Naureen Iqbal

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.

Species Identification and Antifungal Susceptibility Testing of Candida Bloodstream Isolates from Population-Based Surveillance Studies in Two U.S. Cities from 2008 to 2011

ABSTRACT Between 2008 and 2011, population-based candidemia surveillance was conducted in Atlanta, GA, and Baltimore, MD. Surveillance had been previously performed in Atlanta in 1992 to 1993 and in Baltimore in 1998 to 2000, making this the first population-based candidemia surveillance conducted over multiple time points in the United States. From 2,675 identified cases of candidemia in the current surveillance, 2,329 Candida isolates were collected. Candida albicans no longer comprised the majority of isolates but remained the most frequently isolated species (38%), followed by Candida glabrata (29%), Candida parapsilosis (17%), and Candida tropicalis (10%). The species distribution has changed over time; in both Atlanta and Baltimore the proportion of C. albicans isolates decreased, and the proportion of C. glabrata isolates increased, while the proportion of C. parapsilosis isolates increased in Baltimore only. There were 98 multispecies episodes, with C. albicans and C. glabrata the most frequently encountered combination. The new species-specific CLSI Candida MIC breakpoints were applied to these data. With the exception of C. glabrata (11.9% resistant), resistance to fluconazole was very low (2.3% of isolates for C. albicans, 6.2% for C. tropicalis, and 4.1% for C. parapsilosis). There was no change in the proportion of fluconazole resistance between surveillance periods. Overall echinocandin resistance was low (1% of isolates) but was higher for C. glabrata isolates, ranging from 2.1% isolates resistant to caspofungin in Baltimore to 3.1% isolates resistant to anidulafungin in Atlanta. Given the increase at both sites and the higher echinocandin resistance, C. glabrata should be closely monitored in future surveillance.

FKS Mutations and Elevated Echinocandin MIC Values among Candida glabrata Isolates from U.S. Population-Based Surveillance

ABSTRACT Candida glabrata is the second leading cause of candidemia in the United States. Its high-level resistance to triazole antifungal drugs has led to the increased use of the echinocandin class of antifungal agents for primary therapy of these infections. We monitored C. glabrata bloodstream isolates from a population-based surveillance study for elevated echinocandin MIC values (MICs of ≥0.25 μg/ml). From the 490 C. glabrata isolates that were screened, we identified 16 isolates with an elevated MIC value (2.9% of isolates from Atlanta and 2.0% of isolates from Baltimore) for one or more of the echinocandin drugs caspofungin, anidulafungin, and micafungin. All of the isolates with elevated MIC values had a mutation in the previously identified hot spot 1 of either the glucan synthase FKS1 (n = 2) or FKS2 (n = 14) gene. No mutations were detected in hot spot 2 of either FKS1 or FKS2. The predominant mutation was mutation of FKS2-encoded serine 663 to proline (S663P), found in 10 of the isolates with elevated echinocandin MICs. Two of the mutations, R631G for FKS1 and R665G for FKS2, have not been reported previously for C. glabrata. Multilocus sequence typing indicated that the predominance of the S663P mutation was not due to the clonal spread of a single sequence type. With a rising number of echinocandin therapy failures reported, it is important to continue to monitor rates of elevated echinocandin MIC values and the associated mutations.

Role of FKS Mutations in Candida glabrata: MIC Values, Echinocandin Resistance, and Multidrug Resistance

ABSTRACT Candida glabrata is the second leading cause of candidemia in U.S. hospitals. Current guidelines suggest that an echinocandin be used as the primary therapy for the treatment of C. glabrata disease due to the high rate of resistance to fluconazole. Recent case reports indicate that C. glabrata resistance to echinocandins may be increasing. We performed susceptibility testing on 1,380 isolates of C. glabrata collected between 2008 and 2013 from four U.S. cities, Atlanta, Baltimore, Knoxville, and Portland. Our analysis showed that 3.1%, 3.3%, and 3.6% of the isolates were resistant to anidulafungin, caspofungin, and micafungin, respectively. We screened 1,032 of these isolates, including all 77 that had either a resistant or intermediate MIC value with respect to at least one echinocandin, for mutations in the hot spot regions of FKS1 and FKS2, the major mechanism of echinocandin resistance. Fifty-one isolates were identified with hot spot mutations, 16 in FKS1 and 35 in FKS2. All of the isolates with an FKS mutation except one were resistant to at least one echinocandin by susceptibility testing. Of the isolates resistant to at least one echinocandin, 36% were also resistant to fluconazole. Echinocandin resistance among U.S. C. glabrata isolates is a concern, especially in light of the fact that one-third of those isolates may be multidrug resistant. Further monitoring of U.S. C. glabrata isolates for echinocandin resistance is warranted.

Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin

ABSTRACT The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 μg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-μg disks, voriconazole 1- and 10-μg disks, two sources of caspofungin 5-μg disks [BBL and Oxoid], and posaconazole 5-μg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h (Aspergillus fumigatus, A. flavus, and A. niger), and 48 h (other species); and (iii) the posaconazole 5-μg disk, voriconazole 1-μg disk, itraconazole 10-μg disk (for all except zygomycetes), BBL caspofungin 5-μg disk, and amphotericin B 10-μg (zygomycetes only).

Correlation of Genotype and In Vitro Susceptibilities of Cryptococcus gattii Strains from the Pacific Northwest of the United States

ABSTRACT Cryptococcus gattii emerged in North America in 1999 as a human and veterinary pathogen on Vancouver Island, British Columbia. The emergent subtype, VGIIa, and the closely related subtype VGIIb can now be found in the United States in Washington, Oregon, and California. We performed multilocus sequence typing and antifungal susceptibility testing on 43 isolates of C. gattii from human patients in Oregon, Washington, California, and Idaho. In contrast to Vancouver Island, VGIIa was the most frequent but not the predominant subtype in the northwest United States. Antifungal susceptibility testing showed statistically significant differences in MICs between the subtypes. This is the first study to apply antifungal susceptibility testing to C. gattii isolates from the Pacific Northwest and the first to make direct comparisons between subtypes.

Cryptococcus gattii Infection: Characteristics and Epidemiology of Cases Identified in a South African Province with High HIV Seroprevalence, 2002–2004

We describe 46 Cryptococcus gattii-infected persons identified by population-based surveillance conducted in South Africa. Most patients with C. gattii infection presented with meningitis. The mortality rate during hospitalization was 36%. We found no significant differences between persons with and persons without C. gattii infection with regard to clinical presentation, acquired immunodeficiency syndrome diagnosis, concomitant conditions, or prior opportunistic infections. C. gattii isolates had low MICs to the tested antifungal drugs.

Preliminary Laboratory Report of Fungal Infections Associated with Contaminated Methylprednisolone Injections

ABSTRACT In September 2012, the Centers for Disease Control and Prevention (CDC) initiated an outbreak investigation of fungal infections linked to injection of contaminated methylprednisolone acetate (MPA). Between 2 October 2012 and 14 February 2013, the CDC laboratory received 799 fungal isolates or human specimens, including cerebrospinal fluid (CSF), synovial fluid, and abscess tissue, from 469 case patients in 19 states. A novel broad-range PCR assay and DNA sequencing were used to evaluate these specimens. Although Aspergillus fumigatus was recovered from the index case, Exserohilum rostratum was the primary pathogen in this outbreak and was also confirmed from unopened MPA vials. Exserohilum rostratum was detected or confirmed in 191 specimens or isolates from 150 case patients, primarily from Michigan (n = 67 patients), Tennessee (n = 26), Virginia (n = 20), and Indiana (n = 16). Positive specimens from Michigan were primarily abscess tissues, while positive specimens from Tennessee, Virginia, and Indiana were primarily CSF. E. rostratum antifungal susceptibility MIC50 and MIC90 values were determined for voriconazole (1 and 2 μg/ml, respectively), itraconazole (0.5 and 1 μg/ml), posaconazole (0.5 and 1 μg/ml), isavuconazole (4 and 4 μg/ml), and amphotericin B (0.25 and 0.5 μg/ml). Thirteen other mold species were identified among case patients, and four other fungal genera were isolated from the implicated MPA vials. The clinical significance of these other fungal species remains under investigation. The laboratory response provided significant support to case confirmation, enabled linkage between clinical isolates and injected vials of MPA, and described significant features of the fungal agents involved in this large multistate outbreak.

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.