PCR assays have not reached the same level of acceptance for the detection of human fungal pathogens as for other micro-organisms, mainly because the low number of micro-organisms challenges the detection limits of PCR. Therefore, whereas meta-analyses focusing on clinical validation suggest interest in adding PCR results to the diagnostic workup for invasive fungal disease (IFD) along with clinical evaluation, CT scans, classical mycology and antigen detection, no consensual PCR method has emerged. Compared with the end-point format of the 1990s, real-time quantitative PCR is a major breakthrough. This format prevents contamination with previously amplified products, provides the yield of amplification, allows for developing consensus procedures and should therefore be the only format used. An internal control is now mandatory to avoid false-negative results. Primer design strongly impacts on the objectives: pan-fungal primers can provide false-positive results due to environmental fungal DNA contamination; conversely, species-specific primers miss infections caused by untargeted fungi. Unresolved issues include the best specimens to be used; serum is currently preferred to blood because of the ease of the DNA extraction step. Work is in progress to establish standards at least for Aspergillus PCR, and the implementation of quality controls should help centres to improve assays. Eventually, the classical analysis of biomarker performance does not consider the evolving risk factors and changing treatments during IFD, which can lead to variable conclusions. New statistical methods such as event history analysis should be considered.

Abstract Invasive candidiasis (IC) is a significant cause of morbidity and mortality. Diagnosis relies on culture-based methods, which lack sensitivity and delay diagnosis. We conducted a systematic review assessing the diagnostic accuracy of PCR-based methods to detect Candida spp. directly in blood samples. We searched electronic databases for prospective or retrospective cohort and case-control studies. Two reviewers abstracted data independently. Meta-analysis was performed using a hierarchical logistic regression model. Random-effects metaregression was performed to assess the effects of study methods and infection characteristics on sensitivity or specificity values. We included 54 studies with 4,694 patients, 963 of whom had proven/probable or possible IC. Perfect (100%) sensitivity and specificity for PCR in whole-blood samples was observed when patients with cases had candidemia and controls were healthy people. When PCR was performed to evaluate patients with suspected invasive candidiasis, the pooled sensitivity for the diagnosis of candidemia was 0.95 (confidence interval, 0.88 to 0.98) and the pooled specificity was 0.92 (0.88 to 0.95). A specificity of >90% was maintained in several analyses considering different control groups. The use of whole-blood samples, rRNA, or P450 gene targets and a PCR detection limit of 10 CFU/ml were associated with improved test performance. PCR positivity rates among patients with proven or probable IC were 85% (78 to 91%), while blood cultures were positive for 38% (29 to 46%). We conclude that direct PCR using blood samples had good sensitivity and specificity for the diagnosis of IC and offers an attractive method for early diagnosis of specific Candida spp. Its effects on clinical outcomes should be investigated.

Fungal diseases kill more than 1.5 million and affect over a billion people. However, they are still a neglected topic by public health authorities even though most deaths from fungal diseases are avoidable. Serious fungal infections occur as a consequence of other health problems including asthma, AIDS, cancer, organ transplantation and corticosteroid therapies. Early accurate diagnosis allows prompt antifungal therapy; however this is often delayed or unavailable leading to death, serious chronic illness or blindness. Recent global estimates have found 3,000,000 cases of chronic pulmonary aspergillosis, ~223,100 cases of cryptococcal meningitis complicating HIV/AIDS, ~700,000 cases of invasive candidiasis, ~500,000 cases ofPneumocystis jiroveciipneumonia, ~250,000 cases of invasive aspergillosis, ~100,000 cases of disseminated histoplasmosis, over 10,000,000 cases of fungal asthma and ~1,000,000 cases of fungal keratitis occur annually. Since 2013, the Leading International Fungal Education (LIFE) portal has facilitated the estimation of the burden of serious fungal infections country by country for over 5.7 billion people (>80% of the world population). These studies have shown differences in the global burden between countries, within regions of the same country and between at risk populations. Here we interrogate the accuracy of these fungal infection burden estimates in the 43 published papers within the LIFE initiative.

