Genetic analysis of emerging fungal pathogens: Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic fungus that mainly causes fatal disseminated trichosporonosis, especially in immunocompromised patients. T. asahii infection has been reported in Thailand, but few studies of this fungus have been published. Therefore, this study investigated the genetic diversity of 51 clinical strains of T. asahii from urine samples in Thailand. We sequenced and characterized the beta-1-tubulin (TUB1), copper-exporting ATPase (ATP), phosphate carrier protein (PHCP), and topoisomerase-1 (TOP1) genes. In addition, intergenic spacer 1 (IGS1) sequences from our previous studies were investigated. The numbers of haplotypes were 3, 3, 2, 2, and 2 for IGS1, TUB1, ATP, PHCP, and TOP1, respectively. The results suggested a relatively low level of genetic diversity among the strains. The findings illustrated that IGS1, TUB1, ATP, PHCP, and TOP1 can be collectively used as an alternative molecular typing tool for investigating the population diversity and structure of T. asahii.

SARS-CoV-2 Variants by Whole-Genome Sequencing in a University Hospital in Bangkok: First to Third COVID-19 Waves.

BACKGROUND: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants emerged globally during the recent coronavirus disease (COVID-19) pandemic. From April 2020 to April 2021, Thailand experienced three COVID-19 waves, and each wave was driven by different variants. Therefore, we aimed to analyze the genetic diversity of circulating SARS-CoV-2 using whole-genome sequencing analysis. METHODS: A total of 33 SARS-CoV-2 positive samples from three consecutive COVID-19 waves were collected and sequenced by whole-genome sequencing, of which, 8, 10, and 15 samples were derived from the first, second, and third waves, respectively. The genetic diversity of variants in each wave and the correlation between mutations and disease severity were explored. RESULTS: During the first wave, A.6, B, B.1, and B.1.375 were found to be predominant. The occurrence of mutations in these lineages was associated with low asymptomatic and mild symptoms, providing no transmission advantage and resulting in extinction after a few months of circulation. B.1.36.16, the predominant lineage of the second wave, caused more symptomatic COVID-19 cases and contained a small number of key mutations. This variant was replaced by the VOC alpha variant, which later became dominant in the third wave. We found that B.1.1.7 lineage-specific mutations were crucial for increasing transmissibility and infectivity, but not likely associated with disease severity. There were six additional mutations found only in severe COVID-19 patients, which might have altered the virus phenotype with an inclination toward more highly pathogenic SARS-CoV-2. CONCLUSION: The findings of this study highlighted the importance of whole-genome analysis in tracking newly emerging variants, exploring the genetic determinants essential for transmissibility, infectivity, and pathogenicity, and helping better understand the evolutionary process in the adaptation of viruses in humans.

Characterization of Virulence Factors in Candida Species Causing Candidemia in a Tertiary Care Hospital in Bangkok, Thailand.

Candidemia is often associated with high mortality, and Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis are common causes of this disease. The pathogenicity characteristics of specific Candida spp. that cause candidemia in Thailand are poorly understood. This study aimed to characterize the virulence factors of Candida spp. Thirty-eight isolates of different Candida species from blood cultures were evaluated for their virulence properties, including exoenzyme and biofilm production, cell surface hydrophobicity, tissue invasion, epithelial cell damage, morphogenesis, and phagocytosis resistance; the identity and frequency of mutations in ERG11 contributing to azole-resistance were also determined. C. albicans had the highest epithelial cell invasion rate and phospholipase activity, with true hyphae formation, whereas C. tropicalis produced the most biofilm, hydrophobicity, protease activity, and host cell damage and true hyphae formation. ERG11 mutations Y132F and S154F were observed in all azole-resistant C. tropicalis. C. glabrata had the most hemolytic activity while cell invasion was low with no morphologic transition. C. glabrata was more easily phagocytosed than other species. C. parapsilosis generated pseudohyphae but not hyphae and did not exhibit any trends in exoenzyme production. This knowledge will be crucial for understanding the pathogenicity of Candida spp. and will help to explore antivirulence-based treatment.

Molecular characterization and antibiotic resistance of Staphylococcus aureus isolated from clinical specimens in an urban university hospital in Bangkok, Thailand.

