FATAL SYSTEMIC FUNGAL INFECTION IN EASTERN BONGO ANTELOPE (TRAGELAPHUS EURYCERUS ISAACI): SIX CASES.

Over a span of 6 yr, six adult eastern bongo antelope (Tragelaphus eurycerus isaaci) from a single institution died due to systemic mycotic infections. All animals were of the same genetic lineage and in good body condition at the time of death. Gross findings in all cases included multifocal white-to-tan nodules up to 10 cm in diameter that were most numerous in the heart, lung, and kidney. Histologic examination identified these nodules as foci of granulomatous inflammation containing branching, septate, broad, undulating fungal elements. Identification of the fungal species was pursued using PCR with sequencing, immunohistochemistry, and culture. Multiple fungal species were identified using the various modalities, and commonality of species identification was limited to Cladosporium sp. in four of the cases. The clinical and postmortem findings in these cases were identical and were considered to be the same infectious disease. The Cladosporium sp. was considered a candidate as an emerging fatal infectious agent in this population of bongo antelopes. In all of these cases, death was attributed to conduction abnormalities associated with the cardiac lesions or euthanasia.

Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA.

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Clinical Characteristics, Health Care Utilization, and Outcomes Among Patients in a Pilot Surveillance System for Invasive Mold Disease-Georgia, United States, 2017-2019.

Background: Invasive mold diseases (IMDs) cause severe illness, but public health surveillance data are lacking. We describe data collected from a laboratory-based, pilot IMD surveillance system. Methods: During 2017-2019, the Emerging Infections Program conducted active IMD surveillance at 3 Atlanta-area hospitals. We ascertained potential cases by reviewing histopathology, culture, and Aspergillus galactomannan results and classified patients as having an IMD case (based on European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group [MSG] criteria) or a non-MSG IMD case (based on the treating clinician's diagnosis and use of mold-active antifungal therapy). We described patient features and compared patients with MSG vs non-MSG IMD cases. Results: Among 304 patients with potential IMD, 104 (34.2%) met an IMD case definition (41 MSG, 63 non-MSG). The most common IMD types were invasive aspergillosis (n = 66 [63.5%]), mucormycosis (n = 8 [7.7%]), and fusariosis (n = 4 [3.8%]); the most frequently affected body sites were pulmonary (n = 66 [63.5%]), otorhinolaryngologic (n = 17 [16.3%]), and cutaneous/deep tissue (n = 9 [8.7%]). Forty-five (43.3%) IMD patients received intensive care unit-level care, and 90-day all-cause mortality was 32.7%; these outcomes did not differ significantly between MSG and non-MSG IMD patients. Conclusions: IMD patients had high mortality rates and a variety of clinical presentations. Comprehensive IMD surveillance is needed to assess emerging trends, and strict application of MSG criteria for surveillance might exclude over one-half of clinically significant IMD cases.

Performance evaluation of fungal DNA PCR amplification from formalin-fixed paraffin-embedded tissue for diagnosis: Experience of a tertiary reference laboratory.

BACKGROUND: Diagnosis of invasive fungal infections from formalin-fixed paraffin-embedded (FFPE) tissues by PCR amplification is a developing technology. One of the difficulties of establishing a validated protocol for this testing is that the gold standard, culture, is much less sensitive than the test being validated. OBJECTIVES: To validate FFPE PCR as a refence laboratory identification methodology in the absence of abundant gold standard specimens. METHODS: In this validation, PCR from FFPE tissue was compared to other diagnostic methods for genus/species identification. Four different groups of correlative data from FFPE tissues were used to validate this procedure. Thirteen specimens had culture or serology results and FFPE PCR results, 49 specimens had both immunohistochemistry (IHC) identification and FFPE PCR results, 118 specimens had histological evidence of fungal elements, 64 of which also had FFPE PCR results, and 36 fungal mock tissues or fungal negative tissues were used. RESULTS: The sensitivity determined from the tissues with positive fungal histopathology was 54%. The specificity of the cases for which there were both culture and FFPE PCR results was 100%. For the correlation with IHC, the specificity was 98%. For the mock tissues and fungal negative tissues, the calculated analytical sensitivity was 94%, specificity was 95%, and accuracy was 94%. CONCLUSIONS: By uniquely combining various data sources, this study provides a comprehensive framework for how validation can be achieved in the absence of a gold standard and outlines the excellent performance of PCR from FFPE tissue, despite relatively the low sensitivity when compared to histopathology.

Molecular typing of clinical and environmental isolates of Cryptococcus gattii species complex from southern California, United States.

BACKGROUND: Two species complexes (SC) cause the majority of human Cryptococcus infections: Cryptococcus neoformans SC and Cryptococcus gattii SC. Infection is typically thought to be acquired following environmental exposure. In an urban setting, parks and other public spaces are a likely source of contact with C. gattii SC. OBJECTIVES: The goals of this study were to describe the genetic diversity of C. gattii SC in the California environment, to determine the extent of environmental exposure in publicly accessed areas and to correlate the genotypes of environmental C. gattii SC isolates with those from patients in southern California. METHODS: Specimens from trees and soil from 13 parks and public areas of seven California counties were examined for C. gattii SC isolates. Isolates were sequence typed and compared to sequence types from human clinical isolates from the same area. RESULTS: Multilocus sequence typing identified C. gattii sensu stricto (VGI molecular type) as well as Cryptococcus bacillisporus (VGIII molecular type). Several C. bacillisporus but none of the C. gattii sensu stricto isolates shared sequence types with human clinical isolates from southern California. CONCLUSIONS: C. gattii SC colonies exist in some California public parks. The presence of identical STs in environmental and human isolates of C. bacillisporus is suggestive of an arboreal origin of human infections. Two new tree species were documented as hosts for C. gattii SC in California, adding to the four species previously identified.