Abstract Although fungal infections contribute substantially to human morbidity and mortality, the impact of these diseases on human health is not widely appreciated. Moreover, despite the urgent need for efficient diagnostic tests and safe and effective new drugs and vaccines, research into the pathophysiology of human fungal infections lags behind that of diseases caused by other pathogens. In this Review, we highlight the importance of fungi as human pathogens and discuss the challenges we face in combating the devastating invasive infections caused by these microorganisms, in particular in immunocompromised individuals.

The Candida haemulonii species complex is currently known as C. haemulonii groups I and II. Here we describe C. haemulonii group II as a new species, Candida duobushaemulonii sp. nov., and C. haemulonii var. vulnera as new a variety of C. haemulonii group I using phenotypic and molecular methods. These taxa and other relatives of C. haemulonii (i.e., Candida auris and Candida pseudohaemulonii) cannot be differentiated by the commercial methods now used for yeast identification. Four isolates (C. haemulonii var. vulnera) differed from the other isolates of C. haemulonii in the sequence of the internal transcribed spacer (ITS) regions of the nuclear rRNA gene operon. The new species and the new variety have a multiresistant antifungal profile, which includes high MICs of amphotericin B (geometric mean MIC, 1.18 mg/liter for C. haemulonii var. vulnera and 2 mg/liter for C. duobushaemulonii sp. nov) and cross-resistance to azole compounds. Identification of these species should be based on molecular methods, such as sequence analysis of ITS regions and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

During the past 20 yr, the population of immunocompromized patients at risk of developing invasive fungal infections (IFIs) has increased, and there has been a shift in fungal epidemiology, with more infections caused by non-Aspergillus molds and yeasts, which are often resistant to one or more antifungal drugs. Traditional diagnostic methods, such as culture and the histopathology of infected tissue, often fail to detect IFIs until the later stages. Furthermore, invasive diagnostic methods to obtain tissue may be contraindicated in severely ill patients; even when tissue is available, the morphology of several filamentous fungi is identical, or the cultures may fail to grow the pathogen. Recently developed non-invasive diagnostic techniques, such as tests for serum markers and polymerase chain reaction assays, may allow for earlier and more accurate diagnoses crucial in the effort to reduce morbidity and the risk of mortality. This article reviews current approaches to diagnosis and treatment, focusing on how an early and accurate diagnosis can guide treatment and improve outcomes. Strategies for improving the management of IFIs also are discussed.

Summary Cryptococcal meningitis (CM) is a life-threatening mycosis primarily occurring in HIV-infected individuals. Recently, non-HIV-infected hosts were increasingly reported to form a considerable proportion. However, the majority of the reported studies on the diagnosis of CM patients were performed on HIV-infected patients. For evaluation of various diagnostic approaches for CM in non-HIV-infected patients, a range of conventional and molecular assays used for diagnosis of CM were verified on 85 clinical CSFs from non-HIV-infected CM patients, including India ink staining, culture, a newly developed loop-mediated isothermal amplification (LAMP), the lateral flow assay (LFA) of cryptococcal antigen detection and a qPCR assay. The LFA had the highest positive detection rate (97.6%; 95% CI, 91.8–99.7%) in non-HIV-infected CM patients, followed by the LAMP (87.1%; 95% CI, 78.0–93.4%), the qPCR (80.0%; 95% CI, 69.9–87.9%), India ink staining (70.6%; 95% CI, 59.7–80.0%) and culture (35.3%; 95% CI, 25.2–46.4%). All culture positive specimens were correctly identified by the LFA.

by Anuradha Chowdhary, Cheshta Sharma, Jacques F. Meis

As the mortality associated with invasive Candida infections remains high, it is important to make optimal use of available diagnostic tools to initiate antifungal therapy as early as possible and to select the most appropriate antifungal drug. A panel of experts of the European Fungal Infection Study Group (EFISG) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) undertook a data review and compiled guidelines for the clinical utility and accuracy of different diagnostic tests and procedures for detection of Candida infections. Recommendations about the microbiological investigation and detection of candidaemia, invasive candidiasis, chronic disseminated candidiasis, and oropharyngeal, oesophageal, and vaginal candidiasis were included. In addition, remarks about antifungal susceptibility testing and therapeutic drug monitoring were made.