Little is known about the properties of the current strains of Staphylococcus aureus associated with human infections in Thailand. This study examined the rate of resistance to various antimicrobial agents, prevalence of virulence genes, and biofilm formation ability of 60 clinical S. aureus isolates from a single Thai hospital. Moreover, the Staphylococcus protein A gene (spa) type was determined among methicillin-resistant S. aureus (MRSA) isolates. Most methicillin-susceptible S. aureus isolates were susceptible to antimicrobials, whereas all MRSA isolates were resistant to erythromycin and clindamycin. The major virulence genes among the isolates were hla (100%), sec (26.7%), and hlb (20%). Meanwhile, 46.7% and 1.7% of the strains exhibited low-grade and high-grade biofilm formation, respectively. Our findings revealed the presence of spa types among MRSA isolates were: t032 (37.5%, 6/16), t088 (25%, 4/16), t001 (12.5%, 2/16), t008 (6.25%, 1/16), t034 (6.25%, 1/16), t439 (6.25%, 1/16), and t1928 (6.25%, 1/16). These findings will be useful for future research on anti-virulence therapies and the epidemiology of the strains circulating in our hospital.

Differential Interaction between Invasive Thai Group B Streptococcus Sequence Type 283 and Caco-2 Cells.

The emergence in Southeast Asia of invasive group B Streptococcus (GBS) infections in adults by sequence type (ST) 283 is suggested to be associated with fish consumption. Genotyping of 55 GBS clinical isolates revealed that 33/44 invasive isolates belonged to ST283/capsular polysaccharide type (CPS) III. This included 15/16 isolates recovered from younger adults aged 16-36 years. Seven ST283/CPSIII isolates from the blood, cerebrospinal fluid, or joint fluid were selected by the patient's age at random to perform interaction studies with intestinal epithelial Caco-2 monolayers. The invasion efficiency profiles from this study classified these isolates into two groups; a higher invasion efficiency group 1 recovered from patients aged between 23 and 36 years, and a lower invasion efficiency group 2 recovered from the elderly and neonate. Intracellular survival tests revealed that only group 1 members could survive inside Caco-2 cells up to 32 h without replication. Additionally, all isolates tested were able to traverse across polarized Caco-2 monolayers. However, the timing of translocation varied among the isolates. These results indicated the potential of GBS invasion via the gastrointestinal tract and showed phenotypic variations in invasiveness, intracellular survival, and translocation efficiency between genetically closely related ST283 isolates infecting young adults and those infecting the elderly.

Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand.

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T. asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 µg ml-1, 0.5-8 µg ml-1, and 0.01-0.25 µg ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production.

Whole genome sequence of pan drug-resistant clinical isolate of Acinetobacter baumannii ST1890.

Acinetobacter baumannii is an opportunistic gram-negative bacteria typically attributed to hospital-associated infection. It could also become multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) during a short period. Although A. baumannii has been documented extensively, complete knowledge on the antibiotic-resistant mechanisms and virulence factors responsible for pathogenesis has not been entirely elucidated. This study investigated the drug resistance pattern and characterized the genomic sequence by de novo assembly of PDR A. baumannii strain VJR422, which was isolated from a catheter-sputum specimen. The results showed that the VJR422 strain was resistant to any existing antibiotics. Based on de novo assembly, whole-genome sequences showed a total genome size of 3,924,675-bp. In silico and conventional MLST analysis of sequence type (ST) of this strain was new ST by Oxford MLST scheme and designated as ST1890. Moreover, we found 10,915 genes that could be classified into 45 categories by Gene Ontology (GO) analysis. There were 1,687 genes mapped to 34 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The statistics from Clusters of Orthologous Genes (COG) annotation identified 3,189 genes of the VJR422 strain. Regarding the existence of virulence factors, a total of 59 virulence factors were identified in the genome of the VJR422 strain by virulence factors of pathogenic bacteria databases (VFDB). The drug-resistant genes were investigated by searching in the Comprehensive Antibiotic Resistance Database (CARD). The strain harbored antibiotic-resistant genes responsible for aminoglycoside, ß-lactam-ring-containing drugs, erythromycin, and streptogramin resistance. We also identified resistance-nodulation-cell division (RND) and the major facilitator superfamily (MFS) associated with the antibiotic efflux pump. Overall, this study focused on A. baumannii strain VJR422 at the genomic level data, i.e., GO, COG, and KEGG. The antibiotic-resistant genotype and phenotype as well as the presence of potential virulence associated factors were investigated.

