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ABSTRACT Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo. IMPORTANCE Blastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces. We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans , and the related species C. dubliniensis , and C tropicalis , suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C . albicans isolates, 10 C . dubliniensis isolates, and 78 C . tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species., Importance: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis , thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans , C. dubliniensis , and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity., Competing Interests: The authors declare no conflict of interest.

Measurement of antibody content and function after a viral illness is important for diagnosis and selection of the best convalescent plasma (CP) units for passive immunization. Zhang et al. (mBio 14:e03523-22, 2013, https://doi.org/10.1128/mbio.03523-22) analyzed over 19,000 coronavirus disease 2019 (COVID-19) CP (CCP) samples from the early days of the COVID-19 pandemic and reported a moderately strong correlation between antibody amount and neutralizing titer. Strikingly, about one-third of the samples had little or no neutralizing activity. The results provide a detailed glimpse of the humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in immunologically naive humans and reveal major differences in the quality of CP units collected for passive therapy before antibody screening. Heterogeneity in CCP quality undoubtedly contributed to the variable therapeutic efficacy. Analysis of the COVID-19 serology data suggest that, for the next infectious disease emergency, the best approach after quick establishment of methods for robust antibody-level stratification would be to use CP units in the top quintile of antibody content and neutralizing capacity., Competing Interests: The authors declare no conflict of interest.

Convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), but the antibody characteristics that contribute to efficacy remain poorly understood. This study analyzed plasma samples from 126 eligible convalescent blood donors in addition to 15 naive individuals, as well as an additional 20 convalescent individuals as a validation cohort. Multiplexed Fc Array binding assays and functional antibody response assays were utilized to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody composition and activity. Donor convalescent plasma samples contained a range of antibody cell- and complement-mediated effector functions, indicating the diverse antiviral activity of humoral responses observed among recovered individuals. In addition to viral neutralization, convalescent plasma samples contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity against SARS-CoV-2. Plasma samples from a fraction of eligible donors exhibited high activity across all activities evaluated. These polyfunctional plasma samples could be identified with high accuracy with even single Fc Array features, whose correlation with polyfunctional activity was confirmed in the validation cohort. Collectively, these results expand understanding of the diversity of antibody-mediated antiviral functions associated with convalescent plasma, and the polyfunctional antiviral functions suggest that it could retain activity even when its neutralizing capacity is reduced by mutations in variant SARS-CoV-2. IMPORTANCE Convalescent plasma has been deployed globally as a treatment for COVID-19, but efficacy has been mixed. Better understanding of the antibody characteristics that may contribute to its antiviral effects is important for this intervention as well as offer insights into correlates of vaccine-mediated protection. Here, a survey of convalescent plasma activities, including antibody neutralization and diverse effector functions, was used to define plasma samples with broad activity profiles. These polyfunctional plasma samples could be reliably identified in multiple cohorts by multiplex assay, presenting a widely deployable screening test for plasma selection and investigation of vaccine-elicited responses., (Copyright © 2021 Natarajan et al.)

Accurate serological assays to detect antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to characterize the epidemiology of SARS-CoV-2 infection and identify potential candidates for coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donation. This study compared the performances of commercial enzyme immunoassays (EIAs) with respect to detection of IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAbs). The diagnostic accuracy of five commercially available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) for detection of IgG or total antibodies to SARS-CoV-2 was evaluated using cross-sectional samples from potential CCP donors who had prior molecular confirmation of SARS-CoV-2 infection ( n  = 214) and samples from prepandemic emergency department patients without SARS-CoV-2 infection ( n  = 1,099). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority ( n  = 16 [7.5%]) had been hospitalized due to COVID-19; 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. Performed according to the protocols of the manufacturers to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff value of ≥160 as the reference representing a positive test result ( n  = 140 CCP donors), the empirical area under the receiver operating curve for each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Commercial EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAb titers. Some but not all commercial EIAs may be useful in the identification of individuals with high nAb titers among convalescent individuals., (Copyright © 2021 American Society for Microbiology.)