Paracoccidioidomycosis is a deep mycosis Caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future. Copyright 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Abstract Traditional culture-based methods have incompletely defined the microbial landscape of common recalcitrant human fungal skin diseases, including athlete's foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms and provides a home for diverse commensal microbiota. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also have major roles in microbial community stability, human health and disease. Genomic methodologies to identify fungal species and communities have been limited compared with those that are available for bacteria. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes. Here we sequenced and analysed fungal communities of 14090009skin sites in 10090009healthy adults. Eleven core-body and arm sites were dominated by fungi of the genus Malassezia, with only species-level classifications revealing fungal-community composition differences between sites. By contrast, three foot sites--plantar heel, toenail and toe web--showed high fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that physiologic attributes and topography of skin differentially shape these two microbial communities. These results provide a framework for future investigation of the contribution of interactions between pathogenic and commensal fungal and bacterial communities to the maintainenace of human health and to disease pathogenesis.

Abstract Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.

Advances in molecular technology show great potential for the rapid detection and identification of fungi for medical, scientific and commercial purposes. Numerous targets within the fungal genome have been evaluated, with much of the current work using sequence areas within the ribosomal DNA (rDNA) gene complex. This section of the genome includes the 18S, 5 8S and 28S genes which code for ribosomal RNA (rRNA) and which have a relatively conserved nucleotide sequence among fungi. It also includes the variable DNA sequence areas of the intervening internal transcribed spacer (ITS) regions called ITS1 and ITS2. Although not translated into proteins, the ITS coding regions have a critical role in the development of functional rRNA, with sequence variations among species showing promise as signature regions for molecular assays. This review of the current literature was conducted to evaluate clinical approaches for using the fungal ITS regions as molecular targets. Multiple applications using the fungal ITS sequences are summarized here including those for culture identification, phylogenetic research, direct detection from clinical specimens or the environment, and molecular typing for epidemiological investigations. The breadth of applications shows that ITS regions have great potential as targets in molecular-based assays for the characterization and identification of fungi. Development of rapid and accurate amplification-based ITS assays to diagnose invasive fungal infections could potentially impact care and improve outcome for affected patients.

Abstract Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans , Candida glabrata , Candida tropicalis, the Candida parapsilosis group , Trichosporon asahii, and Trichosporon mucoides . These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 100 and 10302cells02mL611 in a contaminated dairy product within 102h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products.

Candidemia, the commonest invasive fungal infection, is associated with high morbidity and mortality in developing countries, though the exact prevalence is not known due to lack of systematic epidemiological data from those countries. The limited studies report a very high incidence of candidemia and unique epidemiology with a different spectrum ofCandidaspecies. The recent global emergence of multi-drug resistantCandida aurisis looming large as an important threat in hospitalized patients of developing countries. While managing candidemia cases in those countries several challenges are faced, which include poor infrastructure; compromised healthcare and infection control practices; misuse and overuse of antibiotics and steroids; lack of awareness in fungal infections; non-availability of advance diagnostic tests and antifungal drugs in many areas; poor compliance to antifungal therapy and stewardship program. Considering the above limitations, innovative strategies are required to reduce mortality due to candidemia in adults and neonates. In the present review, we have unraveled the challenges of candidemia faced by low resource countries and propose a ten part strategy to reduce mortality due candidemia.

