Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction.

BACKGROUND: Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis. METHODS: Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced. RESULTS: Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species. CONCLUSION: Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Antigen host response differences between the animal-type strain and human-clinical Pythium insidiosum isolates used for serological diagnosis in Thailand.

The detection of Pythium insidiosum-specific-immunoglobulin-G antibody (Pi-Ab) with enzyme-linked immunosorbent assay (ELISA) test depends on the source of antigen. In this study, the Pi-Ab levels in 140 serum samples from patients with pythiosis were evaluated by ELISA using antigens from 10 P. insidiosum clinical isolates in comparison with antigen from the equine-standard-type strain. The ELISA values (EVs), calculated from antibody levels from serum of patients with pythiosis or other infections versus healthy controls, were significantly higher in the test with clinical-isolates antigen than the standard-equine-type strain (6.0 ± 2.6 vs 4.0 ± 1.7, respectively; P < .0001). ELISA with antigen from human source might be more proper diagnosis test.

Feline cutaneous histoplasmosis: The first case report from Thailand.

A 2-year-old, neutered female domestic shorthaired cat presented with a history of multiple papules and nodules on pinnae, nodules on the nose, and chronic wound at the lateral surface of left radial area for four months. Skin biopsy demonstrated moderate numbers of small, oval-to-round, single-walled yeasts inside the macrophages. In addition, PCR confirmed the sequence of Histoplasma capsulatum. This is the first case report of feline cutaneous histoplasmosis in Thailand.

Incidence and species distribution of candidaemia in Asia: a laboratory-based surveillance study.

The epidemiology of candidaemia varies between hospitals and geographic regions. Although there are many studies from Asia, a large-scale cross-sectional study across Asia has not been performed. We conducted a 12-month, laboratory-based surveillance of candidaemia at 25 hospitals from China, Hong Kong, India, Singapore, Taiwan and Thailand. The incidence and species distribution of candidaemia were determined. There were 1601 episodes of candidaemia among 1.2 million discharges. The overall incidence was 1.22 episodes per 1000 discharges and varied among the hospitals (range 0.16-4.53 per 1000 discharges) and countries (range 0.25-2.93 per 1000 discharges). The number of Candida blood isolates and the total number of fungal isolates were highly correlated among the six countries (R² = 0.87) and 25 hospitals (R² = 0.77). There was a moderate correlation between incidence of candidaemia and the intensive care unit (ICU)/total bed ratio (R² = 0.47), although ICUs contributed to only 23% of candidaemia cases. Of 1910 blood isolates evaluated, Candida albicans was most frequently isolated (41.3%), followed by Candida tropicalis (25.4%), Candida glabrata (13.9%) and Candida parapsilosis (12.1%). The proportion of C. tropicalis among blood isolates was higher in haemato-oncology wards than others wards (33.7% versus 24.5%, p 0.0058) and was more likely to be isolated from tropical countries than other Asian countries (46.2% versus 18.9%, p 0.04). In conclusion, the ICU settings contribute, at least in part, to the incidence variation among hospitals. The species distribution is different from Western countries. Both geographic and healthcare factors contribute to the variation of species distribution.

Childhood cryptococcosis: an increasing problem in the era of AIDS.

The authors reported 8 children with cryptococcosis from King Chulalongkorn Memorial Hospital from 1991 to 2000. Five patients were older than five years. The two common underlying diseases were HIV/AIDS (5 cases) and systemic lupus erythematosus (2 cases). Seven cases had been observed in the past four years, four of these in 2000. One patient developed disseminated disease and two patients died. In the era of HIV/AIDS and due to the fact that HIV-infected children are tending to live longer, we may encounter a higher occurrence of this opportunistic fungus in children.

Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey.

A total of 356 clinical isolates of the encapsulated basidiomycetous fungus Cryptococcus neoformans var. neoformans, obtained from Australia, Argentina, Brazil, India, Italy, New Zealand, Papua New Guinea, South Africa, Thailand and the USA, were analyzed to lay the basis for a comprehensive evaluation of the global genetic structure of C. neoformans. Two polymerase chain reaction (PCR)-based typing techniques were standardized: PCR fingerprinting using a single primer specific to minisatellite or microsatellite DNA, and randomly amplified polymorphic DNA (RAPD) analysis using two combinations of three 20- to 22-mer random primers. Previous studies showed that the resultant profiles are reproducible and stable over time. Identical results were obtained in two different laboratories and by different scientists in the same laboratory. Both typing techniques separated the isolates into four major groups (VNI and VNII, serotype A; VNIII, serotype A/D; and VNIV, serotype D). The majority (78%) of isolates belonged to VNI, compared with 18% VNII, 1% VNIII and 3% VNIV. All US isolates could be differentiated by a unique, strain-specific PCR fingerprint or RAPD pattern in contrast to most of the non-US isolates, which showed a substantially higher degree of genetic homogeneity, with some clonality, in different parts of the world. Isolates obtained from the same patient at different times and from different body sites, had identical banding patterns. Both typing techniques should provide powerful tools for epidemiological studies of medically important fungi.

Analysis of the chromosomal localization of the repetitive sequences (RPSs) in Candida albicans.

