Alireza Abdolrasouli

Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing

ABSTRACT A rapid and global emergence of azole resistance has been observed in the pathogenic fungus Aspergillus fumigatus over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves mutations in the cyp51A gene, which encodes a protein targeted by triazole antifungal drugs. Whole-genome sequencing (WGS) was performed for high-resolution single-nucleotide polymorphism (SNP) analysis of 24 A. fumigatus isolates, including azole-resistant and susceptible clinical and environmental strains obtained from India, the Netherlands, and the United Kingdom, in order to assess the utility of WGS for characterizing the alleles causing resistance. WGS analysis confirmed that TR34/L98H (a mutation comprising a tandem repeat [TR] of 34 bases in the promoter of the cyp51A gene and a leucine-to-histidine change at codon 98) is the sole mechanism of azole resistance among the isolates tested in this panel of isolates. We used population genomic analysis and showed that A. fumigatus was panmictic, with as much genetic diversity found within a country as is found between continents. A striking exception to this was shown in India, where isolates are highly related despite being isolated from both clinical and environmental sources across >1,000 km; this broad occurrence suggests a recent selective sweep of a highly fit genotype that is associated with the TR34/L98H allele. We found that these sequenced isolates are all recombining, showing that azole-resistant alleles are segregating into diverse genetic backgrounds. Our analysis delineates the fundamental population genetic parameters that are needed to enable the use of genome-wide association studies to identify the contribution of SNP diversity to the generation and spread of azole resistance in this medically important fungus. IMPORTANCE Resistance to azoles in the ubiquitous ascomycete fungus A. fumigatus was first reported from clinical isolates collected in the United States during the late 1980s. Over the last decade, an increasing number of A. fumigatus isolates from the clinic and from nature have been found to show resistance to azoles, suggesting that resistance is emerging through selection by the widespread usage of agricultural azole antifungal compounds. Aspergillosis is an emerging clinical problem, with high rates of treatment failures necessitating the development of new techniques for surveillance and for determining the genome-wide basis of azole resistance in A. fumigatus. Resistance to azoles in the ubiquitous ascomycete fungus A. fumigatus was first reported from clinical isolates collected in the United States during the late 1980s. Over the last decade, an increasing number of A. fumigatus isolates from the clinic and from nature have been found to show resistance to azoles, suggesting that resistance is emerging through selection by the widespread usage of agricultural azole antifungal compounds. Aspergillosis is an emerging clinical problem, with high rates of treatment failures necessitating the development of new techniques for surveillance and for determining the genome-wide basis of azole resistance in A. fumigatus.

The Serum Opsonin L-ficolin Is Detected in Lungs of Human Transplant Recipients Following Fungal Infections and Modulates Inflammation and Killing of Aspergillus fumigatus

BACKGROUND Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. METHODS Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. RESULTS L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). CONCLUSIONS L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs.

H‐ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H‐Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H‐ficolin in fungal defence. Additionally, we investigated the presence of H‐ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H‐Ficolin exhibited demonstrable binding to A. fumigatus conidia via l‐fucose, d‐mannose and N‐acetylglucosamine residues in a calcium‐ and pH‐dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H‐ficolin opsonization manifested an increase in interleukin‐8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen‐activated protein kinase MAPK kinase 1/2, p38 MAPK and c‐Jun N‐terminal kinase. Finally, H‐ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H‐ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H‐ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus–host interactions and modulated immune responses.

In vitro efficacy of disinfectants utilised for skin decolonisation and environmental decontamination during a hospital outbreak with Candida auris

Candida auris has caused nosocomial infections and transmissions within hospital settings. As little is known about the efficacy of skin and environmental decontamination products to kill C. auris, this study investigated the in vitro activity of chlorine, chlorhexidine, iodine povidone and vaporised hydrogen peroxide products against C. auris. H2O2 vapour showed 96.6%‐100% effective killing of C. auris. All isolates were inhibited by chlorhexidine gluconate concentrations at 0.125%‐1.5% and for iodinated povidone at 0.07%‐1.25%. Other species of Candida were also killed at 1000 ppm chlorine except C. parapsilosis which failed to be killed at 3 minutes contact time. We conclude that chlorhexidine gluconate, iodinated povidone, chlorine and H2O2 vapour demonstrate effective killing activity against C. auris at concentrations used in clinical practice.

Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris

Candida auris was first described in 2009, and it has since caused nosocomial outbreaks, invasive infections, and fungaemia across at least 19 countries on five continents. An outbreak of C. auris occurred in a specialized cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak in the UK, involving a total of 72 patients. To understand the genetic epidemiology of C. auris infection both within this hospital and within a global context, we sequenced the outbreak isolate genomes using Oxford Nanopore Technologies and Illumina platforms to detect antifungal resistance alleles and reannotate the C. auris genome. Phylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin; the outbreak showed similar genetic diversity to that of the entire clade, and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations add to a growing body of research on multiple antifungal drug targets in this organism. Multiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission and implement outbreak control measures.

SEROPREVALENCE AND RISK FACTORS ASSOCIATED WITH Toxoplasma gondii INFECTION AMONG RURAL COMMUNITIES IN NORTHERN IRAN

SUMMARY Toxoplasmosis is the fourth most common cause of hospitalization and the second cause of death due to food-borne infections. We conducted a cross-sectional study to determine the prevalence, disease awareness and risk factors associated with toxoplasmosis among rural communities in Northern Iran. Data were obtained from serological testing and from participants questionnaires and were analyzed using a logistic regression. Of the 630 participants, 465 (73.8%), and 12 (1.9%) had IgG and both IgG and IgM anti-Toxoplasma gondii antibodies, respectively. In the logistic regression analysis, T. gondii seropositivity was associated with the following factors: age, occupation, consumption of undercooked meat, and of unwashed raw vegetables or fruits (p < 0.001). Our study showed a high prevalence of T. gondii infection in the general population of Northern Iran. A health program is needed to increase the public awareness of toxoplasmosis, and its associated risk factors.

A case of endemic syphilis, Iran.

