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In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence. When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled. When the complementary probe was used, labelling was higher, reaching the 3% level.

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis cariniiin clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequrences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage (BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

Aims-Recent studies suggest that Helicobacter pylori is an invasive enteropathogen. However, the efficiency with which this pathogen invades mammalian cells remains unknown. Therefore, this study was designed to investigate the invasion frequencies of HEp-2 cells by clinical strains of H pylori.Methods-An acridine orange assay and cultured HEp-2 cell monolayers were used to determine the HEp-2 cell penetration frequencies of 17 clinical isolates and one American Type Culture Collection (ATCC) strain of H pylori, and single clinical strains of Yersinia enterocolitica, Shigella flexneri, and a non-invasive ATCC Escherichia coli strain.Results-The acridine orange assay demonstrated that invasion frequencies of HEp-2 cells by all H pylori isolates were significant and, in most instances, exceeded those for the S flexneri strain and equalled those for the Y enterocolitica strain. The assay also showed that internalised H pylori organisms remained viable for at least six hours, the maximum time that bacteria and HEp-2 cells were co-incubated.Conclusions-These results may have important implications for treatment and prevention strategies for this gastric pathogen. Furthermore, the acridine orange assay may be useful for assessing, in vitro, the ability of conventional and newer antibiotics, alone or in combination, to kill intracellular H pylori organisms.

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.

The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.

Sensitivity, specificity, simplicity, speed, and economy fairly well describe the desirable attributes of any clinical diagnostic test. In the mycology laboratory, these conditions unevenly apply to detection and identification of various organisms. They are adequate for the routinely encountered pathogens, but for the opportunistic and emerging pathogens the situation is not optimal. This occurs for a variety of reasons not the least of which is the large number of potential species involved. It also occurs because the morphological and physiological characteristics used for analysis are complex, frequently slow to appear, sometimes variable within a species, and usually require significant experience to evaluate. In contrast , molecular tests, once established, avoid these problems by focusing on a single parameter. This parameter is the detection of a nucleotide sequence that is known to be unique for a given species of organism. Prior work by Carl Woese [1] and his colleagues has established that ribosomal DNA sequences have evolved slowly enough that species specific sequences appear to exist for every living creature. Since all cellular organisms use ribosomes for protein synthesis, one can detect and identify organisms by analysis of ribosomal genes. Many have used this logic to find probes for limited sets of fungi using a wide spectrum of approaches. However, what is needed for the clinical laboratory is a common approach to greatly simplify execution and validation of assays. Sandhu et al. [2] recently described a combination of molecular methods which allow one to detect and identify a large spectrum of fungal species. The combination of methods consists of treating samples with a broad spectrum lytic reagent to release DNA. Next, a uniquely hypervariable region of the 28S ribosomal gene is amplified by the polymerase chain reaction (PCR) using DNA primers that are universally specific for fungi. The amplified DNA is characterized to a species level by hybridiza-tion with a species specific nucleotide probe using a single assay condition for all probes, or it may be characterized by direct DNA sequencing. The entire procedure can be done in one to three days depending on the method chosen for characterizing the amplicon. The important points are that a common set of processing and assay conditions are used for all samples irrespective of their source and the method works equally well with culture and clinical specimens. The sensitivity of our molecular method is as low as one organism. However, in practice it must …

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

We have previously reported that a significant percentage (44%) of isoniazid-resistantMycobacterium tuberculosisstrains carry an arginine to leucine mutation in codon 463 (R463L) in the catalase-peroxidase gene (katG). For the current study, we compared the utility of one mutation screening method, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, with a reference method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to detect this mutation. The PCR-SSCP method detects mutations by electrophoretic mobility shifts of single-stranded DNA in nondenaturing polyacrylamide gels. The RFLP method detects a loss in anMspI restriction site which occurs when the R463L is present. Eighty oneM. tuberculosisstrains, including the wild type strain H37Rv, with isoniazid susceptibility in the range <0·12 to >32 μg ml−1were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-MspI RFLP reference method. Of 81M. tuberculosisstrains analysed, 13 showed mobility shifts by the PCR-SSCP method and all of those strains carried the R463L as detected by the PCR-MspI RFLP method. All of the remaining 54 strains had PCR-SSCP and PCR-MspI RFLP results identical to the wild type (R463)M. tuberculosisstrain, H37Rv. It is concluded that the described PCR-SSCP is a reliable method for screeningM. tuberculosisstrains for thekatGR463L mutation.

