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Purpose: A definitive diagnosis of invasive oral aspergillosis can be difficult because the culturing of tissue samples frequently fails to isolate Aspergillus species. In addition, the mycelial elements of Aspergillus species seen in tissue sections are histopathologically indistinguishable from those of non-Aspergillus species. We analyzed the usefulness of a DNA probe directed against the alkaline proteinase (ALP) gene of Aspergillus fumigatus for the identification of Aspergillus species by the in situ hybridization (ISH) technique in patients with oral mycosis., Patients and Methods: The ALP probe was tested on tissue specimens from 16 patients with hematologic malignancies who had invasive, orofacial fungal infections and a positive culture for one of the following organisms: Aspergillus species in 13 patients (A. flavus in 10, A. terreus in 2, and A. fumigatus in 1), and Exophiala dermatitis, Trichoderma longibrachiatum, and Candida albicans in 1 patient each. In situ hybridization with the ALP probe was performed using formalin-fixed, paraffin-embedded tissue samples., Results: The ALP probe showed a strong reaction with specimens from all 13 patients who had culture-proven aspergillosis specimens attributable to A. flavus, A. terreus, and A. fumigatus. On the other hand, the ALP probe showed no cross-reactivity with other fungi (Exophiala dermatitis, Trichoderma longibrachiatum, and Candida albicans)., Conclusion: These findings indicate that ISH using an ALP probe may increase the accuracy of diagnosing invasive oral aspergillosis in immunocompromised patients, and facilitate the provision of adequate antifungal treatment.

An in-situ hybridisation (ISH) technique to detect Aspergillus fumigatus in infected tissues was developed in which 568-bp, 333-bp and 154-bp PCR products of the alkaline proteinase gene were employed. Dot-blot hybridisation with the 568-bp probe on a membrane containing genomic DNA from several different fungi including A. flavus, A. niger, Penicillium spp., Mucor racemosus or Pseudallescheria boydii gave negative results. ISH was done on formalin-fixed, paraffin-embedded pulmonary tissues from rats infected with A. fumigatus and renal tissues from mice infected with A. fumigatus, A. flavus or A. niger. The 568-bp probe reacted strongly in ISH with both A. fumigatus and A. flavus, and weakly with A. niger. The 333-bp probe also reacted in ISH with A. fumigatus and A. flavus, although the intensity was weaker. However, in ISH with the 154-bp probe, there was no positive signal with any Aspergillus spp. These results demonstrate that A. fumigatus and A. flavus can be specifically detected in infected tissues by ISH with the 568-bp probe. This technique could be applicable to clinical specimens for molecular diagnosis of aspergillus infections.