Accurate malaria diagnosis has dual roles in identification of symptomatic persons for effective malaria treatment and also enumeration of asymptomatic persons who contribute to the epidemiologic determinants of transmission. Three currently used diagnostic tests, microscopy, rapid diagnostic tests (RDTs), and real-time PCR, all have different sensitivities and specificities, which are parasite density dependent. Here, we compare their concordance among 451 febrile episodes in a cohort of 2,058 children and adults followed over 6 months in a region in central Tanzania with hypoendemic malaria. Microscopy, a histidine-rich protein-based RDT, and two different real-time PCR gene probes detected Plasmodium falciparum in 20, 54, 41, and 78 episodes of fever, respectively. They had complete concordance in only 9 episodes. Real-time PCR with an 18S probe was more sensitive than with a mitochondrial probe for cytochrome b despite higher copy numbers of mitochondrial DNA. Both PCR yields were increased 4-fold by glycogen/acetate precipitation with low-speed centrifugation. Duplicate PCR increases low-density malaria detection. RDT had the highest number of unique positives, presumably from persistent antigen despite the absence of parasites, although RDT did not detect 3 parasitemias with over 1,000 parasites/μl. In a latent class analysis, real-time PCR had significantly higher sensitivity than did microscopy or RDT. Agreement between real-time PCR, RDT, and microscopy was highest in March and April, when both the P. falciparum parasite rate and parasite densities are highest. Real-time PCR is more sensitive and specific than RDT and microscopy in low-prevalence, low-parasite-density settings.

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.

Diethyldithiocarbamate (DETC) and pyrrolidine dithiocarbamate (PDTC) were highly active against tubercle bacilli, with MICs of 8 microg/ml and 0.13 microg/ml, respectively. DETC and PDTC were active against old cultures, enhanced pyrazinamide or pyrazinamide/rifampin activity, and had serum inhibitory titers of 1:2 and 1:4, respectively, in mice given 100 mg/kg orally.

The present study investigated the role of the Candida dubliniensis CdCDR1 and CdCDR2 genes in the development of fluconazole resistance. The C. dubliniensis CdCDR1 gene was 92% identical at the nucleotide sequence level to the corresponding C. albicans gene. However, 58% (14 of 24) of C. dubliniensis genotype 1 isolates tested harbored a nonsense mutation in the CdCDR1 open reading frame that converted codon 756 (TAT) to a TAG translational stop codon. Analysis of five of these C. dubliniensis isolates by Western immunoblotting showed that they expressed a truncated 85-kDa CdCdr1p compared to the full-length 170-kDa CdCdr1p. Expression of CdCDR1 alleles from six C. dubliniensis isolates in a pdr5 Saccharomyces cerevisiae strain revealed that CdCDR1 alleles from three isolates that encoded truncated proteins were unable to confer resistance to drugs and antifungals. However, reassignment of the TAG sequence at codon 756 to TAT (encoding tyrosine) in an allele from strain CD36 conferred the ability to mediate resistance to multiple drugs. Fluconazole-resistant isolates of C. dubliniensis harboring functional alleles of CdCDR1 were found to exhibit two- to ninefold-higher levels of CdCDR1 mRNA than did matched fluconazole-susceptible isolates. By comparison, levels of CdMDR1 expression ranged from approximately 50- to 100-fold greater in resistant isolates. Fluconazole resistance was also identified in isolates harboring nonfunctional CdCDR1 alleles, but resistance in these isolates was only associated with increased CdMDR1 expression. Targeted disruption of two functional alleles of CdCDR1 in a fluconazole-resistant derivative of C. dubliniensis that overexpressed both CdCDR1 and CdMDR1 revealed that although CdCDR1 was important for mediating reduced susceptibility to itraconazole and ketoconazole, there was no affect on fluconazole susceptibility in the double mutant. Evidence presented in this study reveals that CdCDR1 is not essential for the development of fluconazole resistance in C. dubliniensis.

The methods currently available for the identification of the pathogenic yeast Candida dubliniensis all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschhäuser, Mycoses 42:521-524; 1999) of 14 C. dubliniensis and 11 C. albicans isolates, it was suggested that the ability of C. dubliniensis to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative C. albicans. In the present investigation, we examined the colony morphology and chlamydospore production of 130 C. dubliniensis and 166 C. albicans isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the C. dubliniensis and C. albicans isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the C. albicans isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the C. dubliniensis isolates produced chlamydospores on SA and CAF, respectively. All of the C. albicans isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30 degrees C, while 97.7% of the C. dubliniensis isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the C. albicans and 93.8% of the C. dubliniensis isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between C. dubliniensis and C. albicans, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.

