Amino acid-derived 1,2-benzisothiazolinone derivatives as novel small-molecule antifungal inhibitors: identification of potential genetic targets.

We have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-derived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC(50) values against a panel of fungal pathogens. These compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activity against Candida spp., Cryptococcus neoformans, and several dermatophytes, while one is fungicidal to Aspergillus fumigatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against azole-, polyene-, and micafungin-resistant strains of Candida spp. A genetics-based approach, along with phenotype analysis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen utilized a homozygous deletion collection of approximately 4,700 Saccharomyces cerevisiae mutants. We identified mutants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistant ace2 mutant clustered within a category of genes related directly or indirectly to mitochondrial functions. In Candida albicans, the functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a respiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less active when cells are grown anaerobically.

Phaeohyphomycosis due to Alternaria infectoria: a single-center experience with utility of PCR for diagnosis and species identification.

The term phaeohyphomycosis refers to a rare group of fungal infections characterized by the presence of dark-walled hyphae or yeast-like cells in affected tissues. Herein, we report on the clinical and epidemiological characteristics of six cases of phaeohyphomycosis due to Alternaria spp. that occurred in our hospital over a 30-month period (from January 2008 to June 2010). Interestingly, whereas histopathological examinations were positive and fungal cultures yielded molds in all cases, mycological identification using conventional phenotypic methods was never possible despite prolonged incubation of the isolates. Identification of Alternaria infectoria species complex was obtained for each isolate by amplification and sequencing of the internal transcribed spacer of the ribosomal DNA (ITS rDNA). All patients had favourable outcomes following the introduction of azole-based antifungal therapy. This case series describes the clinical course of these six patients and highlights the utility of molecular identification to help in the identification of the etiologic agent when classical mycological methods have failed.

Fatal invasive infection with fungemia due to Microascus cirrosus after heart and lung transplantation in a patient with cystic fibrosis.

Scopulariopsis species are rarely but increasingly recognized as opportunistic pathogens in immunocompromised patients. We report on a patient suffering from cystic fibrosis who developed disseminated fungal infection due to a rare Scopulariopsis species, Microascus cirrosus, after heart and lung transplantation. Despite antifungal combination therapy with voriconazole and caspofungin, the patient died 4 weeks after transplantation. Diagnostic difficulties and optimal management of disseminated Scopulariopsis/Microascus infections are discussed.

Disseminated Scedosporium/Pseudallescheria infection after double-lung transplantation in patients with cystic fibrosis.

We report a case of disseminated Scedosporium/Pseudallescheria infection due to Pseudallescheria boydii sensu stricto after lung transplantation in a patient with cystic fibrosis. Dissemination occurred under voriconazole. Despite surgery and combination therapy with voriconazole, caspofungin, and terbinafine, the patient died 8 months after transplantation. Previously reported cases are reviewed.

Successful triple combination therapy of disseminated absidia corymbifera infection in an adolescent with osteosarcoma.

Mucormycosis are opportunistic infections mostly observed in immunocompromised patients. We report the case of a 13-year-old girl who suffered a systemic mucormycosis without presenting the usual risk factors. She was undergoing antineoplastic chemotherapy for advanced osteosarcoma of the femur with an uncommunicative pathologic fracture and pulmonary metastasis. Absidia corymbifera was isolated from skin lesions at the primary tumor site. She subsequently developed fungal pulmonary localizations and blood vessel thrombosis. Surgical treatment together with systemic, high doses of liposomal amphotericin B, posaconazole, and caspofungin cured the local infection and controlled systemic lesions. Unfortunately, the break in chemotherapy led to pulmonary metastasis progression.

Importance of diagnostics in epidemic and pandemic preparedness.

Diagnostics are fundamental for successful outbreak containment. In this supplement, 'Diagnostic preparedness for WHO Blueprint pathogens', we describe specific diagnostic challenges presented by selected priority pathogens most likely to cause future epidemics. Some challenges to diagnostic preparedness are common to all outbreak situations, as highlighted by recent outbreaks of Ebola, Zika and yellow fever. In this article, we review these overarching challenges and explore potential solutions. Challenges include fragmented and unreliable funding pathways, limited access to specimens and reagents, inadequate diagnostic testing capacity at both national and community levels of healthcare and lack of incentives for companies to develop and manufacture diagnostics for priority pathogens during non-outbreak periods. Addressing these challenges in an efficient and effective way will require multiple stakeholders-public and private-coordinated in implementing a holistic approach to diagnostics preparedness. All require strengthening of healthcare system diagnostic capacity (including surveillance and education of healthcare workers), establishment of sustainable financing and market strategies and integration of diagnostics with existing mechanisms. Identifying overlaps in diagnostic development needs across different priority pathogens would allow more timely and cost-effective use of resources than a pathogen by pathogen approach; target product profiles for diagnostics should be refined accordingly. We recommend the establishment of a global forum to bring together representatives from all key stakeholders required for the response to develop a coordinated implementation plan. In addition, we should explore if and how existing mechanisms to address challenges to the vaccines sector, such as Coalition for Epidemic Preparedness Innovations and Gavi, could be expanded to cover diagnostics.

