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Cryptococcus neoformans is an important pathogenic fungus causing cryptococcosis in humans and animals. This pathogen is usually found in avian droppings. Zoonotis infection occurs by inhalation of the fungus into the respiratory tract and causes pneumonia. The fungus can disseminate to the central nervous system and cause meningitis which can be life threatening. For active surveillance and prevention of C. neoformans in Nakhon Ratchasima Zoo, Thailand, either from bird to bird or bird to person, the present study investigated the incidence of C. neoformans in droppings of various captive birds in this zoo in April 2021. Dropping samples were collected from 29 avian species in various taxonomic groups which comprised cranes (5 species), pheasants (8 species), hornbills (4 species), parrots (4 species), storks (3 species), pelican (1 species), vulture (1 species), and flightless birds including emu, cassowary, and penguin (1 species each). A total of 67 fresh avian dropping samples were isolated on Sabouraud dextrose agar (SDA) with chloramphenicol and sunflower seed extract. No colony of C. neoformans was found in all samples. Nevertheless, active surveillance for C. neoformans should be performed continuously. [ABSTRACT FROM AUTHOR]

Objective: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7., Methods: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485., Results: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis., Conclusion: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Despite the strong association between Cryptococcus neoformans infection and the Human immunodeficiency virus (HIV) status of patients globally, most cryptococcosis cases in Far East Asia occur in non-HIV individuals. Molecular epidemiological studies, using multilocus sequence typing (MLST), have shown that more than 95% of cryptococcal strains belong to a specific subtype of VNI. However, this association has never been specifically examined in other parts of Asia. Therefore, in this study, we investigated the VNIc/ST5 genotype distribution among cryptococcosis patients in Thailand. Fifty-one C. neoformans isolates were collected from clinical samples in Siriraj Hospital, Bangkok, Thailand. The strains were predominantly isolated from HIV-positive patients (88.57%) and all were molecular type VNI MATα. An MLST analysis identified five sequence types (ST) in Siriraj Hospital, of which ST4 (45.10%) and ST6 (35.29%) were most common, and ST5 (15.69%), ST32 (1.96%), and ST93 (1.96) were less common. Contrary to reports from Far East Asia, ST5 was predominantly (83.33%) found in HIV patients (P = 0.657), and there was no significant change in the prevalence of ST5 over the past 10 years (P = 0.548). A further analysis of comorbidities showed higher morbidity and delays in the cryptococcal diagnosis in patients with tuberculosis coinfection or without HIV. Our study suggests that although the Thai population is genetically closely related to the Far East Asian population, ST5 is not associated with non-HIV status in Thailand. Therefore, this association may not be related to the host’s genetic background. However, its mechanism remains unclear. [ABSTRACT FROM AUTHOR]

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand's dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%) , C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported. Author Summary: The most common fungal pathogen of the central nervous system, members of the Cryptococcus neoformans/C. gattii species complex, causes life-threatening infection in humans with either normal or compromised immunity worldwide. The molecular epidemiology of this fungus in developing countries is highly under-investigated. Thus, three standard molecular typing methods were performed on 498 cryptococcal strains isolated from humans, the environment and animals in Thailand. As in developed countries, C. neoformans was found to be correlated with HIV infected patients and C. gattii infected humans with apparent normal immune status. Combining current and previous data, a total of 20 sequence types (ST) were identified suggesting a greater spectrum of major molecular types and genotypes of the C. neoformans/C. gattii species complex in Thailand than previously reported. Moreover, the discovery of the minor genotype (ST7) of the Vancouver Island outbreak in Thailand indicates that Asia may be an important stepping stone in its global expansion. This study gives an insight in the molecular epidemiological picture of this important fungal pathogen and necessitates further detailed studies. [ABSTRACT FROM AUTHOR]

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported. [ABSTRACT FROM AUTHOR]