A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists. Random amplified polymorphic DNA (RAPD) or restriction enzyme analysis (REA) are useful to determine the source of an outbreak, but more reproducible and discriminatory methods such as Southern hybridization and pulsed field gel electrophoresis (PFGE) may be required. When applied to some nosocomial Candida infections, multiple strains and species have been identified. Microevolution of yeast species occurs and epidemiologically related isolates may show minor pattern differences, creating uncertainty as to whether they are distinct strains. Approximately 1000 isolates of Aspergillus fumigatus from environmental and clinical sources were typed by REA probed with an A. fumigatus-specific retrotransposon-like sequence. Patients with no symptom of aspergillosis may carry several strains, whereas patients with pulmonary aspergillosis may carry one or two strains; nocosomial transmission of aspergillosis was proven in 39% of the patients studied; any given environmental strain can be infectious; the environmental population of A. fumigatus is extremely diverse and no specific niche was found in the hospital. A PCR assay was designed to target conserved 18S-ribosomal DNA (rDNA) sequences shared by most fungi and a 687 bp product was amplified from 25 medically important fungal species. Studies with blood, cerebrospinal fluid and sputum specimens from patients with mycoses indicated that the PCR assay is more sensitive in diagnosing invasive fungal infections than blood culture methods. More specific identification is obtainable with genus/species-specif c probes designed from within the PCR-amplified sequences for C. albicans, C. krusei, C. lusitaniae, Pneumocystis carinii, Cryptococcus neoformans, Aspergillus/Penicillium spp. and C. glabrata/Saccharomyces cerevisiae. A. fumigatus and A. niger were differentiated by denaturing gradient gel electrophoresis. In situ hybridization (ISH) detected a 648 bp fragment of the 18S rDNA of C. neoformans and a 568 bp fragment of the alkaline proteinase gene of A. fumigatus in tissues from experimentally infected animals. In ISH, the entire process can be automated, making this procedure rapid and easy. The difficulty in establishing a diagnosis of invasive candidiasis has prompted the quest for a clinically useful PCR test for candidaemia. The universal fungal oligonucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S ad 28S rDNA subunits, and the ITS2 region. Although rRNA genes are highly conserved, the ITS regions are distinctive. DNA probes were designed from ITS2 that were specific for 16 different Candida species. Simple, rapid sample preparation was suitable for PCR analysis of BacT/Alert blood culture bottles. Sample preparation, PCR, and EIA detection of the amplicon from five different Candida species was accomplished in 7 h, 2.5 days sooner than by conventional culture methods. As well as saving time, minor yeast species among a major species, or among bacteria, were simultaneously detected. PCR-EIA using a microtitration plate format had sensitivity 10-times greater than that obtained with ethidium bromide-stained agarose gels. Taqman combines in one step PCR, probe hybridization, and fluorescent signal generation. Taqman PCR had sensitivity equivalent to PCR-EIA and required only 5 h, including sample preparation.

Aspergillosis is an opportunistic infection caused by pathogenic Aspergillus species (spp.) and is a major hazard for immunocompromised patients and even for non-immunocompromised individuals. Clinical diagnosis of aspergillosis, especially invasive pulmonary aspergillosis (IPA) is difficult and is largely presumptive, typically based on spiking fevers not responding to antibiotics in a patient with the risk factors. It is well known that Aspergillus spp. can be only infrequently cultured from clinical specimens, and that the cultural examination is laborious and time-consuming. Moreover, positive culture from bronchoalveolar lavage or sputa is indicative, but not proof of infection. The criterion for diagnosis of pulmonary infection by aspergilli requires repeated isolation of the same species of Aspergillus from respiratory specimens. There have been some successful attempts using serological assays to detect circulating antibodies to Aspergillus spp. in the noninvasive form of the disease, but these are generally negative in an acute phase IPA patient. A currently available serodiagnostic kit, Pastrex Aspergillus is limited in clinical usefulness because of low sensitivity and specificity in spite of being simple and rapid. Contamination of clinical specimens with various saprophytic filamentous fungi other than aspergilli also often give false positive. Diagnostic methods using such molecular biological techniques, as polymerase chain reaction (PCR) have recently been employed to identify DNA from a number of pathogens when diagnostic means are limited. PCR is known as the most sensitive and specific technique by which to detect a specific DNA sequence. In this paper we have reviewed new genetic methods of diagnosing aspergillosis including PCR and in situ hybridization.

A polymerase chain reaction (PCR) method capable of detecting both Aspergillus fumigatus infections, pulmonary non-fumigatus Aspergillus species (spp.) and Penicillium spp. from clinical specimens was established. The primer pair was designed on the basis of the sequence of the 18S-ribosomal RNA gene of A. fumigatus and P. notatum. A 385 bp product was successfully amplified by this PCR method from all of 12 medically important Aspergillus spp. and Penicillium spp. (38 strains), but not from human, calf, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), any of 14 medically important yeastlike fungal species tested (32 strains) including Candida albicans, several non-albicans Candida and Saccharomyces cerevisiae, Cryptococcus neoformans, Mucor spp. or Pneumocystis carinii. This specificity was subsequently confirmed by Southern hybridization analysis. The established PCR can detect such a small amount as 1 pg of A. fumigatus DNA by staining the PCR product with ethidium bromide. With sputum specimens from clinically diagnosed aspergilloma patients, this PCR technique was demonstrated to be a more sensitive diagnostic method for Aspergillus infections than conventional culture techniques.