Identification of a Novel Pathogen Using Family-Wide PCR: Initial Confirmation of COVID-19 in Thailand.

In resource-limited countries, early detection of novel pathogens is often challenging, due to financial and technical constraints. This study reports the efficacy of family-wide polymerase chain reaction (PCR) in screening, detecting, and identifying initial cases of the novel SARS-CoV-2 in Thailand. Respiratory secretions were collected from suspected individuals traveling from Wuhan, China to Thailand at the beginning of January 2020. Family-wide PCR assays yielded positive results for coronavirus in one traveler within 12 h on January 8, 2020. Nucleotide sequences (290 bp) showed 100% similarity to SARS-CoV-2. The whole genome sequence was further characterized by Next Generation Sequencing (NGS) for confirmation. Combining family-wide PCR, as a rapid screening tool, with NGS, for full genome characterization, could facilitate early detection and confirmation of a novel pathogen and enable early containment of a disease outbreak.

Early transmission patterns of coronavirus disease 2019 (COVID-19) in travellers from Wuhan to Thailand, January 2020.

We report two cases of coronavirus disease 2019 (COVID-19) in travellers from Wuhan, China to Thailand. Both were independent introductions on separate flights, discovered with thermoscanners and confirmed with RT-PCR and genome sequencing. Both cases do not seem directly linked to the Huanan Seafood Market in Hubei but the viral genomes are identical to four other sequences from Wuhan, suggesting early spread within the city already in the first week of January.

Three new basidiomycetous yeasts, Pseudozyma alboarmeniaca sp. nov., Pseudozyma crassa sp. nov. and Pseudozyma siamensis sp. nov. isolated from Thai patients.

We previously reported the first isolation of Pseudozyma species from the blood of Thai patients. In this study, three additional new Pseudozyma species were isolated from clinical specimens from Thai patients. The Pseudozyma species showed relatively low sensitivity to azole antifungal agents. The names proposed for these isolates are Pseudozyma alboarmeniaca (DMST 17135(T) = JCM 12454(T ) = CBS 9961(T)), Pseudozyma crassa (DMST 17136(T ) = JCM 12455(T) = CBS 9959(T)) and Pseudozyma siamensis (DMST 17137(T ) = JCM 12456(T) CBS 9960(T)), where DMST is Department of Medical Sciences Culture Collection, JCM is Japan Collection of Microorganisms and CBS is Centraalbureau voor Schimmelcultures.

Efficiency of a rapid test for detection of tetrodotoxin in puffer fish.

The selling and importing of puffer fish species and their products was banned in Thailand in 2002, because of possible neurotoxic effects. However, the sale of their flesh is still happening in Thai markets. Standard methods for toxin quantification (HPLC and LC-MS) have significant limitations, therefore a lateral flow, immuno-chromatographic test (TTX-IC) was developed as a tool for rapid detection of toxin. A total of 750 puffer fishes (387 Lagocephalus lunaris(LL), and 363 Lagocephalus spadiceus (LS)) and 100 edible fishes were caught in Thailand from June 2011-February 2012. Screening of TTX from their flesh by TTX-IC revealed that 69 samples (17.8%) of LL possessed TTX at dangerous levels but LS and edible fishes did not. A selected 339 samples were quantified by LC-MS/MS, showing 50 LL possessed TTX at dangerous levels. Comparison of results with LC-MS/MS showed the TTX-IC to have 94.0% sensitivity and 92.4% specificity. The TTX-IC will be a useful tool for TTX screening of a large number of samples, reducing the testing required by LC-MS/MS, thus reducing costs. All positive cases found should be confirmed by standard methods.

Evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcosis.

BACKGROUND: Cryptococcosis is a common opportunistic infection of human immunodeficiency virus (HIV)-infected individuals mostly occurring in resource-limited countries. This study compares the performance of a recently developed lateral flow immunoassay (LFA) to blood culture and enzyme immunoassay (EIA) for the diagnosis of cryptococcosis. METHODS: Archived sera from 704 HIV-infected patients hospitalized for acute respiratory illness in Thailand were tested for cryptococcal antigenemia using EIA. All EIA-positive and a subset of EIA-negative sera were tested by LFA, with results recorded after 5 and 15 minutes incubation. Urine from patients with LFA- and EIA-positive sera was tested by LFA. Antigen results from patients with positive cryptococcal blood cultures were compared. RESULTS: Of 704 sera, 92 (13%) were positive by EIA; among the 91 EIA-positive sera tested by LFA, 82 (90%) and 87 (96%) were LFA positive when read after 5 and 15 minutes, respectively. Kappa agreement of EIA and LFA for sera was 0.923 after 5 minutes and 0.959 after 15 minutes, respectively. Two of 373 EIA-negative sera were LFA positive at both time points. Of 74 urine specimens from EIA-positive patients, 52 (70.3%) were LFA positive. EIA was positive in 16 of 17 sera from blood culture-positive patients (94% sensitivity), and all sera were positive by LFA (100% sensitivity). CONCLUSIONS: A high level of agreement was shown between LFA and EIA testing of serum. The LFA is a rapid, easy-to-perform assay that does not require refrigeration, demonstrating its potential usefulness as a point-of-care assay for diagnosis of cryptococcosis in resource-limited countries.