Candida dubliniensis is a recently discovered yeast species principally associated with carriage and disease in the oral cavities of human immunodeficiency virus (HIV)-infected individuals. To date the majority of isolates of this species have been identified in Europe and North America. In this study, five Candida isolates recovered from separate HIV-negative hospitalized patients in Jerusalem, Israel, were presumptively identified as C. dubliniensis on the basis of their dark green coloration when grown on CHROMagar Candida medium. Their identification was confirmed by a variety of techniques, including carbohydrate assimilation profiles, absence of growth at 45 degrees C, positive reaction with C. dubliniensis-specific antibodies as determined by indirect immunofluorescence analysis, and positive amplification with C. dubliniensis-specific PCR primers. All five strains were shown to be susceptible to a range of antifungal agents, including fluconazole. One of the five isolates was recovered from urine specimens, while the remaining four were recovered from upper respiratory tract and oral samples. While none of the patients was HIV positive, all were receiving broad-spectrum antibacterials at the time isolates of C. dubliniensis were obtained from clinical specimens. This study describes the first isolates of C. dubliniensis from the Middle East and confirms that this yeast can be associated with carriage and infection in the absence of HIV infection.

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans, including the ability to form germ tubes and chlamydospores. These similarities have caused significant problems in the identification of C. dubliniensis by the average clinical mycology laboratory. To facilitate the differentiation of these species, we investigated the growth of 120 isolates of C. dubliniensis and 98 C. albicans isolates at 42 and 45 degrees C on Emmons' modified Sabouraud glucose agar (SGA) and 10 isolates of each species in yeast-peptone-dextrose broth. None of the C. dubliniensis isolates grew on the agar or in the broth medium at 45 degrees C, while 11 isolates were capable of growing on SGA at 42 degrees C. In contrast, all of the C. albicans isolates but one grew at 45 degrees C on or in either medium. These reproducible results clearly demonstrate that the incubation of isolates suspected to be C. dubliniensis or C. albicans at 45 degrees C provides a simple, reliable, and inexpensive method for the differentiation of the two species.

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.

Six separate human immunodeficiency virus-positive patients with cryptococcal meningitis were each found to have been infected with a unique strain of Cryptococcus neoformans on the basis of genomic DNA finger-printing analysis with the microsatellite sequence-containing oligonucleotide probe (GGAT)4 and by random amplification of polymorphic DNA. Two patients (A and B) experienced a recurrent episode of infection. Between 12 and 16 single-colony isolates recovered from primary isolation media (> 50% of C. neoformans colonies recovered) from cerebrospinal fluid specimens were fingerprinted from both patients during each episode. The fingerprints of both isolate collections from patient B were very similar, although minor polymorphisms were evident in both sets of profiles. The fingerprints of the isolate collection from the initial episode of infection in patient A were also identical to each other, apart from minor polymorphisms, but they were clearly different from the corresponding profiles of the isolate collection from the recurrent episode, the latter of which were completely identical, apart from minor polymorphisms in a single isolate. Furthermore, prolonged storage and in vitro subculture of the isolates did not alter the fingerprint profiles. These results provided convincing evidence that patients A and B were each infected with a single C. neoformans strain during each episode of infection and that in patient B, the same strain persisted and caused both episodes, while in patient A, a different strain was responsible for each episode. The prevalence of polymorphisms in multiple single-colony isolates from both patients also suggested that C. neoformans populations may undergo microevolution.

Oligonucleotide fingerprinting of genomic DNA from oral isolates of four different Candida species other than C. albicans and atypical chlamydospore-positive isolates from human immunodeficiency virus (HIV)-positive individuals and AIDS patients was investigated as a means for differentiating between isolates within individual species. Oligonucleotides composed of simple repetitive sequence motifs, including (GACA)4, (GATA)4, (GGAT)4, (GTG)5, and (GT)8, all yielded fingerprints suitable for strain segregation of 8 C. tropicalis isolates, 12 Torulopsis (Candida) glabrata isolates, 8 atypical Candida isolates, and, except for (GATA)4, 2 C. krusei probe in turn and so generate several distinct DNA fingerprints of the same DNA sample. However, none of the probes yielded fingerprints suitable for strain segregation with three C. parapsilosis isolates. The (GATA)4 probe was also used to detect restriction fragment length polymorphisms among a genetically closely related group of atypical Candida isolates on primary isolation from an additional HIV-infected patient. These chlamydospore-positive atypical Candida isolates were sucrose positive, were of C. albicans serotype A, hybridized weakly with the C. albicans-specific mid-repeat sequence probe 27A, and yielded fingerprint profiles by random polymorphic DNA analysis that were distinct from those derived from C. albicans isolates. The C. stellatoidea ex-type strain NCPF 3108 was indistinguishable from the atypical Candida isolates in all these tests and also yielded an identical carbohydrate and nitrogen source assimilation profile by using the ID 32C yeast identification system.