The location and organization of repetitive sequences, members of the RPS family, which are sequences specific to Candida albicans, were determined on each chromosome of C. albicans strain FC18. Using pulsed-field gel electrophoresis, we separated seven fractions from eight chromosomes. Each chromosome was cleaved by BamHI and XhoI to excise the RPSs, which were then detected by hybridization with an RPS probe. All chromosomes except chromosome 4 carried RPSs, and these RPSs were located within a limited region on each chromosome. From the digestion of each chromosome with SfiI and probing with the RPSs, we found that these recognition sites within the RPS region were conserved among all RPS-containing chromosomes. For further characterization of the RPSs, the locations and the boundary regions of the RPSs were examined on chromosome 6 of strain FC18 as a model chromosome. Using the restriction enzymes SfiI, SmaI, XhoI, BamHI, MluI and NruI, we constructed a semi-macro physical map of the RPSs and their boundary regions on this chromosome. We also determined which part of the RPS was adjacent to each boundary by using sub-fragments of RPS as probes. The physical configuration around the RPSs and their boundary regions are presented. The results obtained should be useful for future analysis of the function of these regions.

Diversity of tandemly repetitive sequences due to short periodic repetitions in the chromosomes of Candida albicans.

In a previous study, a repeated sequence, RPS1, was cloned from the genomic DNA of Candida albicans. It was 2.1 kb in length and was tandemly repeated in a limited region of almost all of the chromosomes. In this study, we examined and characterized the diversity of the repeating structure of the RPS units were of 2.1, 2.3, 2.5, and 2.9 kbp in length after digestion of the genomic DNA with SmaI and 2.1 and 2.3 kbp after digestion with PstI, with the differences being multiples of approximately 0.2 kbp. Moreover, one or two types of RPS unit were present specifically on each chromosome. We cloned 14 RPS units from the mixed DNA of chromosomes 1 and 2 and 59 RPS units from chromosome 6. These RPS units were classified into four types by their SfiI digestion profiles and chromosomal origins. Sequence comparisons revealed a tandem arrangement of internal, small repeating units of 172 bp. This unit of repetition was designated alt (C. albicans tandem repeating unit). The size of RPS units was variable, with sizes representing a series of increments of approximately 0.2 kbp that corresponded to the alt sequence. By contrast, the sequences other than the tandem repeats of alts were highly conserved, with homology of more than 98% among all cloned RPS units. These results suggested that RPS plays an important role in the organization and function of the chromosomes of C. albicans even though the actual function of RPS has not yet been clarified. Structural features of RPS that contains the repeated alt sequence are discussed in relation to human alpha-satellite DNA with its tandem repeats of about 170 bp that are similar in size to alt, the repetition of which is responsible for the variations in the size of the higher-order repeats.

Clonal size-variation of rDNA cluster region on chromosome XII of Saccharomyces cerevisiae.

Using pulsed-field gel electrophoresis (PFGE), we have demonstrated clonal variation in the size of chromosome XII in a diploid strain of Saccharomyces cerevisiae X2180-2D. The sizes of the two chromosome XII homologues were very different: 2600 (L-type) and 1450 kb (S-type). The frequency with which we detected clonal size variation in the diploid, compared to that of the parental clones, was about 15-50% of the progeny clones and the range of the size variation of the homologues was 2580-2680 kb (L-type) and 1340-1500 kb (S-type), respectively. The homologue of the L-type appeared to be more frequently variable than that of the S-type. The size variation was shown to be derived from size changes in the rDNA cluster region, which is present in chromosome XII, by digesting the chromosome with XhoI, whose cutting site is not present in a rDNA repeat unit, and hybridizing to rDNA probes. The clonal size variation was also investigated in haploids from spores after meiosis. The L-type and S-type chromosomes segregated 2:2 in an ascus and the sizes of all the S-type chromosomes were shifted up, compared to the original diploid, though the L-type ones were stable. The S-type sizes of 1340, 1450 and 1780 kb in the original diploids changed into the ranges of 1475-1610 kb, 1520-1680 kb and 1820-2010 kb, respectively, in the segregants. Furthermore, we observed that the size of S-type chromosomes in haploid cells was gradually increasing in mitosis during successive subcultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of chromosome number and size by pulsed-field gel electrophoresis (PFGE) in medically important Candida species.

The chromosomal DNAs of eight medically important Candida species, C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata, were analysed by pulsed-field gel electrophoresis under various conditions. The corresponding bands in the gels were assigned by three kinds of DNA probe which hybridized to DNA of all the species: rDNA, TUB2 and PEP4. The best conditions for separating the chromosomal DNAs were investigated and the numbers and molecular sizes of the chromosome bands were determined for each species. The chromosomal DNAs of the species were separated into 5-14 bands ranging in size from 0.5 to 4.5 Mb. Based on the quantification of the chromosome band intensities using a laser fluorescent gel scanner, the chromosome numbers were estimated. The apparent average total number of chromosomes per cell was 16 for C. albicans, 16 for C. stellatoidea, 12 for C. tropicalis, 14 for C. parapsilosis, 8 for C. krusei, 8 for C. guilliermondii, 18 for C.kefyr, and 14 for C. glabrata; the total chromosomal DNA size of each species per cell was calculated at about 31 Mb, 33 Mb, 31 Mb, 26 Mb, 20 Mb, 12 Mb, 29 Mb and 14 Mb, respectively.