Roles of the multiplex real-time PCR assay and ß-D-glucan in a high-risk population for intra-abdominal candidiasis (IAC).

Multiplex quantitative real-time PCR (MRT-PCR) using blood can improve the diagnosis of intra-abdominal candidiasis (IAC). We prospectively studied 39 patients with suspected IAC in the absence of previous antifungal therapy. Blood cultures, MRT-PCR, and ß-D-glucan (BDG) in serum were performed in all patients. IAC was defined according to the 2013 European Consensus criteria. For MRT-PCR, the probes targeted the ITS1 or ITS2 regions of ribosomal DNA. Candidaemia was confirmed only in four patients (10%), and IAC criteria were present in 17 patients (43.6%). The sensitivity of MRT-PCR was 25% but increased to 63.6% (P = .06) in plasma obtained prior to volume overload and transfusion; specificity was above 85% in all cases. BDG performance was improved using a cutoff > 260 pg/ml, and improvement was not observed in samples obtained before transfusion. In this cohort of high risk of IAC and low rate of bloodstream infection, the performance of non-culture-based methods (MRT-PCR or BDG) was moderate but may be a complementary tool given the limitations of diagnostic methods available in clinical practice. Volume overload requirements, in combination with other factors, decrease the accuracy of MRT-PCR in patients with IAC.

African histoplasmosis: new clinical and microbiological insights.

African histoplasmosis is defined as the fungal infection caused by Histoplasma capsulatum var. duboisii (Hcd). Studies focused on distinguishing Hcd and H. capsulatum var. capsulatum (Hcc), which coexist in Africa, are scarce or outdated, and African strains are continuously underrepresented. In this work, 13 cases of African patients with histoplasmosis diagnosed in the Spanish Mycology Reference Laboratory have been reviewed showing that 77% had disseminated disease and AIDS as underlying disease although Hcd infection has been classically considered a rare presentation in AIDS patients. Strains isolated from these patients and other clinical and reference strains were studied by assessing classical identification methods and performing a three-loci multi-locus sequence analysis (MLSA). Classical identification methods based on biochemical tests and measurement of yeast size proved to be useless in distinguishing both varieties. The MLSA defined an African cluster, with a strong statistical support, that included all strains with African origin. Finally, mating type was also determined by using molecular methods revealing an unequal mating type distribution in African strains. In conclusion, historical statements and classical identification methods were useless to distinguish between varieties, whereas molecular analyses revealed that all strains with African origin grouped together suggesting that traditional classification should be revised. Further investigation is required in order to unravel traditional concepts about Hcd infection and support results obtained in this work.

Clinical validation of a multiplex real-time PCR assay for detection of invasive candidiasis in intensive care unit patients.

BACKGROUND: New techniques, such as those based on multiplex quantitative real-time PCR (MRT-PCR), can improve the detection of invasive candidiasis (IC). METHODS: We prospectively studied 63 intensive care unit patients with suspected IC and 40 healthy controls. Blood cultures and MRT-PCR were performed at day 0 and +2, +7, +14 and +21 days in all patients. In addition, ß-d-glucan (BDG) and Candida albicans germ tube antibody (CAGTA) were quantified. RESULTS: IC was confirmed in 27 patients. Colonization was significantly higher in patients with IC (96% versus 64%, P = 0.002). The sensitivity, specificity, positive predictive value and negative predictive value of MRT-PCR for the diagnosis of IC were 96.3%, 97.3%, 92.8% and 98.7%, respectively. The positive predictive value and specificity were significantly higher for MRT-PCR than for BDG and CATGA. MRT-PCR performed very well, especially in deep-seated IC (sensitivity 90.9% versus 45.4% for blood culture; P = 0.06). As regards the most appropriate clinical sample for DNA amplification, in this study whole blood and serum presented similar results. CONCLUSIONS: MRT-PCR appears to be a useful test for confirming a diagnosis of IC in critically ill patients, especially in those with deep-seated disease. Its high sensitivity and positive predictive value make it a much more efficient tool for the management of IC than other diagnostic procedures and clinical scores.

Performance of panfungal--and specific-PCR-based procedures for etiological diagnosis of invasive fungal diseases on tissue biopsy specimens with proven infection: a 7-year retrospective analysis from a reference laboratory.

A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species.

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease.

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection.

Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species.

Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.

Incidence of zygomycosis in transplant recipients.