The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity.

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.

Genotyping and antifungal drug susceptibility of the pathogenic yeast Trichosporon asahii isolated from Thai patients.

Trichosporonosis due to Trichosporon asahii is a life-threatening infection with a very poor prognosis. We analyzed the genotype of intergenic transcribed spacer (IGS) region 1 of the rRNA gene and determined the drug susceptibility of 101 T. asahii isolates obtained from Thai patients to collect basic information on trichosporonosis in Thailand. Of the five genotypes in the IGS region identified in this study, types 1 and 3 were predominant in Thailand. The distribution in Thailand differs from that in other countries, suggesting that there is a geographic substructure among T. asahii clinical isolates. Voriconazole appeared to be the most active drug.

Comparative evaluation of Trichosporon asahii susceptibility using ASTY colorimetric microdilution and CLSI M27-A2 broth microdilution reference methods.

The in vitro activity ofAMPH-B, 5-FC, FLCZ, MCZ, ITCZ, and VRCZ against 50 isolates of T. asahii was determined using CLSI M27-A2 microdilution and ASTY colorimetric methods. Observed agreement ranged from 96 to 100% according to the drug. Overall, the agreement between two methods was 97.7%. The ASTY colorimetric method was thus determined to be comparable to the CLSI reference method when testing the susceptibility of T. asahii to a variety of antifungal agents.

Real-time PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis.

Deep-seated trichosporonosis due to Trichosporon asahii is life-threatening and has high mortality. A real-time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real-time PCR assay may be used for diagnosing deep-seated trichosporonosis due to T. asahii.

Candida pseudohaemulonii Sp. Nov., an amphotericin B-and azole-resistant yeast species, isolated from the blood of a patient from Thailand.

Candida haemulonii(types I and II) is rarely isolated from clinical specimens. We isolated a strain that is phylogenetically close to C. haemulonii from the blood of a Thai patient, and named it C. pseudohaemulonii sp. nov. (CBS 10099T = JCM 12453T = DMST 17134T). The new species and C. haemulonii types I and II were resistant to amphotericin B and azole agents but were susceptible to a 1,3-beta-D-glucan synthetase inhibitor, micafungin, and 5-flucytosine. The species were easily distinguished using an ID32 yeast identification kit. The taxonomic description of C. pseudohaemulonii sp. nov. is presented.

Characterization of clinical isolates of pathogenic Nocardia strains and related actinomycetes in Thailand from 1996 to 2003.

In Thailand from 1996 to 2003, 171 strains of pathogenic aerobic actinomycetes from clinical specimens were isolated. Of those strains, 134 were mycolic acid containing actinomycetes, including 96 strains of Nocardia species. Others included 10 strains of Gordonia, 14 strains of Rhodococcus, and 22 strains of Mycobacterium. One strain each of the genera Tsukamurella and Corynebacterium were also isolated. Also identified were 27 strains of non-mycolic acid containing actinomycetes. Our identification studies of 96 strains of Nocardia species showed that significant pathogens in Thailand were N. beijingensis (18 strains), N. cyriacigeorgica (13 strains), and N. farcinica (34 strains); the most prevalent species was N. farcinica (35.4%). We also isolated four strains of N. asiatica, five strains of N. asteroides sensu stricto, four strains of N. nova, seven strains of N. otitidiscaviarum, eight strains of N. transvalensis, and two strains of N. pseudobrasiliensis.

The first isolation of ustilaginomycetous anamorphic yeasts, Pseudozyma species, from patients' blood and a description of two new species: P. parantarctica and P. thailandica.

The genus Pseudozyma is ustilaginomycetous anamorphic yeasts, and are mainly isolated from plants. We isolated three Pseudozyma strains from the blood of patients in Thailand. While one isolate was identified as P. antarctica by rDNA sequence analysis, the other two were considered to be new species and were named P. parantarctica and P. thailandica. The three isolates proved to be resistant to 5-flucytosine, and P. thailandica was also resistant to fluconazole and itraconazole. As far as we know, this is the first isolation of Pseudozyma strains from humans. The two new species are described.