A study to assess the efficacy of fractional carbon dioxide laser with topical platelet-rich plasma in the treatment of androgenetic alopecia.

Owing to the inadequate response to and limitations of the approved medications for androgenetic alopecia, novel therapies are warranted to enhance outcomes. The objective of this paper is to ascertain the effectiveness of fractional carbon dioxide laser followed by platelet-rich plasma application on hair regrowth and to compare the two laser settings. Seven participants were enrolled in this half-head pilot study based on different laser pulse energy and density values (12 mJ, 800 spots/cm2 and 22 mJ, 400 spots/cm2). Ten treatment sessions were performed every 2 weeks with 12-week follow-up. The evaluation methods were based on hair density from standardized phototrichograms, global photographic assessment, and patient satisfaction. Significantly increased total and terminal hair densities were observed during treatment in both groups as compared with baselines. The mean total hair density has weekly increased significantly with high pulse energy (1.42 hair/cm2) as compared to low pulse energy (1.04 hair/cm2) throughout the study period (p = 0.023; 4.89 hair/cm2). Global photographs and patient satisfaction scores demonstrated improvement, but only the latter showed statistical significance (p = 0.029). The average pain intensity scores were not different between the groups (p = 0.760) all over the procedure. The adverse events were minor and well tolerated. This synergistic treatment remarkably leads to hair restoration and high patient satisfaction. The parameters of 22-mJ beam energy with 400 spots/cm2 density are appropriate and superior to 12-mJ beam energy with 800 spots/cm2. Albeit higher energy, no more pain is indicated in the high pulse energy group. Trial registration number (TRN): TCTR20180619004, Thai Clinical Trials Registry (TCTR) since 2018-05-15 13:39:48.

Species Distribution, Antifungal Susceptibility, and Molecular Epidemiology of Candida Species Causing Candidemia in a Tertiary Care Hospital in Bangkok, Thailand.

Candida species represent a common cause of bloodstream infection (BSI). Given the emergence of non-albicans Candida (NAC) associated with treatment failure, investigations into the species distribution, fungal susceptibility profile, and molecular epidemiology of pathogens are necessary to optimize the treatment of candidemia and explore the transmission of drug resistance for control management. This study evaluated the prevalence, antifungal susceptibility, and molecular characteristics of Candida species causing BSI in a tertiary-level hospital in Bangkok, Thailand. In total, 54 Candida isolates were recovered from 49 patients with candidemia. C. tropicalis was the most prevalent species (33.3%), followed by C. albicans (29.6%). Most Candida species were susceptible to various antifungal agents, excluding C. glabrata and C. tropicalis, which had increased rates of non-susceptibility to azoles. Most C. glabrata isolates were non-susceptible to echinocandins, especially caspofungin. The population structure of C. albicans was highly diverse, with clade 17 predominance. GoeBURST analysis of C. tropicalis revealed associations between genotype and fluconazole resistance in a particular clonal complex. The population structure of C. glabrata appeared to have a low level of genetic diversity in MLST loci. Collectively, these data might provide a fundamental database contributing to the development of novel antifungal agents and diagnostic tests.

Multi-locus Sequence Typing and Whole Genome Sequence Analysis of Cryptococcus neoformans Isolated from Clinical Specimens in Vajira Hospital, Bangkok, Thailand.

The basidiomycete yeast Cryptococcus neoformans causes disease in immunocompromized patients. Whole genome sequencing (WGS) technology provides insights into the molecular epidemiology of C. neoformans. However, the number of such studies is limited. Here we used WGS and multilocus sequence typing (MLST) to determine the genetic diversity of C. neoformans isolates and genetic structures of their populations among patients admitted to a single hospital in Bangkok, Thailand. Seven isolates from six patients collected during 1 year were identified as C. neoformans sensu stricto according to colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nucleotide sequence analysis of internal transcribed sequences. These isolates were sensitive to the antifungal drugs amphotericin B, fluconazole, 5-flucytosine, voriconazole, itraconazole and posaconazole and were mating type α and molecular type VNI. MLST analysis identified ST4, ST5 and ST6. We further employed WGS to determine the genetic diversity and relationships of C. neoformans isolated here combined with C. neoformans sequences data acquired from a public database (n = 42). We used the data to construct a phylogenetic tree. WGS provided additional genomics data and achieved high discriminatory power for identifying C. neoformans isolates isolated in Thailand. This report further demonstrates the applicability of WGS analysis for conducting molecular epidemiology and provides insight into the genetic diversity of C. neoformans isolates from one hospital in Thailand.