Abstract: Longan, Dimocarpus longan Lour., contains polyphenolic compounds which exhibit several pharmacological properties. This study aims to evaluate antifungal activities of longan fruit extract in comparison to its active compounds. The results showed that longan seed exhibited antifungal activity against the opportunistic yeasts (Candida species and Cryptococcus neoformans). In contrast, longan pulp and whole fruit did not demonstrate any inhibitory effects. Ellagic acid showed the most potent antifungal activity followed by corilagin and gallic acid, respectively. Ellagic acid inhibited Candida parapsilosis and C. neoformans more effectively than Candida krusei and also some Candida albicans clinical strains. Baidam cultivar possessed higher antifungal activity (MIC=500–4000μg/ml) as it contained higher contents of ellagic acid and gallic acid than Edor (MIC=1000–8000μg/ml). For antibacterial activity, only corilagin and gallic acid possessed weak to moderate inhibitory effects against Staphylococcus aureus and Streptococcus mutans, respectively. Longan seed was then applied in the oral care products. Longan effervescent granule (5% extract) significantly reduced adhesion of C. albicans to acrylic strips. Mouthwash containing 0.5% extract exhibited good antifungal activity compared to a commercial product. These findings indicated that longan seed extract and its polyphenolic compounds can be used as an antifungal agent in oral care products for the treatment of opportunistic yeast infection. [Copyright &y& Elsevier]

The aim of this study was to identify baseline prognostic factors for poor clinical outcome of HIV-associated cryptococcal meningitis. We conducted a trial in Thailand and the USA comparing low- and high-dose concomitant use of amphotericin B and fluconazole for HIV-associated cryptococcal meningitis to amphotericin B followed by fluconazole. Subjects who were either alive and cerebrospinal fluid (CSF) culture-positive or dead were considered to have a poor outcome. At day 14, baseline characteristics associated with poor outcome included: low weight, high CSF cryptococcal antigen (CrAg) titre and low CSF white blood cell (WBC) count. At day 70, the associated baseline characteristics included: CSF CrAg titre >1:1024 and low Karnofsky performance status. Overall, consistent with published findings, low weight, high CSF CrAg titre and low CSF WBC counts at baseline were predictors for poor clinical outcome. In addition, we found that low Karnofsky performance status was predictive of poor outcome. Prompt management with appropriate antifungal therapy for this particular group of patients may improve the outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Background: International clinical trials can provide scientific and logistic benefits in spite of the many challenges. Determining whether a country, especially a developing country, is an appropriate location for the research should include in-country consultation and partnering to assess its social value for the population; that treatments are relevant for the population under study; and that the research infrastructure and ethical oversight are adequate. Collaboration increases the likelihood of study success and helps ensure that benefits accrue to recruited populations and their community. Purpose: This paper describes our experiences on a bi-national study and may provide guidance for those planning to engage in future collaborations. Methods: A Thai and United States team collaborated to develop and implement a phase II clinical trial for HIV-associated cryptococcal meningitis to assess safety and tolerability of combination therapy vs. standard treatment. Clinical and cultural differences, regulatory hurdles and operational issues were addressed before and during the study to ensure a successful collaboration between the 2 groups. Results: The international multicenter study allowed for more rapid enrollment, reduced costs to complete the study, sharing of the benefits of research, greater generalizability of results and capacity building in Thailand; quality metrics in Thailand were equivalent to or better than those in the U.S. Conclusions: Conducting successful clinical trials internationally requires early and ongoing collaboration to ensure the study meets sites'' requirements and expectations, conforms to varying national regulations, adheres to data quality standards and is responsive to the health needs of studied populations. [Copyright &y& Elsevier]