Early diagnosis of fungal infections and the implementation of appropriate treatment represent major issues for clinicians, nowadays. Histopathological demonstration of microorganisms in tissue specimens or growth of fungal agents in culture media is still considered the “gold standard”, but obtaining such specimens may be difficult. Several groups have investigated serological assays for cell wall elements unique to fungal organisms in serum or other body fluids to improve diagnostics in patients with haematological malignancies or undergoing haematopoietic stem-cell transplantation. In this review we have concentrated on the currently available assays allowing for detection of highly immunogenic components of fungal cell wall: galactomannan, mannan, and also (1→3)-β-D-glucan. Rapid serological tests appear to be useful for screening high-risk haematological patients, since they allow for the early diagnosis of invasive fungal infections, including infections with the most common pathogens such as Aspergillus and Candida . Based on current literature, factors increasing the probability of obtaining false-positive or false-negative results detected by each test were also analysed and tabulated.

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas64 and Bruker Biotyper64, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas64; 1360/1383 for Bruker Biotyper64) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.

Members of the Cryptococcus species complex ( C. neoformans and C. gattii ) are opportunistic pathogens responsible for frequently fatal cases of meningoencephalitis. These yeasts have been classified into five serotypes. Serotypes A and D are assigned to C. neoformans var. grubii and C. neoformans var. neoformans , respectively, Serotype AD strains are hybrids and serotype B and C strains are considered to belong to the related but distinct species C. gattii . Previous studies have identified 'serotype-associated' alleles of several genes in the Cryptococcus species complex. We developed a loop-mediated isothermal DNA amplification method using CAP59 allele-specific primers to identify the serotypes A, D and B/C of the Cryptococcus species complex.

The diagnosis of invasive aspergillosis (IA) in patients with hematologic disorders is not straightforward; lack of sensitive and specific noninvasive diagnostic tests remains a major obstacle for establishing a precise diagnosis. In a series of 362 consecutive high-risk treatment episodes that were stratified according to the probability of IA based on recently accepted case definition sets, the potential for diagnosis of serial screening for circulating galactomannan (GM), a major aspergillar cell wall constituent was validated. After incorporating postmortem findings to allow a more accurate final analysis, this approach proved to have a sensitivity of 89.7% and a specificity of 98.1%. The positive and negative predictive values equaled 87.5% and 98.4%, respectively. False-positive reactions occurred at a rate of 14%, although this figure might be overestimated due to diagnostic uncertainty. More or less stringent criteria of estimation could highly influence sensitivity, which ranged from 100% to 42%; the impact on other test statistics was far less dramatic. All proven cases of IA, including 23 cases confirmed after autopsy only, had been detected before death, although serial sampling appeared to be necessary to maximize detection. The excellent sensitivity and negative predictive value makes this approach suitable for clinical decision making. Unfortunately, given the species-specificity of the assay, some emerging non-Aspergillus mycoses were not detected. In conclusion, serial screening for GM, complemented by appropriate imaging techniques, is a sensitive and noninvasive tool for the early diagnosis of IA in high-risk adult hematology patients.

(1-->3)-beta-D-Glucan is one of the major structural components of fungi, and it seems that it can be detected by the fractionated (1-->3)-beta-D-glucan-sensitive component from a Limulus lysate, factor G. We evaluated the concentration of (1-->3)-beta-D-glucan by using factor G and other fungal antigens in 24 patients with clinical evidence of mycosis and 36 healthy subjects. The mean concentration of (1-->3)-beta-D-glucan in the plasma of the healthy subjects was found to be 2.7 +/- 1.9 pg/ml (range, < 6.9 pg/ml), and it was found to be substantially higher in all 11 patients with candidemia (mean, 2,207.4 pg/ml; range, 325.4 to 8,449.0 pg/ml). Eight of those 11 patients with candidemia (73%) were positive for the Cand-Tec heat-labile candida antigen and only 3 patients (27%) were positive for mannan antigen. Three patients with invasive pulmonary aspergillosis were positive for galactomannan and had, in addition, high concentrations of (1-->3)-beta-D-glucan (mean, 323.3 pg/ml; range, 27.0 to 894.0 pg/ml). All 10 patients with cryptococcosis (including 2 patients with probable cryptococcosis) were positive for cryptococcal antigen by the Eiken latex test; however, (1-->3)-beta-D-glucan levels were not elevated in these patients (mean, 7.0 pg/ml; range, < 16.5 pg/ml). Our results indicated that (1-->3)-beta-D-glucan levels are elevated in patients with candidiasis and aspergillosis but not in those with cryptococcosis.