Fatal coinfection with Legionella pneumophila serogroup 8 and Aspergillus fumigatus.

Legionella pneumophila is an important cause of community-acquired and nosocomial pneumonia. We report on a patient who simultaneously developed L. pneumophila serogroup 8 pneumonia and Aspergillus fumigatus lung abscesses. Despite appropriate treatments, Aspergillus disease progressed with metastasis. Coinfections caused by L. pneumophila and A. fumigatus remain exceptional. In apparently immunocompetent patients, corticosteroid therapy is a key risk factor for aspergillosis.

Aspergillus fumigatus endocarditis of the mitral valve in a heart transplant recipient: a case report.

Aspergillus endocarditis is a rare event after heart transplantation. We report a case of Aspergillus fumigatus endocarditis after orthotopic heart transplantation. The patient was treated with a combination of voriconazole and caspofungin without valve replacement and survived for 168 days after the diagnosis. Previously reported cases are reviewed.

Performance characteristics of the new ARCHITECT Toxo IgG and Toxo IgG Avidity assays.

The ARCHITECT Toxo IgG and IgG Avidity assays have been developed as a fully automated panel for immune status determination and acute infection exclusion. Resolved relative specificity and sensitivity of the ARCHITECT Toxo IgG assay were 99.6% (1359/1365) and 99.7% (1096/1099) as determined on pregnant females, blood donor, and diagnostic specimens. Seroconversion sensitivity of the ARCHITECT assay was comparable with the AxSYM Toxo IgG assay. The ARCHITECT Toxo IgG Avidity assay detected 100.0% (124/124) of acute phase specimens (<4 months after infection) as low avidity, whereas the Vidas Toxo IgG Avidity assay detected 98.9% (89/90) as low avidity. In summary, the ARCHITECT Toxo IgG assay, using recombinant antigens, showed excellent specificity and sensitivity for acute phase as well as past infection specimens. The ARCHITECT Toxoplasmosis panel can be reliably used to rule out acute Toxoplasma gondii infection in pregnant women.

Field-based evidence for the linkage of pfcrt and pfdhfr drug-resistant malaria genotypes and clinical profiles of severe malaria in Niger.

Drug resistance has been shown to increase malaria mortality and morbidity in both community- and hospital-based studies. We investigated the association between two Plasmodium falciparum drug resistance-related molecular markers and clinical profiles of severe malaria in children hospitalised in Niger. PCR-RFLP analysis showed that the codon 108 mutation of the pfdhfr gene was positively linked to severe malarial anaemia. These findings are consistent with persistent parasite infection leading to unbalanced anaemia in young children. No significant relationship was found between the molecular markers and hypoglycaemia or hyperparasitaemia. Conversely, the pfcrt T76 mutation was found to be negatively associated with cerebral malaria and neurological symptoms, such as convulsions and coma. These results have implications for the strain-specific virulence hypothesis and for parasite fitness and evolution. Our findings are discussed in regard to the local malaria transmission level.

Clinical evaluation of the BioFire FilmArray® BioThreat-E test for the diagnosis of Ebola Virus Disease in Guinea.

BACKGROUND: The recent West Africa Ebola outbreak highlighted the need to provide access to rapid, safe and reliable Ebola Virus Disease diagnostics. OBJECTIVES: The objective of this field study was to assess the clinical performance of the FilmArray® BioThreat-E test for the detection of Ebola Zaïre virus in whole blood in symptomatic patients suspected of Ebola Virus Disease in Conakry (Guinea) from March to July 2015. STUDY DESIGN: The BioThreat-E test was compared to the two RT-PCRs, using serum, implemented at Donka Hospital in the emergency context: an in-house developed quantitative one-step RT-PCR adapted from the Weidmann technique, and the RealStar® Filovirus RT-PCR Kit 1.0 (Altona-Diagnostics). We also assessed the performance of this assay in noninvasive specimens (urine and saliva) to detect infected patients. RESULTS: Of 135 patients enrolled and eligible for performance assessment on whole blood, the sensitivity was 95.7% [95% CI: 85.5-99.5] and specificity 100% [95% CI: 95.9-100]. Of the 37 symptomatic infected patients able to provide saliva and/or urine samples, 34 of the 35 saliva samples and all 3 of the urine samples were positive with the BioThreat-E test. CONCLUSIONS: This study showed that the FilmArray BioThreat-E test performs comparably to conventional molecular tests under field conditions, providing results and interpretation in approximately 1h. Due to its operational characteristics, it can be easily deployed in the field during an epidemic and could also be a useful tool for post-outbreak surveillance.

Genetic Screens for Determination of Mechanism of Action.

