Mark D. Lindsley

Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes

ABSTRACT Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA fromHistoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii,Cryptococcus neoformans, five Candidaspecies, and Pneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA from H.capsulatum, B.dermatitidis, C. immitis,P. brasiliensis, and P.marneffei but not with DNA from nondimorphic fungi. Specific probes for H. capsulatum,B. dermatitidis, C.immitis, P. brasiliensis,P. marneffei, S.schenckii, C. neoformans, and P. carinii hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for theB. dermititidis probe used againstC. immitis DNA and for theH. capsulatum probe used againstCandida albicans DNA. However, the C.immitis probe did not cross-react with B.dermititidis DNA, nor did the dimorphic probe hybridize with C. albicans DNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.

Phylogenetic Diversity and Microsphere Array-Based Genotyping of Human Pathogenic Fusaria, Including Isolates from the Multistate Contact Lens-Associated U.S. Keratitis Outbreaks of 2005 and 2006

ABSTRACT In 2005 and 2006, outbreaks of Fusarium keratitis associated with soft contact lens use occurred in multiple U.S. states and Puerto Rico. A case-control study conducted by the Centers for Disease Control and Prevention (CDC) showed a significant association between infections and the use of one particular brand of lens solution. To characterize the full spectrum of the causal agents involved and their potential sources, partial DNA sequences from three loci (RPB2, EF-1α, and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 91 corneal and 100 isolates from the patients environment (e.g., contact lens and lens cases). We also sequenced a 1.8-kb region encoding the RNA polymerase II second largest subunit (RPB2) from 126 additional pathogenic isolates to better understand how the keratitis outbreak isolates fit within the full phylogenetic spectrum of clinically important fusaria. These analyses resulted in the most robust phylogenetic framework for Fusarium to date. In addition, RPB2 nucleotide variation within a 72-isolate panel was used to design 34 allele-specific probes to identify representatives of all medically important species complexes and 10 of the most important human pathogenic Fusarium in a single-well diagnostic assay, using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the three most common species comprised 55% of CDCs corneal and environmental isolates and that the corneal isolates comprised 29 haplotypes distributed among 16 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination.

Evaluation of a Newly Developed Lateral Flow Immunoassay for the Diagnosis of Cryptococcosis

This study, evaluating the performance of a novel cryptococcal lateral flow immunoassay, shows that the assay performs as well as available diagnostic methods is economical, rapid, and easy to perform; and as such can be a point of care test in resource limited settings.

Gastrointestinal Basidiobolomycosis in Arizona: Clinical and Epidemiological Characteristics and Review of the Literature

Gastrointestinal basidiobolomycosis (GIB) is an unusual fungal infection that is rarely reported in the medical literature. From April 1994 through May 1999, 7 cases of GIB occurred in Arizona, 4 from December 1998 through May 1999. We reviewed the clinical characteristics of the patients and conducted a case-control study to generate hypotheses about potential risk factors. All patients had histopathologic signs characteristic of basidiobolomycosis. Five patients were male (median age, 52 years; range, 37--59 years) and had a history of diabetes mellitus (in 3 patients), peptic ulcer disease (in 2), or pica (in 1). All patients underwent partial or complete surgical resection of the infected portions of their gastrointestinal tracts, and all received itraconazole postoperatively for a median of 10 months (range, 3--19 months). Potential risk factors included prior ranitidine use and longer residence in Arizona. GIB is a newly emerging infection that causes substantial morbidity and diagnostic confusion. Further studies are needed to better define its risk factors and treatment.

Coccidioidomycosis among workers at an archeological site, northeastern Utah.

In 2001, an outbreak of acute respiratory disease occurred among persons working at a Native American archeological site at Dinosaur National Monument in northeastern Utah. Epidemiologic and environmental investigations were undertaken to determine the cause of the outbreak. A clinical case was defined by the presence of at least two of the following symptoms: self-reported fever, shortness of breath, or cough. Ten workers met the clinical case definition; 9 had serologic confirmation of coccidioidomycosis, and 8 were hospitalized. All 10 were present during sifting of dirt through screens on June 19; symptoms began 9–12 days later (median 10). Coccidioidomycosis also developed in a worker at the site in September 2001. A serosurvey among 40 other Dinosaur National Monument workers did not find serologic evidence of recent infection. This outbreak documents a new endemic focus of coccidioidomycosis, extending northward its known geographic distribution in Utah by approximately 200 miles.