Background Acute meningitis is a medical emergency which needs immediate assessment and treatment. Knowing the epidemiology of acute meningitis may guide the physician for promptly empirical therapy as well as minimize morbidity and mortality. In Thailand, there are few studies regarding acute meningitis in adults and most of them have not been updated. We aimed to determine etiology, clinical manifestations, cerebrospinal fluid (CSF) findings and outcomes of patients with acute meningitis. Methods A retrospective cohort study was conducted among adult (age > 15 years) patients with acute meningitis who were treated at Ramathibodi Hospital between 2013 and 2017. The list of the patients was retrieved from the hospital database using the International Classification of Diseases, 10th revision (ICD-10) codes. Comparisons of clinical presentations and laboratory investigations between patients with bacterial meningitis and those with non-bacterial meningitis were analyzed. Results A total of 89 patients were included. Of all, 48 (53.9%) patients were men and median age (interquartile range; IQR) was 49 (32.1–63.8) years. The most common coexisting condition was HIV infection (30%), receiving prednisolone (16.9%), and diabetes mellitus (15.7%). Causes of acute meningitis were Cryptococcus neoformans (37%), bacteria (31.5%), Mycobacterium tuberculosis (27%), and virus (4.5%). Common clinical presentations were fever (74%), headache (70.8%), and confusion (31.5%). Older age, higher proportion of patients with diabetes, lower proportion of HIV infection, higher median white blood cells (WBCs) in complete blood count (CBC), lower median platelet, higher median aspartate aminotransaminase, higher median alkaline phosphatase, higher median WBCs, and polymorphonuclear neutrophils (PMNs) percentage in CSF were found in patients with acute bacterial meningitis. By multivariate logistic regression, predicting factors of acute bacterial meningitis were WBCs in CBC (OR 1.02 per 100 cells/mm3 increased; 95% CI 1.01–1.04, P = 0.001), WBCs in CSF (OR 1.04 per 10 cells/mm3 increased; 95% CI 1.01–1.07, P = 0.012) and PMNs percentage in CSF (OR 1.21 per 5% increased; 95% CI 1.07–1.37, P = 0.002). Overall, the median (IQR) duration of hospitalization was 23 (11–29) days. A total of 26 (29%) patients had a complication, such as septic shock, hydrocephalus, seizure, and brain edema. The mortality rate was 7.9%. Conclusion In this setting, the most common cause of acute meningitis in adults is cryptococcosis. In addition, tuberculosis is not uncommon. Awareness of update epidemiology may guide the physicians to initiate appropriate antimicrobial therapy. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

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.

Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.

The present study was conducted to investigate the effects of a natural product from honeybees, named propolis, against Cryptococcus neoformans and its effect in the expression of putative virulence factors, such as capsular polysaccharides, melanin production and urease enzyme. Ethanol extract propolis (EEP) was first tested for its anti-cryptococcal activity and explored its impact on virulence factors in both phenotypes and enzyme activities. Moreover, the cryptococcal virulence genes were investigated using real time RT-PCR. The MIC value of EEP, 1 mg ml -1 , displayed potent inhibition of C. neoformans cell viability. Of note is the high efficacy of sub-MIC concentrations (ranging from 0.5 to 0.125 mg ml -1 ) in decreasing the production of capsule, melanin, as well as laccase and urease enzyme activities. Importantly, EEP exhibited statistically decrease in the expression of gene-encoded virulence factors. In conclusion, EEP mediates C. neoformans growth inhibition and virulence factors by reducing the gene-encoding virulence-associated proteins and, thereby, disrupting the morphologic presence and attenuating their virulence. This study introduced EEP as regards anti-cryptococcal virulence factors activities; therefore, EEP would provide alternative ways of controlling the pathogenicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background:  Cryptococcal meningitis (CM) is a leading cause of death in individuals infected with human immunodeficiency virus (HIV). Identifying factors associated with mortality informs strategies to improve outcomes., Methods:  Five hundred one patients with HIV-associated CM were followed prospectively for 10 weeks during trials in Thailand, Uganda, Malawi, and South Africa. South African patients (n = 266) were followed for 1 year. Similar inclusion/exclusion criteria were applied at all sites. Logistic regression identified baseline variables independently associated with mortality., Results:  Mortality was 17% at 2 weeks and 34% at 10 weeks. Altered mental status (odds ratio [OR], 3.1; 95% confidence interval [CI], 1.7-5.9), high cerebrospinal fluid (CSF) fungal burden (OR, 1.4 per log10 colony-forming units/mL increase; 95% CI, 1.0-1.8), older age (>50 years; OR, 3.9; 95% CI, 1.4-11.1), high peripheral white blood cell count (>10 × 10(9) cells/L; OR, 8.7; 95% CI, 2.5-30.2), fluconazole-based induction treatment, and slow clearance of CSF infection were independently associated with 2-week mortality. Low body weight, anemia (hemoglobin <7.5 g/dL), and low CSF opening pressure were independently associated with mortality at 10 weeks in addition to altered mental status, high fungal burden, high peripheral white cell count, and older age. In those followed for 1 year, overall mortality was 41%. Immune reconstitution inflammatory syndrome occurred in 13% of patients and was associated with 2-week CSF fungal burden (P = .007), but not with time to initiation of antiretroviral therapy (ART)., Conclusions:  CSF fungal burden, altered mental status, and rate of clearance of infection predict acute mortality in HIV-associated CM. The results suggest that earlier diagnosis, more rapidly fungicidal amphotericin-based regimens, and prompt immune reconstitution with ART are priorities for improving outcomes.