To the Editor: Endemic syphilis, also known as bejel, is a nonvenereal treponematosis with onset in early childhood; the disease is caused by the bacterium Treponema pallidum subsp. endemicum. Until the 1970s, the disease was endemic to many parts of the world, including the Middle East; aggressive treatment programs abated its prevalence, but such programs have since ceased. Transmission occurs through contact with infectious lesions on the skin and mucous membranes and with contaminated drinking vessels (1). We report a case of bejel in a young boy in Iran, manifested by gummatous ulcerating lesions of the face. In November 2010, a 14-year-old Iranian boy was brought by his grandfather to our private infectious diseases clinic in Tehran, Iran, because of cutaneous lesions on his face, which had increased progressively over 9 months. This adolescent had spent his childhood in Izeh, in the southwest region of Iran. He had 5 healthy siblings, and the family medical history was unremarkable. He reported experiencing a rash in childhood without mucous membrane involvement but had no history of joint or bone pain. Examination revealed disfiguring gummatous lesions infiltrating the skin of the nose, glabella, and forehead, with clustered nodules in the left interciliary region (Figure). No other abnormality was found. He denied any sexual contacts, and there were no stigmata of congenital syphilis. Skin biopsy was refused. Tuberculin skin test results were negative. Full blood count, erythrocyte sedimentation rate, and C-reactive protein level were within reference ranges. The Venereal Disease Research Laboratory test result was positive (titer >640), and a fluorescent treponemal antibody absorption test result was strongly reactive. Because of the positive serologic test results and a preliminary diagnosis of benign tertiary syphilis, the patient was treated with 2.4 million units of benzathine penicillin G, by intramuscular injection, once per week for 3 weeks. The ulcerations completely resolved, and an atropic scar and peripheral hyperpigmentation developed over the 3-week period. The patient did not return for follow-up examination. Figure Disfiguring infiltration of the nose, glabella, and forehead with clustered nodules in left interciliary region of boy with endemic syphilis, Iran, 2010. Serologic tests cannot distinguish between bejel and venereal syphilis. For this patient, lack of history of primary chancre, absence of cardiovascular and neurologic complications in the chronic stage of the infection, absence of history of any sexual activity, and socioeconomic background are suggestive of the nonvenereal subspecies. Because the boy had no syphilitic stigmata and his siblings were healthy, congenital syphilis is unlikely. By the early 1970s, the global prevalence of endemic treponematoses (pinta, bejel, and yaws) had been reduced from 50 million to 2.5 million cases because of widespread use of long-acting, injectable penicillin in the 1950s and 1960s, led by the World Health Organization (WHO) and the United Nations Children’s Fund (2). However, penicillin mass treatment campaigns were not maintained and, as a result, the disease has reemerged. In 1995, WHO estimated the total number of treponematoses cases (infectious, latent, and late-stage) to be 2.6 million worldwide, including 460,000 infectious cases (3). Most of these were cases of yaws in Africa and Southeast Asia. Bejel predominantly affects children <15 years of age. Poor personal hygiene and overcrowding facilitate transmission of infection (1,4). Manifestation as primary lesions is rare; secondary lesions or rashes are common and are usually succeeded by a period of latency. Angular stomatitis, papules, mucous patches, and macules on the moist areas of the body are the most typical manifestations. Condylomata lata, similar to those seen in venereal syphilis and yaws, can occur. If late-stage disease develops, it usually affects the skin, the long bones of the legs, and the cartilage. Cartilage damage may result in severe destruction of the nose and palate (gangosa). Whether bejel is transmitted congenitally is unknown (1,4). Bejel was known to be endemic to the Middle East and was prevalent in Iraq and in the Bedouin population in Saudi Arabia until the 1980s (5,6). In 1995, it was diagnosed in 3 children and their father in southeastern Turkey, an area where no cases of bejel had been reported for >30 years (7). In 1954, 1 epidemiologic study of bejel in Iran reported a prevalence of 23%–34% in 4 remote villages of Khousistan (8), near the home of the case-patient in the current study. Since 1954, no cases in Iran have been reported to WHO. This case report shows that bejel continues to be transmitted among isolated, poor rural communities in Iran. Our patient was living in a remote, rural district of low socioeconomic status, and his community had almost no access to medical facilities. WHO recently convened a meeting to discuss a new initiative for the eradication of yaws, after it was demonstrated that a single oral dose of azithromycin was as effective as injected penicillin in the treatment of this disease (9,10). Bejel should be equally susceptible to eradication, but only if health services are made available to poor rural communities in areas where the disease is endemic. We recommend that countries in which this disease was formerly declared endemic initiate surveillance programs with the goal of eradication if new cases are found.

Subcutaneous Phaeohyphomycosis Cyst Associated with Medicopsis romeroi in an Immunocompromised Host

An 88-year-old man, receiving prednisolone for sarcoidosis, presented with a discrete keratotic lesion on the dorsum of his right hand following the placement of an intravenous cannula a month prior to its appearance. Medicopsis romeroi was isolated from the tissue and identified by sequencing the internal transcribed spacer region ITS-1 and the D1-2 fragment of the 28S rDNA gene. Histopathological examination showed fungal hyphae in the internal inflammatory cells layer and within the histocyte-macrophage layer, highly suggestive of deep mycosis. The patient was successfully treated with surgical excision of the cyst. M. romeroi exhibited high MIC values for echinocandin drugs in vitro, but appeared susceptible to newer triazole agents, amphotericin B and terbinafine. This is the first report of a subcutaneous phaeohyphomycotic cyst occurring following the placement of an intravenous cannula. This report highlights the potential role of M. romeroi as an emerging cause of deep, non-mycetomatous infection in immunocompromised patients.

Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory

Aims Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. Methods A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. Results MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. Conclusions MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods.

Bilateral Conjunctivitis Due to Trichomonas vaginalis without Genital Infection: an Unusual Presentation in an Adult Man

ABSTRACT We report an unusual case of extragenital infection with Trichomonas vaginalis of the conjunctiva of a 32-year-old man. Only one other similar case has been reported in the English language literature. The present report reinforces the widening pathologic spectrum of trichomonads in humans, especially in the context of emerging extragenital infections.