Srisurang Tantimavanich

Pathogenicity of basidiospores of Filobasidiella neoformans var. neoformans

Basidiospores of Filobasidiella neoformans var. neoformans (progeny of Cryptococcus neoformans MT 100.1 x VR 45980) were able to induce cryptococcosis in Swiss albino mice if inoculated by intraperitoneal injection, nasal instillation or nasal spraying. The latter method, with the aid of a jet nebulizer, was first adopted to imitate the natural entrance of infectious particles. Using this method the small number of basidiospores (7 x 10(3)) could induce cryptococcosis in mice, while the higher number of the parental laboratory-grown yeast cells (1.5 x 10(6)) did not produce infections. By nasal instillation Cyclophosphamide (Cy)-treated mice were more susceptible to the basidiospores, showing 80% cryptococcosis (eight of 10). Seven of the eight infected mice had disseminated cryptococcosis. Immunocompetent mice were more resistant to basidiospore infection than Cy-treated mice, as 40% of that group developed only pulmonary cryptococcosis; none had disseminated infection. Thus, we propose that basidiospores are one form of the infectious propagules of F. neoformans var. neoformans which can cause cryptococcosis, particularly in immunocompromised people.

Phenotypic Switching and Genetic Diversity of Cryptococcus neoformans

ABSTRACT Niger seed agar was used as a primary plating medium for the isolation of Cryptococcus neoformans from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans, C. neoformans may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.

Preferentially different mechanisms of inactivation of 9p21 gene cluster in liver fluke―related cholangiocarcinoma

Cholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15(INK4b), p16(INK4a), and p14(ARF) have been reported in many human cancers but have not hitherto been studied in liver fluke-related cholangiocarcinoma, particularly genetic and epigenetic effects on protein expression. We investigated loss of heterozygosity and microsatellite instability and performed fine mapping of the chromosomal region 9p21-pter in 94 microdissected cholangiocarcinoma samples using polymerase chain reaction based-microsatellite markers. Methylation and protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was determined using methylation-specific polymerase chain reaction and immunohistochemistry, respectively. Genetic and epigenetic alterations, including loss of protein expression, were correlated with clinicopathological data. Fine mapping at 9p21-pter showed a distinctive region between D9S286 and D9S1752 of common loss. Methylation frequency was 40.2% for p14(ARF), 48.9% for p15(INK4b), and 28.3% for p16(INK4a). Loss of protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was 30.9%, 58%, and 81.5%, respectively. Both p14(ARF) methylation and allelic loss at 9p21 were associated with loss of p14(ARF) expression. Poor prognosis was associated with loss of p16(INK4a) expression. In conclusion, mechanisms of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) in liver fluke-related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14(ARF), whereas p16(INK4a) and p15(INK4b) inactivation occurs through genetic and both genetic and epigenetic events, respectively.

Disseminated Cladophialophora bantiana infection in an idiopathic thrombocytopenic purpura patient: a case report.

Cladophialophora bantiana, a dematiaceous fungus (previously known as Cladosporium trichoides, Cladosporium bantiana, Xylohypha bantiana), was first described in a culture-proven case of cerebral phaeohyphomycosis by Binford et al. (Am J Clin Pathol 1952; 22: 535–42) in 1952. The fungus is well recognised as a neurotropic fungus, which usually causes fatal cerebral phaeohyphomycosis (Dixon DM et al., Chemotherapy 1987; 33: 129–40; Roche M et al., J Infect 2005; 51: e285–8). Cladophialophora bantiana is the most frequently isolated species of cerebral phaeohyphomycosis (Revankar SG et al., Clin Infect Dis 2004; 38: 206–16). However, it is extremely rare for this pathogen to cause a disseminated infection. Prior to our patient, there has been only one report of disseminated C. bantiana infection throughout the world (Keyser A et al., J Heart Lung Transplant 2002; 21: 503–5). We report the second case of disseminated C. bantiana infection in an idiopathic thrombocytopenic purpura (ITP) patient; this patient presented with cutaneous, pulmonary and cerebral lesions.

Electrophoretic karyotypes of C. neoformans serotype A recovered from Thai patients with AIDS.

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)4. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013–2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics. Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously.

Construction of chimeric antibody binding green fluorescent protein for clinical application

A chimeric antibody-binding green fluorescent protein (ZZGFPuv) was successfully constructed and applied as a powerful tool for immunological diagnosis. A gene encoding two repetitive sequences of Z-domain, derivative of IgG-binding B domain of staphylococcal protein A, was fused in-frame to the N-terminus of gfpuv gene. The chimeric gene was subsequently transformed and expressed in various strains of E. coli. Expression of chimeric protein in E. coli strain HB101 resulted in a protein translocation from cytoplasm to periplasmic space and cultivation medium. The chimeric ZZGFPuv could be purified using either IgG Sepharose column or immobilized metal (Cu 2+ ) affinity chromatography. The purified protein migrated in non-denaturing SDS-PAGE as two major bands. A fluorescent band was located at 36 kDa while another band at 48 kDa exhibited non-fluorescence. The fluorescent band was isolated and assessed for IgG-binding via fluorescent emission. The lowest amount of IgG that could be detected by dot immunobinding assay was approximately 630 ng. Indirect immunofluorescent assay for a serological detection of leptospirosis was performed by using the chimeric ZZGFPuv as IgG detector. A strong fluorescent intensity as comparable to that of the fluorescein isothiocyanate (FITC) conjugated system was significantly detected. All these findings support a high feasibility to apply the chimeric Ab- binding GFP for clinical applications in the future.

Halogenated benzoate derivatives of altholactone with improved anti-fungal activity.

Abstract Altholactone exhibited the anti-fungal activity with a high MIC value of 128 μg ml−1 against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1–15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 μg ml−1, while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11–13) displayed potent activity against S. cerevisiae with MIC values of 1 μg ml−1. In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.