatment.

Sonication of Explanted Prosthetic Components in Bags for Diagnosis of Prosthetic Joint Infection Is Associated with Risk of Contamination

ABSTRACT Explanted orthopedic implants from 54 patients with aseptic failure and 24 patients with prosthetic knee or hip infection were sonicated in polyethylene bags. The sensitivities of periprosthetic tissue and sonicate fluid cultures for the diagnosis of prosthetic joint infection were 54% and 75%, whereas the specificities were 98% and 87%, respectively. Sonication in bags improved bacterial recovery from the surface of orthopedic implants; however, it lacked specificity, due to bag leakage.

Detection of Bacillus anthracis DNA by LightCycler PCR

ABSTRACT Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

ABSTRACT We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionellaspecies directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both aLegionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the “gold standard” method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection byLegionella genus LC-PCR, 100 and 100%;Legionella genus detection by DFA assay, 33 and 100%; andL. pneumophila detection by L. pneumophilaspecies-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophilaspecies-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionellaspecies from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multipleLegionella species in lung tissue. WS staining,Legionella genus LC-PCR, and L. pneumophilaspecies-specific ISH were useful as rapid tests with lung tissue.

Prevention of Infection in Critically Ill Patients by Selective Decontamination of the Digestive Tract

OBJECTIVE To determine whether selective decontamination of the digestive tract using oral and nonabsorbable antimicrobial agents and parenteral cefotaxime prevents infection in critically ill patients. DESIGN Randomized, controlled trial without blinding. SETTING Surgical trauma and medical intensive care units in a tertiary referral hospital. PATIENTS One hundred fifty patients admitted to surgical trauma and medical intensive care units during a 3-year interval, whose condition suggested a prolonged stay (greater than 3 days). INTERVENTION Patients were randomly allocated to an experimental group (n = 75) that received cefotaxime, 1 g intravenously every 8 hours for the first 3 days only, and oral, nonabsorbable antibiotics (gentamicin, polymyxin, and nystatin by oral paste and oral liquid) for the entire stay in the intensive care unit. Control patients (n = 75) received usual care. MEASUREMENTS The number of infections, total hospital days, and deaths, as well as the number of days in intensive care unit, were recorded. RESULTS Control patients experienced more infections (36 compared with 12, P = 0.04), including bacteremias (14 compared with 4, P = 0.05) and pulmonary infections (14 compared with 4, P = 0.03). Although total hospital days, days in intensive care, and the overall death rate all were lower in the treatment group, these differences were not statistically significant. Clinically important complications of selective decontamination of the digestive tract were not encountered. CONCLUSIONS Selective decontamination of the digestive tract decreases subsequent infection rates, especially by gram-negative bacilli, in selected patients during long-term stays in the intensive care unit.

Clinical and Epidemiological Features of Enterococcus casseliflavus/flavescens and Enterococcus gallinarum Bacteremia: A Report of 20 Cases

The clinical significance of intrinsically vancomycin-resistant enterococci is not yet fully established, as these organisms are infrequently recovered from clinical specimens. We report our experience with 20 cases of Enterococcus gallinarum and Enterococcus casseliflavus/flavescens bacteremia in humans from 1992 through 1998. Sixteen cases of bacteremia were caused by E. gallinarum. Underlying conditions were present in 19 (95%) of the patients and included malignancy, receipt of transplant, and Carolis disease. Polymicrobial bacteremia was present in 9 patients (45%). E. gallinarum and E. casseliflavus/flavescens, although they are infrequently isolated from clinical specimens, may cause serious invasive infections.

Pulmonary disease in the immunocompromised host (first of two parts)

With few exceptions, pulmonary complications in the immunocompromised host will proceed to death unless the clinician intercedes. The differential diagnosis of diffuse pulmonary disease in this setting includes (1) infection, most commonly from opportunistic organisms; (2) recurrence or extension of the basic underlying disease process to involve the lungs; (3) adverse pulmonary reaction to drugs; (4) a new, unrelated disease process such as cardiac pulmonary edema or pulmonary emboli; and (5) any combination of these categories. Up to a third of these patients have two or more complications, such as pneumonitis from two different opportunistic organisms or an opportunistic infection and a drug-induced pulmonary complication. An understanding of the host defense that is compromised enables the clinician to narrow the differential diagnosis. The most common types of impairment of defense mechanisms are reductions in the number of granulocytes, B-lymphocytes, or T-lymphocytes, and not uncommonly, two or all three of these types of cells are involved. Impairment of each of these cell types is associated with an increased frequency of infection by a particular group of organisms. Consequently, the clinician can be somewhat selective if empiric therapy is being considered. In the immunocompromised patient, most pulmonary complications, including drug-induced pulmonary disease and pulmonary emboli, are associated with fever that mimics an infection. Up to 25% of the pulmonary complications in these patients are noninfectious.

Comparison of LightCycler PCR, Rapid Antigen Immunoassay, and Culture for Detection of Group A Streptococci from Throat Swabs

ABSTRACT We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (n = 58) than either culture (n = 55) or the Directigen immunoassay (n = 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted “gold standard,” bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (n = 7) or the Directigen method (n = 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (P = 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs; P < 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs; P < 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time (∼1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).

Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cultures from Cystic Fibrosis Patients

ABSTRACT The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting Gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).