Objective: To compare molecular techniques with conventional diagnostic methods for evaluating nosocomial transmission of multidrug-resistant tuberculosis (MDR-TB).Design: We conducted a 12-week postexposure inception cohort study of health-care personnel who had been exposed to a patient with MDR-TB.Material and Methods: In addition to baseline and follow-up tuberculin skin tests and chest roentgenography, meekly pulmonary specimens were evaluated by (1) auramine-rhodamine fluorescent staining, (2) culture for mycobacteria, and (3) polymerase chain reaction (PCR) to amplify IS6110, a nucleic acid insertion sequence unique to the Mycobacterium tuberculosis complex.Results: The index patient's isolate of M. tuberculosis showed a mutation in codon 531 of the RNA polymerase beta subunit (rpoB) gene of M. tuberculosis, which is associated with rifampin resistance and considered a marker for this MDR-TB strain. All pulmonary and gastric specimens from study participants had negative auramine stains and cultures for mycobacteria. One person, however, had separate specimens with repeatedly positive PCR results for IS6110 sequences, but the specimens contained a wildtype M. tuberculosis rpoB codon 531 dissimilar from the index patient's strain.Conclusion: Although both molecular and conventional testing showed that no exposed person was infected with the MDR-TB strain, molecular test results were available sooner and seemed more sensitive for detecting M. tuberculosis in one exposed person, presumably in a preinfection or ''colonized'' stage. Molecular methods provided information that helped distinguish this person's M. tuberculosis strain from the index patient's MDR-TB strain. Additional prospective studies should assess the value of these molecular techniques in similar clinical settings.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

The complete catalase-peroxidase (katG) gene DNA sequence was determined for 15 strains of Mycobacterium tuberculosis with a wide range of susceptibility to isoniazid. Five of 9 strains with isoniazid MICs > or = 1.0 microgram/mL had one or more missense mutations and all 5 strains had a common G-->T transversion in codon 463, causing the replacement of arginine with leucine and the loss of an NciI or MspI restriction site. None of 6 strains with an isoniazid MIC < 1.0 microgram/mL had mutations affecting codon 463. Restriction analysis of 43 strains with isoniazid MICs > or = 1.0 microgram/mL showed that 19 (44.2%) had lost the NciI-MspI restriction site at the locus of codon 463 while only 1 of 32 strains with isoniazid MICs < or = 1.0 microgram/L had this restriction polymorphism. These results indicate that the mutation arginine-->leucine in codon 463 of the catalase-peroxidase gene occurs in a significant fraction (44.2%) of M. tuberculosis strains with isoniazid MICs > or = 1.0 microgram/mL.

Other workers have found that clinical isolates of Helicobacter pylori exhibit very extensive DNA sequence polymorphisms when they are examined by ribotyping or some other genomic sequence characterization technique. In fact, it is rare to find similar clones, much less identical ones, among isolates. We found that the levels of divergence between the 16S ribosomal DNA sequences of individual organisms and the consensus sequence of the five isolates which we examined ranged from 0.2 to 0.5%. In contrast, other workers have shown that levels of divergence between the 16S ribosomal DNA sequence of H. pylori and the 16S ribosomal DNA sequences of four other Helicobacter species range from 2.7 to 8.0%. Our results show that the H. pylori 16S ribosomal DNA is not very polymorphic and support the conclusion that H. pylori is a unique species.

This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identified by the length of the extension products on gel electrophoresis. This simple procedure eliminates the much-demanding process of sequencing. We term this process Arrested Primer Extension (APE). As a demonstration of the feasibility of this method, we have used APE to speciate a known set of cultured mycobacteria. There should be many other applications of this method.

This chapter focuses on the characterization of recombinant DNA vector by polymerase chain reaction (PCR) analysis of whole cells. The vectors are analyzed for the presence and the orientation of the inserted sequences by restriction enzyme digestion and gel electrophoresis. Short fragments of DNA without convenient restriction sites may require DNA sequencing to obtain this information. The advantage in the use of PCR to analyze recombinant DNA molecules is that it is a rapid and reliable technique. It is a technique that can obviate the need for purification, restriction mapping, or sequencing. In terms of sensitivity, the ability to amplify DNA from single eukaryotic cells is well established. By using PCR analysis with one recombinant specific internal primer and another baculovirus-specific flanking primer, the identity of a suspected plaque can be confirmed without waiting days or weeks to be certain of visual identification.