Utility of (1–3)-β-D-glucan Testing for Diagnostics and Monitoring Response to Treatment During the Multistate Outbreak of Fungal Meningitis and Other Infections

BACKGROUND  The 2012 outbreak of fungal meningitis associated with contaminated methylprednisolone produced by a compounding pharmacy has resulted in >750 infections. An important question facing patients and clinicians is the duration of antifungal therapy. We evaluated (1-3)-β-d-glucan (BDG) as a marker for monitoring response to treatment. METHODS  We determined sensitivity and specificity of BDG testing using the Fungitell assay, by testing 41 cerebrospinal fluid (CSF) specimens from confirmed cases of fungal meningitis and 66 negative control CSF specimens. We also assessed whether BDG levels correlate with clinical status by using incident samples from 108 case patients with meningitis and 20 patients with serially collected CSF. RESULTS  A cutoff value of 138 pg/mL provided 100% sensitivity and 98% specificity for diagnosis of fungal meningitis in this outbreak. Patients with serially collected CSF were divided into 2 groups: those in whom BDG levels declined with treatment and those in whom BDG remained elevated. Whereas most patients with a decline in CSF BDG had clinical improvement, all 3 patients with continually elevated BDG had poor clinical outcomes (stroke, meningitis relapse, or development of new disease). CONCLUSIONS  Our data suggest that measuring BDG in CSF is a highly sensitive test for diagnosis of fungal meningitis in this outbreak. Analysis of BDG levels in serially collected CSF demonstrated that BDG may correlate with clinical response. Routine measurement of BDG in CSF may provide useful adjunctive data for the clinical management of patients with outbreak-associated meningitis.

Detection of fungal DNA in human body fluids and tissues during a multistate outbreak of fungal meningitis and other infections

ABSTRACT Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak.

Nonsporulating Clinical Isolate Identified as Petromyces alliaceus (Anamorph Aspergillus alliaceus) by Morphological and Sequence-Based Methods

ABSTRACT Concerted morphological and sequencing-based strategies revealed the identity of a nonsporulating clinical isolate as Petromyces alliaceus (anamorph Aspergillus alliaceus). This rare Aspergillus sp. was recovered as the etiological agent of invasive pulmonary aspergillosis and had reduced in vitro susceptibilities to amphotericin B and caspofungin, which correlated with clinical failure of therapy.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay To Detect Histoplasma capsulatum Antigenuria in Immunocompromised Patients

ABSTRACT Histoplasma capsulatum infection causes significant morbidity and mortality in human immunodeficiency virus-infected individuals, particularly those in countries with limited access to rapid diagnostics or antiretroviral therapies. The fungus easily disseminates in persons with AIDS, resulting in progressive disseminated histoplasmosis (PDH), which can progress rapidly to death if undiagnosed. The availability of a simple, rapid method to detect H. capsulatum infection in less developed countries where the infection is endemic would dramatically decrease the time to diagnosis and treatment of PDH. We have developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect PDH antigenuria in infected patients. The assay uses polyclonal antibodies against H. capsulatum as both capture and detection reagents, and a standard reference curve is included to quantify antigenuria and ensure reproducibility. We evaluated this assay using specimens collected from patients with AIDS and culture-proven histoplasmosis in a Guatemalan clinic (n = 48), from healthy persons (n = 83), and from patients with other, nonhistoplasmosis diseases (n = 114). The ELISA demonstrated a sensitivity of 81% and a specificity of 95% in detecting H. capsulatum antigen in urine. This assay relies on simple technology that can be performed in institutions with limited resources. Use of this test will facilitate rapid diagnosis of PDH in countries where mortality is high, expediting treatment and likely reducing PDH-related mortality.

High Prevalence of Cryptococcal Infection Among HIV-Infected Patients Hospitalized With Pneumonia in Thailand

Cryptococcal antigenemia was common among human immunodeficiency virus-infected patients hospitalized with acute respiratory illness in Thailand. Few clinical differences were evident between antigenemic and nonantigenemic HIV-infected patients. Cryptococcal infection was a possible etiologic agent of pulmonary disease in many antigenemic patients.