Nancy L. Wengenack

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.

Performance and Cost Analysis of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Routine Identification of Yeast

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification. MALDI-TOF mass spectrometry yielded 96.3% and 84.5% accurate species level identifications (spectral scores, ≥1.8) for 138 common and 103 archived strains of yeast. MALDI-TOF mass spectrometry is accurate, rapid (5.1 min of hands-on time/identification), and cost-effective (

Detection of Coccidioides Species in Clinical Specimens by Real-Time PCR

0.50/sample) for yeast identification in the clinical laboratory.

Dermatophyte Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Coccidioides spp. are dimorphic fungal pathogens endemic to the semiarid regions of North, Central, and South America. Currently, direct smear and culture are the most common means of identifying Coccidioides spp. While these methods offer relatively sensitive and specific means of detecting Coccidioides spp., growth in culture may take up to 3 weeks, potentially delaying the diagnosis and initiation of appropriate antifungal therapy. In addition, growth of the organism represents a significant safety risk to laboratory personnel. The need for a rapid and safe means of diagnosing coccidioidomycosis prompted us to develop a real-time PCR assay to detect Coccidioides spp. directly from clinical specimens. Primers and fluorescent resonance energy transfer (FRET) probes were designed to target the internal transcribed spacer 2 region of Coccidioides. The assays limit of detection is below 50 targets per reaction. An analysis of 40 Coccidioides sp. clinical isolates grown in culture demonstrated 100% sensitivity of the assay. A cross-reactivity panel containing fungi, bacteria, mycobacteria, and viruses was tested and demonstrated 100% specificity for Coccidioides spp. An analysis of 266 respiratory specimens by LightCycler PCR demonstrated 100% sensitivity and 98.4% specificity for Coccidioides spp. compared with culture. Analysis of 66 fresh tissue specimens yielded 92.9% sensitivity and 98.1% specificity versus those of the culture method. The sensitivity of the assay testing 148 paraffin-embedded tissue samples is 73.4%. A rapid method for the detection of Coccidioides spp. directly from clinical material will greatly assist in the timely diagnosis and treatment of patients, while at the same time decreasing the risk of accidental exposure to laboratory personnel.

Real-Time PCR Method for Detection of Encephalitozoon intestinalis from Stool Specimens

ABSTRACT The performance of the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, ≥1.7; species, ≥2.0) and adjusted cutoff values established by this study (genus, ≥1.5; species, ≥1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P = 0.005)- and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.

Real-Time PCR Method for Detection of Zygomycetes

ABSTRACT The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoonintestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 102 to 104 spores/ml of feces, a value which represented a significant improvement over that achieved by staining (≥1.0 × 106 spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

Recombinant Mycobacterium tuberculosis KatG(S315T) Is a Competent Catalase-Peroxidase with Reduced Activity toward Isoniazid

ABSTRACT Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/μl, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.

Tuberculosis and Trimethoprim-Sulfamethoxazole

The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.

Use of Hydrogen Peroxide Vapor for Deactivation of Mycobacterium tuberculosis in a Biological Safety Cabinet and a Room

ABSTRACT The sulfonamides were the first drugs with antituberculous effects. Their use was abandoned and basically forgotten with the advent of streptomycin and isoniazid combination treatment. There is a widespread belief, apparently based on testing a single isolate on questionable media, that Mycobacterium tuberculosis is resistant to trimethoprim-sulfamethoxazole (TMP-SMX). We saw a complex immunocompromised patient with tuberculosis who was initially treated with TMP-SMX without antituberculous drugs and defervesced on this treatment. An isolate of M. tuberculosis from this patient was found to be sensitive to TMP-SMX. We examined how frequently M. tuberculosis is sensitive to TMP-SMX. Isolates were tested for susceptibility to TMP-SMX on supplemented Middlebrook 7H10 plates. We found that 43 of 44 (98%) isolates of M. tuberculosis were susceptible to the combination of ≤1 μg/ml of TMP and 19 μg/ml of SMX (≤1/19 μg/ml). Thus, the vast majority of our M. tuberculosis isolates were susceptible to TMP-SMX at an MIC similar to that for Mycobacterium kansasii, Mycobacterium marinum, and sensitive rapidly growing mycobacteria, organisms successfully treated with TMP-SMX as part of the treatment regimen. It is possible that TMP-SMX may be useful in treating patients with multiple-drug-resistant and extended drug-resistant tuberculosis. We feel that a clinical trial looking at the effectiveness of TMP-SMX as an antituberculous drug is worthwhile.

Formic Acid-Based Direct, On-Plate Testing of Yeast and Corynebacterium Species by Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Mycobacterium tuberculosis is an important human pathogen that is routinely cultured in clinical and research laboratories. M. tuberculosis can contaminate surfaces and is highly resistant to disinfection. We investigated whether hydrogen peroxide vapor (HPV) is effective for the deactivation of M. tuberculosis on experimentally contaminated surfaces in a biological safety cabinet (BSC) and a room. Biological indicators (BIs) consisting of an ∼3-log10 inoculum of M. tuberculosis on stainless steel discs and a 6-log10 inoculum of Geobacillus stearothermophilus were exposed to HPV in BSC time course experiments and at 10 locations during room experiments. In three separate BSC experiments, M. tuberculosis BIs were transferred to growth media at 15-min intervals during a 180-min HPV exposure period. No M. tuberculosis BIs grew following 30 min of HPV exposure. In three separate room experiments, M. tuberculosis and G. stearothermophilus BIs were exposed to HPV for 90, 120, and 150 min, respectively. BIs for both microorganisms were deactivated in all 10 locations following 90 min of HPV exposure. HPV provides an alternative to traditional decontamination methods, such as formaldehyde fumigation, for laboratories and other areas contaminated with M. tuberculosis.