Candida auris, an emerging multi-drug resistant yeast associated with high mortality, has been increasingly reported from outside of the US to cause outbreaks in the hospital settings (1).….

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.

Expert Rev. Proteomics 10(2), 151-164 (2013) MALDI-TOF mass spectrometry (MS) is becoming essential in most clinical microbiology laboratories throughout the world. Its successful use is mainly attributable to the low operational costs, the universality and flexibility of detection, as well as the specificity and speed of analysis. Based on characteristic protein spectra obtained from intact cells - by means of simple, rapid and reproducible preanalytical and analytical protocols - MALDI-TOF MS allows a highly discriminatory identification of yeasts and filamentous fungi starting from colonies. Whenever used early, direct identification of yeasts from positive blood cultures has the potential to greatly shorten turnaround times and to improve laboratory diagnosis of fungemia. More recently, but still at an infancy stage, MALDI-TOF MS is used to perform strain typing and to determine antifungal drug susceptibility. In this article, the authors discuss how the MALDI-TOF MS technology is destined to become a powerful tool for routine mycological diagnostics.

Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory.

A trunk of human cytidylate-phosphate-deoxyguanylate-binding protein/CXXC finger protein 1 (CFP1), immobilized onto an aminohexyl-Sepharose column, can be used as a preanalytical tool for the selective enrichment of bacterial DNA from mixed solutions with high amounts of human background DNA for nucleic acid amplification technique-based detection of pathogens. The transcriptional activator protein exhibits a high affinity for nonmethylated CpG dinucleotide motifs, which are differentially distributed in prokaryotic and higher eukaryotic genomes. The feasibility of the affinity chromatography (AC) step was tested with DNA from severely septic patients. AC using 16S rRNA gene primers substantially increased PCR sensitivity. Approximately 90% of eukaryotic DNA was removed, which significantly increased the signal-to-noise ratio. Threshold cycle values revealed that sensitivity was elevated at least 10-fold. The change in the ratio of bacterial DNA to human DNA increased from 26% to 74% the likelihood of culture-independent PCR-based identification of bacterial presence. Compared to the results seen with blood culture (which is the clinical gold standard for systemic infections, exhibiting 28% positives), the combination of AC and PCR achieves a significant increase in sensitivity and contributes to shortening the time to results for the initiation of guided antibiotic therapy.

Microfluidics, a technology characterized by the engineered manipulation of fluids at the submillimetre scale, has shown considerable promise for improving diagnostics and biology research. Certain properties of microfluidic technologies, such as rapid sample processing and the precise control of fluids in an assay, have made them attractive candidates to replace traditional experimental approaches. Here we analyse the progress made by lab-on-a-chip microtechnologies in recent years, and discuss the clinical and research areas in which they have made the greatest impact. We also suggest directions that biologists, engineers and clinicians can take to help this technology live up to its potential.

Invasive candidiasis remains one of the most serious community and healthcare-acquired infections worldwide. Conventional Candida detection methods based on blood and plate culture are time-consuming and require at least 20900094 days to identify various Candida species. Despite considerable advances for candidiasis detection, the development of simple, compact and portable point-of-care diagnostics for rapid and precise testing that automatically performs cell lysis, nucleic acid extraction, purification and detection still remains a challenge. Here, we systematically review most prominent conventional and nonconventional techniques for the detection of various Candida species, including Candida staining, blood culture, serological testing and nucleic acid-based analysis. We also discuss the most advanced lab on a chip devices for candida detection.