Recently, a remarkable increase in the incidence of zygomycosis has been reported from institutions in the USA and Europe. The use of voriconazole for the treatment of aspergillosis and, less frequently, the use of echinocandins as empirical treatment for invasive fungal infections are thought to be responsible for the increase. In addition, an increased incidence of this infection has been observed in transplant recipients, including both haematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) patients. There are no global surveys on the prevalence or incidence of zygomycosis, but data from individual institutions and countries show that zygomycosis is an emerging infection. The increased incidence of zygomycosis most probably reflects a greater frequency of predisposing factors, such as higher numbers of patients undergoing HSCT and immunosuppressive chemotherapy. In addition, the emergence of rare pathogens as a result of the rise in the use of antifungal therapy against common species can be postulated. Further, the availability of antifungal agents with activity profiles that are more specific for selected fungi increases the necessity of identifying pathogenic fungi; the frequency of Zygomycetes infections may have been underestimated until now because therapeutic decisions did not depend on the precise identification of pathogenic fungi.

Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis.

An increase in immigration from endemic regions has resulted in a number of cases of paracoccidioidomycosis (PCM) being imported into Spain. A molecular diagnostic technique based on real-time PCR was developed for the detection of Paracoccidioides brasiliensis DNA in both culture and patients' clinical samples. A Molecular Beacon probe was used, labelled with FAM and directed at the ITS1 region of ribosomic DNA. The detection limit of the technique developed was 1 fg of fungal DNA per microl of sample. This procedure proved to be very reproducible and specific. The technique was tested with cultures of 12 clinical strains and on samples from two patients with proven PCM. Real-time PCR was positive for all the culture strains, as well as those from both patients. By samples, the technique was positive in sputum and tissue biopsies but less useful on blood samples. Samples were analyzed several months after patient treatment, detecting a small amount of fungal DNA in one respiratory sample. This technique of real-time PCR is a sensitive method for rapid diagnosis of paracoccidioidomycosis and could serve to monitor patients after treatment has begun.

Detection of imported histoplasmosis in serum of HIV-infected patients using a real-time PCR-based assay.

A new real-time PCR-based assay was used for detecting DNA of Histoplasma capsulatum in serum samples collected from four HIV-infected patients with proven histoplasmosis. The assay targeted the ITS1 region of rDNA and its in vitro sensitivity, specificity and reproducibility were evaluated. The technique detected DNA of H. capsulatum in all of the HIV-infected patients with proven histoplasmosis (4/4, 100%). The PCR result was positive for seven of the ten (70%) samples studied. The assay's specificity was determined to be 100%, since the method was negative for 25 other serum samples (10 from patients with proven aspergillosis and 15 from healthy controls). The PCR assay is a new and promising diagnostic alternative and further investigation is warranted.

Targeted gene disruption of the 14-alpha sterol demethylase (cyp51A) in Aspergillus fumigatus and its role in azole drug susceptibility.

The role of Aspergillus fumigatus 14alpha-sterol demethylase (Cyp51A) in azole drug susceptibility was assessed. Targeted disruption of cyp51A in azole-susceptible and -resistant strains decreased MICs from 2- to 40-fold. The cyp51A mutants were morphologically indistinguishable from the wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice.

Glucan synthase complex of Aspergillus fumigatus.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

The nucleotide sequence of Saccharomyces cerevisiae chromosome IV.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined. Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S. cerevisiae chromosome. It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States. The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified. Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs). On average there is one gene approximately every two kilobases. Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes. Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

The sequence of a 20.3 kb DNA fragment from the left arm of Saccharomyces cerevisiae chromosome IV contains the KIN28, MSS2, PHO2, POL3 and DUN1 genes, and six new open reading frames.

We report the sequence of a 20 300 bp DNA fragment from the left arm of Saccharomyces cerevisiae chromosome IV. This segment contains 13 complete open reading frames (ORFs) and part of another ORF, altogether covering 84.2% of the entire sequence, five of which correspond to the previously characterized KIN28, MSS2, PHO2, POL3/CDC2 and DUN1 genes. One putative protein, D2358p, shares considerable homology with an O-sialoglycoprotein endopeptidase from Pasteurella haemolytica serotype A1. The putative product of D2325 contains the characteristic consensus motif of triacylglycerol lipases. D2320p and D2352p have a putative 'leucine-zipper' structure and a RNA-binding region Rnp-1 signature, respectively.

The sequence of a 21.3 kb DNA fragment from the left arm of yeast chromosome XIV reveals LEU4, MET4, POL1, RAS2, and six new open reading frames.

The nucleotide sequence of a fragment of 21 308 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been determined. Analysis of the sequence revealed 13 open reading frames (ORFs) longer than 300 bp, four of which correspond to the previously identified genes LEU4, MET4, POL1 and RAS2. One putative protein, N2160, shares considerable homology (32% identity) with a hypothetical protein encoded by a gene located on chromosome XV as well as with human OCRL protein (36% identity), involved in Lowe's syndrome. N2185 contains ten predicted transmembrane segments and is similar to another putative protein (YKL146) from yeast.