The search for new antifungal drugs and cell targets continues. During the discovery process, mechanism-of-action (MOA) studies are critical to the continued progress of the compound through the pipeline. There are many approaches that can be utilized in understanding the MOA. One of these approaches is a genetic screen utilizing the availability of Saccharomyces cerevisiae mutant libraries. Both null and heterozygous library mutants covering the entire genome of this model yeast are available. The desired phenotype when screening the new compound is either resistance (null mutants) or haploinsufficiency or loss of fitness (heterozygote mutants). Both types of mutants can be clustered by software into common targets that provide clues as to a pathway or other cell process. Below, methods are described for genetic screens.

Epidemiological, clinical and biological features of malaria among children in Niamey, Niger.

BACKGROUND: Malaria takes a heavy toll in Niger, one of the world's poorest countries. Previous evaluations conducted in the context of the strategy for the Integrated Management of Childhood Illness, showed that 84% of severe malaria cases and 64 % of ordinary cases are not correctly managed. The aim of this survey was to describe epidemiological, clinical and biological features of malaria among <5 year-old children in the paediatric department of the National Hospital of Niamey, Niger's main referral hospital. METHODS: The study was performed in 2003 during the rainy season from July 25th to October 25th. Microscopic diagnosis of malaria, complete blood cell counts and measurement of glycaemia were performed in compliance with the routine procedure of the laboratory. Epidemiological data was collected through interviews with mothers. RESULTS: 256 children aged 3-60 months were included in the study. Anthropometrics and epidemiological data were typical of a very underprivileged population: 58% of the children were suffering from malnutrition and all were from poor families. Diagnosis of malaria was confirmed by microscopy in 52% of the cases. Clinical symptoms upon admission were non-specific, but there was a significant combination between a positive thick blood smear and neurological symptoms, and between a positive thick blood smear and splenomegaly. Thrombopaenia was also statistically more frequent among confirmed cases of malaria. The prevalence of severe malaria was 86%, including cases of severe anaemia among < 2 year-old children and neurological forms after 2 years of age. Overall mortality was 20% among confirmed cases and 21% among severe cases. CONCLUSIONS: The study confirmed that malaria was a major burden for the National Hospital of Niamey. Children hospitalized for malaria had an underprivileged background. Two distinctive features were the prevalence of severe malaria and a high mortality rate. Medical and non-medical underlying factors which may explain such a situation are discussed.

Antifungal drug discovery: the process and outcomes.

New data suggest that the global incidence of several types of fungal diseases have traditionally been under-documented. Of these, mortality caused by invasive fungal infections remains disturbingly high, equal to or exceeding deaths caused by drug-resistant tuberculosis and malaria. It is clear that basic research on new antifungal drugs, vaccines and diagnostic tools is needed. In this review, we focus upon antifungal drug discovery including in vitro assays, compound libraries and approaches to target identification. Genome mining has made it possible to identify fungal-specific targets; however, new compounds to these targets are apparently not in the antimicrobial pipeline. We suggest that 'repurposing' compounds (off patent) might be a more immediate starting point. Furthermore, we examine the dogma on antifungal discovery and suggest that a major thrust in technologies such as structural biology, homology modeling and virtual imaging is needed to drive discovery.

Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans.

Several and often combined mechanisms can lead to acquired azole resistance in Candida albicans and subsequent therapeutic failure. The aim of this study was to provide a complete overview of the molecular basis of azole resistance in a set of six C. albicans clinical isolates recovered from patients who failed azole therapy. For this purpose, expression levels of CDR1, MDR1 and ERG11 were investigated by reverse transcription PCR (RT-PCR) together with amplification and sequencing of the genes encoding their transcription factors TAC1, MRR1 and UPC2. In all, the data underline that azole resistance in this set of clinical isolates results from distinct, often combined, mechanisms, being mostly driven by CDR1 and/or MDR1 active efflux. We show that gain-of-function (GOF) mutations in the transcription-factor-encoding genes TAC1, MRR1 and UPC2 are a common event in azole-resistant C. albicans clinical isolates. In addition, together with the finding that these genes are highly permissive to nucleotide changes, we describe several novel mutations that could act as putative GOF mutations involved in fluconazole resistance.

Comparative evaluation of the ARCHITECT Toxo IgG, IgM, and IgG Avidity assays for anti-Toxoplasma antibodies detection in pregnant women sera.

We assessed the performance of the ARCHITECT Toxo IgG, IgM, and IgG Avidity assays against corresponding assays on AxSYM and Vidas using 730 sera from pregnant women. The ARCHITECT Toxo IgG and IgM assays showed a relative sensitivity of 97.5% and 89.9% and a relative specificity of 99.1% and 99.8%, respectively. If IgM sensitivity is calculated only for sera drawn less than 4 months after infection, the relative sensitivity rises to 98.1%. Correlation between the ARCHITECT and Vidas Avidity assays was 0.87 (n = 103). Testing 86 IgG-positive specimens from recent infection (<4 months), we never obtained high avidity results, but 2 specimens were in the gray zone, whereas sera from past infections (>4 months) exhibited high avidity results in 72.5% (137/189) of cases. The method can be used reliably to exclude recent infections in sera with concurrently positive results for IgM and IgG (IgG, >3 IU/mL).

Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings.

We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.