A single strain of a novel ascomycetous yeast species belonging to the genus Candida was isolated from the external ear canal of an inpatient in a Japanese hospital. Analyses of the 26S rDNA D1/D2 domain, nuclear ribosomal DNA ITS region sequences, and chemotaxonomic studies indicated that this strain represents a new species with a close phylogenetic relationship to Candida ruelliae and Candida haemulonii in the Metschnikowiaceae clade. This strain grew well at 40 掳C, but showed slow and weak growth at 42 C. The taxonomic description of Candida auris sp. nov. is proposed (type strain JCM15448 T = CBS10913 T = DSM21092 T ).

Several fungal diseases have become serious threats to human health and life, especially upon the advent of human immunodeficiency virus/AIDS epidemics and of other typical immunosuppressive conditions of modern life. Accordingly, the burden posed by these diseases and, concurrently, by intensive therapeutic regimens against these diseases has increased worldwide. Existing and available rapid tests for point-of-care diagnosis of important fungal diseases could enable the limitations of current laboratory methods for detection and identification of medically important fungi to be surpassed, both in low-income countries and for first-line diagnosis (screening) in richer countries. As with conventional diagnostic methods and devices, former immunodiagnostics have been challenged by molecular biology-based platforms, as a way to enhance the sensitivity and shorten the assay time, thus enabling early and more accurate diagnosis. Most of these tests have been developed in-house, without adequate validation and standardization. Another challenge has been the DNA extraction step, which is especially critical when dealing with fungi. In this paper, we have identified three major research trends in this field: (1) the application of newer biorecognition techniques, often applied in analytical chemistry; (2) the development of new materials with improved physico-chemical properties; and (3) novel bioanalytical platforms, allowing fully automated testing. Keeping up to date with the fast technological advances registered in this field, primarily at the proof-of-concept level, is essential for wise assessment of those that are likely to be more cost effective and, as already observed for bacterial and viral pathogens, may provide leverage to the current tepid developmental status of novel and improved diagnostics for medical mycology.

Abstract Rhodotorula spp. are emergent opportunistic pathogens, particularly in immunocompromised individuals. They have been associated with endocarditis, peritonitis, meningitis endophthalmitis and catheter-associated fungemia. The aim of this study was to review all cases of central venous catheter-related fungemia due to Rhodotorula spp. reported in the literature in order to determine the best management of this uncommon infection. All patients but one in the 88 cases examined had some form of underlying disease including sixty-nine (78.4%) who had cancer. Rhodotorula mucilaginosa was the species most frequently recovered (75%), followed by Rhodotorula glutinis (6%). Amphotericin B deoxycholate was the most common antifungal agent used as treatment and the overall mortality was 9.1% in this review. This fungemia is a rare disease which can be found in immunocompromised and in the intensive care patients. The use of specific antifungal therapy may be associated with an increase in the survival. It should be noted that Rhodotorula spp. is resistant to fluconazole.

Abstract Loop-mediated isothermal amplification (LAMP) is a novel, rapid nucleic acid amplification method with high specificity and sensitivity under isothermal conditions. In this study a LAMP assay for diagnosing Pneumocystis pneumonia (PCP) was developed. Oligonucleotide primers specific for Pneumocystis species were designed corresponding to 18S rRNA gene sequences. The assay, performed for 30 min at 61 degrees C, was capable of detecting 50 copies per tube (2 x 10(3) copies ml(-1)) in 30 min and did not show cross-reactivity to other species of fungi, including the genera Candida, Aspergillus and Cryptococcus. A total of 21 of 24 clinical specimens (sputum and bronchoalveolar lavage fluid) from patients with suspected PCP tested positive using the LAMP assay by real-time fluorescence detection. The results of the LAMP reaction were also observed by real-time turbidity detection and end-point visual turbidity or fluorescence detection. With real-time fluorescence detection, melting curves of the products were effective at distinguishing specific amplification from non-specific amplification or self-amplification. Visual detection was also possible as a rapid and easy assay using only a heat block and a black light.