MixInYeast: A Multicenter Study on Mixed Yeast Infections.

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions.

Suppression of the pathogenicity of Candida albicans by the quorum-sensing molecules farnesol and tryptophol.

This study examines the ability of the quorum-sensing molecules (QSMs) farnesol and tryptophol to induce programmed cell death of the pathogenic fungus Candida albicans, to alter the expression of apoptosis-related genes, and to reduce the pathogenicity and virulence of C. albicans in Galleria mellonella. Our results showed that both farnesol and tryptophol inhibited C. albicans germ tube formation. In the QSM-treated group, the expression levels of the apoptosis genes increased, whereas the expression level of the anti-apoptosis gene decreased. Further, pretreatment of C. albicans with tryptophol or farnesol prior to G. mellonella larval infection significantly enhanced host survival compared with larvae infected with untreated C. albicans. Thus, farnesol and tryptophol may trigger apoptosis of C. albicans in vitro and reduce the virulence of C. albicans in vivo. Although further study is needed to identify the precise mechanisms underlying the antifungal properties of farnesol and tryptophol, these results suggest that QSMs may be effective agents for controlling fungal infection.

Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand.

The Scedosporium apiospermum species complex is an emerging filamentous fungi that has been isolated from environment. It can cause a wide range of infections in both immunocompetent and immunocompromised individuals. We aimed to study the genetic variation and relationships between 48 strains of S. apiospermum sensu stricto isolated from soil in Bangkok, Thailand. For PCR, sequencing and phylogenetic analysis, we used the following genes: actin; calmodulin exons 3 and 4; the second largest subunit of the RNA polymerase II; ß-tubulin exon 2-4; manganese superoxide dismutase; internal transcribed spacer; transcription elongation factor 1α; and beta-tubulin exons 5 and 6. The present study is the first phylogenetic analysis of relationships among S. apiospermum sensu stricto in Thailand and South-east Asia. This result provides useful information for future epidemiological study and may be correlated to clinical manifestation.

Common dermatophytes and in vitro anti-fungal susceptibility testing in patients attending the Dermatological Clinic at the Hospital for Tropical Medicine, Bangkok.

Dermatophytes comprising the genera Trichophyton, Microsporum, and Epidermophyton are important causes of superficial mycoses. The number of infected patients and the distribution of species of these organisms in our hospital were unknown. We therefore aimed to investigate the clinical pattern of dermatophyte infections and to identify the species of these dermatophytes at the Dermatological Clinic of the Hospital for Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok in a 1-year period. Twenty-six patients who had typical dermatophytosis lesions were recruited (27 specimens); 17 were female (65.38%) and 9 (34.62%) were male. The age range of the patients was 16-92 years. In total, nine dermatophyte isolates were identified by macroscopic and microscopic morphological characteristics. We found Microsporum canis (four isolates), Trichophyton mentagrophytes complex (one), Trichophyton rubrum (two), Trichophyton verrucosum (one), and Trichophyton tonsurans (one). The in vitro susceptibility profiles of seven antifungal agents against the nine dermatophytes were as follows (minimum inhibitory concentration ranges in µg/ml): The results were as follows (MIC ranges in µg/ml): ciclopirox, ≤0.06-0.5, griseofulvin ≤0.06-0.5, itraconazole ≤0.002-0.06, posaconazole ≤0.015-0.03, voriconazole ≤0.02-≥1, fluconazole ≤0.08-8, and terbinafine ≤0.01-0.125. This study represents the current state of dermatophyte infections in a metropolitan area of Bangkok, Thailand.

GROWTH-INHIBITORY EFFECTS OF FARNESOL AGAINST SCEDOSPORIUM BOYDII AND LOMENTOSPORA PROLIFICANS.

