Matthew J. Binnicker

Emergence of a New Pathogenic Ehrlichia Species, Wisconsin and Minnesota, 2009

BACKGROUND Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause. METHODS We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis. RESULTS On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris. CONCLUSIONS We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.).

Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens

ABSTRACT The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

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

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.

Detection of Blastomyces dermatitidis and Histoplasma capsulatum from Culture Isolates and Clinical Specimens by Use of Real-Time PCR

ABSTRACT Blastomyces dermatitidis and Histoplasma capsulatum are dimorphic fungi that often cause self-limited respiratory infections. However, they may also cause severe disseminated disease, depending on the level of the exposure to the organism and the host immune status. In addition, patients with infections caused by these fungi may have very similar clinical presentations. Although microbiologic culture is a standard method for detecting these pathogens, their recovery may require days to weeks, and the manipulation of cultures presents a significant safety hazard to laboratory personnel. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect and differentiate B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens. Primers and fluorescence resonance energy transfer hybridization probes were designed to target the histidine kinase and glyceraldehyde-3-phosphate dehydrogenase genes of B. dermatitidis and H. capsulatum, respectively. The analytical sensitivity of the assay was determined to be 100 copies/μl for both fungi. From culture isolates, the assay demonstrated 100% specificity and 100% sensitivity for B. dermatitidis and 100% specificity and 94% sensitivity for H. capsulatum. Detection directly from 797 clinical specimens demonstrated specificities and sensitivities of 99% and 86% for B. dermatitidis and 100% and 73% for H. capsulatum compared with the results for culture. This real-time PCR assay provides a rapid method for the detection of B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens.

Treponema-Specific Tests for Serodiagnosis of Syphilis: Comparative Evaluation of Seven Assays

ABSTRACT The diagnosis of syphilis is challenging and often relies on serologic tests to detect treponemal or nontreponemal antibodies. Recently, the Centers for Disease Control and Prevention and the Association of Public Health Laboratories proposed an update to the syphilis serology testing algorithm, in which serum samples are first tested using a treponema-specific test and positive samples are analyzed with a nontreponemal assay. The goal of this study was to compare the performance of seven treponemal assays (BioPlex 2200 syphilis IgG [Bio-Rad, Hercules, CA], fluorescent treponemal antibody [FTA] assay [Zeus Scientific, Raritan, NJ], Treponema pallidum particle agglutination [TP-PA; Fujirebio Diagnostics, Malvern, PA], Trep-Sure enzyme immunoassay [EIA; Phoenix Biotech, Oakville, Ontario, Canada], Trep-Chek EIA [Phoenix Biotech], Trep-ID EIA [Phoenix Biotech], and Treponema ViraBlot IgG [Viramed Biotech AG, Planegg, Germany]) using serum samples (n = 303) submitted to our reference laboratory. In addition to testing with these 7 assays, all samples were tested by a rapid plasma reagin (RPR) assay and a treponemal IgM Western blot assay (Viramed ViraBlot). Compared to the FTA assay as the gold standard, the evaluated treponemal tests demonstrated comparable levels of performance, with percent agreement ranging from 95.4% (95% confidence interval, 92.3 to 97.3) for the Trep-Sure EIA to 98.4% (96.1 to 99.4) for the Trep-ID EIA. Compared to a “consensus of the test panel” (defined as at least 4 of 7 treponemal tests being in agreement), the percent agreement ranged from 95.7% (92.7 to 97.5) for Trep-Sure to 99.3% (97.5 to 99.9) for Trep-ID. These data may assist clinical laboratories that are considering implementing a treponemal test for screening or confirmatory purposes.

Multiplex Molecular Panels for Diagnosis of Gastrointestinal Infection: Performance, Result Interpretation, and Cost-Effectiveness

ABSTRACT Gastrointestinal disease is a major cause of morbidity and mortality worldwide, especially among young children and immunocompromised patients. Diarrhea may result from infection with a variety of microbial pathogens, including bacteria, viruses, or parasites. Historically, the diagnosis of infectious diarrhea has been made using microscopy, antigen tests, culture, and real-time PCR. A combination of these traditional tests is often required due to the inability to distinguish between infectious etiologies based on the clinical presentation alone. Recently, several multiplex molecular assays have been developed for the detection of gastrointestinal pathogens directly from clinical stool samples. These panels allow for the detection and identification of up to 20 pathogens in as little as 1 h. This review will focus on the multiplex molecular panels that have received clearance from the FDA for the diagnosis of diarrheal disease and will highlight issues related to test performance, result interpretation, and cost-effectiveness of these new molecular diagnostic tools.

Direct Comparison of the Traditional and Reverse Syphilis Screening Algorithms in a Population with a Low Prevalence of Syphilis

ABSTRACT We describe the first direct comparison of the reverse and traditional syphilis screening algorithms in a population with a low prevalence of syphilis. Among 1,000 patients tested, the results for 6 patients were falsely reactive by reverse screening, compared to none by traditional testing. However, reverse screening identified 2 patients with possible latent syphilis that were not detected by rapid plasma reagin (RPR).

Comparison of four assays for the detection of cryptococcal antigen.

ABSTRACT We compared the performance of four assays for the detection of cryptococcal antigen in serum samples (n = 634) and cerebrospinal fluid (CSF) samples (n = 51). Compared to latex agglutination, the sensitivity and specificity of the Premier enzyme immunoassay (EIA), Alpha CrAg EIA, and CrAg lateral flow assay (LFA) were 55.6 and 100%, 100 and 99.7%, and 100 and 99.8%, respectively, from serum samples. There was 100% agreement among the four tests for CSF samples, with 18 samples testing positive by each of the assays.

Direct Detection of Influenza A and B Viruses in Less Than 20 Minutes Using a Commercially Available Rapid PCR Assay

ABSTRACT We compared an FDA-cleared rapid (<20 min) PCR assay (Cobas Liat; Roche Diagnostics) to our routine influenza A and B real-time PCR assay (Simplexa Flu A/B & RSV Direct; Focus Diagnostics) using respiratory swabs (n = 197). The Cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for each target.

Fungal Molecular Diagnostics

This article summarizes the current state of the art in molecular diagnostic methods for the detection and identification of fungi as performed in the mycology laboratory. The advantages and limitations of current molecular testing methods are evaluated, with examples provided from the recent literature. Finally, commentary is provided on the future of molecular fungal diagnostics, including the potential role of multiplex detection assays, microarrays, and the molecular determination of antifungal resistance directly from clinical specimens.