Saksenaea vasiformis Orbital Cellulitis in an Immunocompetent Child Treated With Posaconazole.

An 11-year-old immunocompetent girl presented with two and a half months of progressive right orbital cellulitis, which did not respond to multiple courses of antibiotics or prednisone. A panfungal polymerase chain reaction primer was positive for Saksenaea vasiformis, and she completed 5 months of oral posaconazole therapy after debridement. Saksenaea vasiformis is a rare cause of zygomycosis, and it typically causes skin and soft tissue infection in immunocompetent hosts, particularly after a traumatic injury. The diagnosis should be considered in cases with a protracted course that fail to respond to typical antibiotic therapy. Treatment includes surgical debridement, in additional to antifungal therapy with amphotericin B or posaconazole.

Two cases of fungal keratitis caused by Metarhizium anisopliae.

We present two cases of keratitis due to Metarhizium anisopliae in geographically separated areas of the United States. The isolates were microscopically similar but morphologically different and were identified by ribosomal DNA sequencing. Both isolates had low minimum inhibitory concentration (MIC) values to caspofungin and micafungin, but high MIC values to amphotericin B. The morphologic and antifungal susceptibility differences between the two isolates indicate possible polyphylogeny of the group.

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C. gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C. gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C. gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100 years. While the cause of C. gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events.

Molecular characterization of zoonotic pathogens Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in calves in Algeria.

Little is known on the identity and public health potential of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in farm animals in Algeria. In this study, 102 fecal specimens from pre-weaned dairy calves with or without diarrhea were collected from 19 dairy farms located in 6 provinces. PCR-restriction fragment length polymorphism analysis of the small subunit rRNA gene was used to detect and differentiate Cryptosporidium spp., whereas PCR-sequence analysis of the triosephosphate isomerase gene and ribosomal internal transcribed spacer were used to detect and genotype G. duodenalis and E. bieneusi, respectively. Cryptosporidium was found in 14 specimens, among which 7 had C. parvum, 4 had C. bovis, and 3 had mixed infection of C. parvum and C. bovis or C. bovis and C. andersoni. Subtyping of C. parvum by PCR-sequence analysis of the 60kDa glycoprotein gene identified two zoonotic subtypes IIaA16G2R1 and IIaA17G3R1. G. duodenalis was found in 28 specimens, with 6 having the host-specific assemblage E, 14 having the zoonotic assemblage A (all belonging to A2 subtype), and 8 having mixed assemblages. Six known genotypes of E. bieneusi belonging to Group 2, including I, J, BEB3, BEB4, BEB6 and PtEb XI, were identified in 11 specimens. Diarrhea was mostly associated with the occurrence of C. parvum. Data from this study suggest that human-pathogenic C. parvum subtypes and G. duodenalis and E. bieneusi genotypes are common on dairy farms in Algeria.

Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey.

Molecular characterizations of Cryptosporidium spp. in ruminants reared under traditional animal management systems are scarce and studies conducted thus far have revealed largely an absence of the pathogenic and zoonotic species Cryptosporidium parvum in pre-weaned animals. In this study, we examined Cryptosporidium species and subtype distribution in free-range pre-weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and Cryptosporidium ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known human pathogens, pre-weaned farm animals could play a potential role in the transmission of human cryptosporidiosis.

Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing.

Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (CT) values yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly <14.0) CT values were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing.

Neuraminidase inhibitor susceptibility surveillance of influenza viruses circulating worldwide during the 2011 Southern Hemisphere season.

BACKGROUND: Neuraminidase (NA) inhibitors (NAIs) are currently the only antivirals effective against influenza infections due to widespread resistance to M2 inhibitors. METHODS: Influenza A and B viruses (n = 1079) collected worldwide between April 01, 2011, and September 30, 2011, were assessed for susceptibility to FDA-approved NAIs, oseltamivir and zanamivir, and investigational peramivir, using the fluorescent-based NA-Fluor™ Influenza Neuraminidase Assay Kit. A subset of viruses (n = 98) were tested for susceptibility to the investigational NAI, laninamivir. RESULTS: Influenza A(H1N1)pdm09 viruses (n = 326) were sensitive to all NAIs, except for two (0.6%) with H275Y (N1 numbering; H274Y in N2 numbering) substitution, which exhibited elevated IC50 s for oseltamivir and peramivir, and a third with previously unreported N325K substitution, exhibiting reduced susceptibility to oseltamivir. Influenza A(H3N2) viruses (n = 407) were sensitive to all NAIs. Influenza B viruses (n = 346) were sensitive to all NAIs, except two (0.6%) with H273Y (N1 numbering; H274Y in N2 numbering) substitution, exhibiting reduced susceptibility to oseltamivir and peramivir, and one with previously unreported G140R and N144K substitutions, exhibiting reduced susceptibility to oseltamivir, zanamivir, and peramivir. All influenza A and B viruses were sensitive to laninamivir. It is unknown whether substitutions N325K, G140R, and N144K were present in the virus prior to culturing because clinical specimens were unavailable for testing. CONCLUSIONS: This study summarizes NAI susceptibility of influenza viruses circulating worldwide during the 2011 Southern Hemisphere (SH) season, assessed using the NA-Fluor™ Kit. Despite low resistance to NAIs among tested influenza viruses, constant surveillance of influenza virus susceptibility to NAIs should be emphasized.