Scedosporium boydii and Lomentospora prolificans are filamentous fungi reported to cause infection in immunocompromized individuals. We studied the effect of farnesol to inhibit growth of S. boydii and L. prolificans by measuring colony diameter and determining minimal effective concentration (MEC). S. boydii and L. prolificans were grown on Sabouraud dextrose agar (SDA) at 37oC for 5 days. Conidia were collected and adjusted to a concentration of 104 conidia/ ml. Twenty microliters of conidia suspension was placed in each well of a sixwell plate containing serial dilutions of farnesol (10 µM, 100 µM, 1,000 µM, and 10,000 µM) in SDA. Colony morphology and diameter were observed on days 1, 2, 3, and 4. Farnesol at concentrations of 1,000 µM or higher caused the colony diameter of both S. boydii and L. prolificans to be smaller than untreated controls in a dose-dependent manner. The MEC of farnesol to inhibit growth of both S. boydii and L. prolificans was 3.2 mM. This study reveals the antifungal property of farnesol against S. boydii and L. prolificans, which can be used for further study as an alternative antifungal agent against these fungal infections.

Environmental Screening for the Scedosporium apiospermum Species Complex in Public Parks in Bangkok, Thailand.

The Scedosporium apiospermum species complex, comprising filamentous fungal species S. apiospermum sensu stricto, S. boydii, S. aurantiacum, S. dehoogii and S. minutispora, are important pathogens that cause a wide variety of infections. Although some species (S. boydii and S. apiospermum) have been isolated from patients in Thailand, no environmental surveys of these fungi have been performed in Thailand or surrounding countries. In this study, we isolated and identified species of these fungi from 68 soil and 16 water samples randomly collected from 10 parks in Bangkok. After filtration and subsequent inoculation of samples on Scedo-Select III medium, colony morphological examinations and microscopic observations were performed. Scedosporium species were isolated from soil in 8 of the 10 parks, but were only detected in one water sample. Colony morphologies of isolates from 41 of 68 soil samples (60.29%) and 1 of 15 water samples (6.67%) were consistent with that of the S. apiospermum species complex. Each morphological type was selected for species identification based on DNA sequencing and phylogenetic analysis of the ß-tubulin gene. Three species of the S. apiospermum species complex were identified: S. apiospermum (71 isolates), S. aurantiacum (6 isolates) and S. dehoogii (5 isolates). In addition, 16 sequences could not be assigned to an exact Scedosporium species. According to our environmental survey, the S. apiospermum species complex is widespread in soil in Bangkok, Thailand.

Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity.

When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell-cell communication, microorganisms secrete molecules called quorum-sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast-to-hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

Evaluation of real-time polymerase chain reaction for detection of the 16S ribosomal RNA gene of Mycobacterium tuberculosis and the diagnosis of cervical tuberculous lymphadenitis in a country with a high tuberculosis incidence.

BACKGROUND: Tuberculous lymphadenitis (TBL) is the most common form of extrapulmonary tuberculosis. Currently, the standard diagnostic test for TBL is culture, which takes more than several weeks to yield results. We studied a real-time polymerase chain reaction (PCR) for rapid detection of Mycobacterium tuberculosis in cervical lymph node specimens obtained from patients in a country where the tuberculosis incidence is high. METHODS: Patients with cervical lymphadenopathy were prospectively enrolled between April 2009 and March 2010. Clinical specimens obtained through fine-needle aspiration (FNA) and excisional biopsy were tested for M. tuberculosis by the COBAS TaqMan MTB Test, a real-time PCR assay for detecting the 16S ribosomal RNA gene of M. tuberculosis. Mycobacterial culture and histopathological findings from tissue biopsy specimens were used as a reference standard for sensitivity and specificity calculations. RESULTS: Of 73 patients, 41 received a diagnosis of TBL. For biopsy specimens, the sensitivity of real-time PCR was 63.4%, and the specificity was 96.9%. For FNA specimens, the sensitivity was 17.1%, and the specificity was 100%. The sensitivity of real-time PCR of biopsy specimens was comparable to that of tissue culture but significant lower than that of histopathological examination (P < .01). CONCLUSIONS: Real-time PCR did not increase the yield for rapid diagnosis of TBL.