Fungal Polymerase Chain Reaction Research Articles

EQUAL Aspergillosis Score 2018: An ECMM score derived from current guidelines to measure QUALity of the clinical management of invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis is a serious threat to immunocompromised and critical care patients. Recent detailed guidelines and treatment algorithms lead microbiologists and clinicians in diagnosis and treatment of invasive aspergillosis. Currently, there is no tool available that allows to measure guideline adherence. To develop such a tool, we reviewed current guidelines provided by five scientific societies (European Society for Clinical Microbiology and Infectious Diseases, European Confederation of Medical Mycology, European Respiratory Society, Infectious Diseases Society of America (IDSA), and Infectious Diseases Working Party of the German Society for Hematology and Medical Oncology) and selected the strongest recommendations for management as key components for our scoring tool. We integrated diagnostic measures (chest computed tomography, bronchoalveolar lavage with galactomannan, fungal culture, fungal polymerase chain reaction analysis, species identification, susceptibility testing, histology with silver stain, Periodic acid-Schiff staining, and molecular diagnostics), treatment (antifungal choice and therapeutic drug monitoring), and follow-up computed tomography. The EQUAL Aspergillosis Score 2018 aggregates and weighs the components and provides a tool to support antifungal stewardship and to quantify guideline adherence.

Early Enteral Feeding of Daikenchuto Stimulates Early Bowel Movement With Increased Portal Venous Blood Flow After Living Donor Liver Transplantation

Daikenchuto (DKT), a Japanese Kampo medicine, had been reported to increase small intestinal blood flow after liver resection. The aim of this study was to evaluate the effects of early enteral feeding of DKT on portal venous flow and early bowel movement after living donor liver transplantation (LDLT) in an attempt to clarify whether these effects on bowel motility can prevent bacterial and/or fungal translocation. Materials and MethodsOur prospective study included the consecutive 16 LDLT recipients at Mie University Hospital between June 2006 and September 2009. Sixteen patients were divided into the 2 groups according to enteral feeding starting postoperative day (POD) 1: 8 patients in DKT (15 g/d) administration (DKT group, for 1 week) and 8 patients in tepid water administration (non-DKT group, for 1 week). Portal venous flow, portal venous pressure, presence of fungal infection (serum level of β-D-glucan and fungal polymerase chain reaction assay), time to first food intake, and time to first defecation were serially examined. ResultsPortal venous flow (mean [SD] velocity) was significantly increased in DKT group compared with non-DKT group: 47.5 (12.9) vs 31.8 (15.4) (P = .04) on POD 1, 46.8 (11.5) vs 28.8 (12.5) (P = .03) on POD 3, and 42.3 (17.2) vs 25.2 (9.0) (P = .05) on POD 5. However, mean (SD) portal venous pressures did not significantly change between the 2 groups. Between the 2 groups (DKT vs non-DKT), the day of first oral intake was not significantly different: 6.9 (2.5) vs 11.3 (8.7) (P = .061), but the mean (SD) day of first defecation was significantly shorter in the DKT group: 3.9 (1.1) vs 5.5 (2.6) (P = .02). Although fungal polymerase chain reaction assay was not significantly different between the 2 groups (4 vs 4 positive cases), the mean (SD) serum levels of β-D-glucan were significantly lower in the DKT group than in the non-DKT group: 9.0 (7.4) vs 18.4 (15.9) pg/mL (P = .04). ConclusionEarly enteral feeding of DKT after LDLT increased portal vein blood flow without increasing portal vein pressure and stimulated early bowel movement, which in turn might prevent fungal translocation.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

Role of Molecular Biomarkers in the Diagnosis of Invasive Fungal Diseases in Children.

Invasive fungal diseases are important clinical problems that are often complicated by severe illness and therefore the inability to use invasive measures to definitively diagnose the disease. Tests for a range of fungal biomarkers that do not require an invasive sample-collection procedure have been incorporated into adult clinical practice, but pediatric data and pediatric-specific recommendations for some of these diagnostic tools are lacking. In this review, we summarize the published literature and contemporary strategies for using the biomarkers galactomannan, (1→3)-β-d-glucan, Candida mannan antigen and anti-mannan antibody, and fungal polymerase chain reaction for diagnosing invasive fungal disease in children. Data on biomarker use in neonates and children with cancer, history of hematopoietic stem cell transplant, or primary immunodeficiency are included. Fungal biomarker tests performed on blood, other body fluids, or tissue specimens represent promising adjuncts to the diagnostic armamentarium in populations with a high prevalence of invasive fungal disease, but substantial gaps exist in the correct use and interpretation of these diagnostic tools in pediatric patients.

Infection control on ECMO

Background: Mortality of patients on extracorporeal membrane oxygenation (ECMO) remains high. Diagnosis of infection during extracorporeal life support (ECLS) is still challenging, and prevention strategies vary widely from center to center.1–3 These facts led us to analyze the occurrence rate, site, and organism in our ECLS patients in order to implement infection control measures to reduce the incidence of infections during ECLS.4 Our objective was to analyze our Extracorporeal Life Support Organization (ELSO) registry center data specifically focused on incidence of infection, typical microorganisms, time of manifestation, and site of cultures in the settings of tertiary pediatric cardiac intensive care unit mainly utilizing transthoracic cannulation and VA-ECMO, and compare with the ELSO database.1Methods: We conducted a retrospective study analyzing 25 neonatal and pediatric ECMO cases in relation to infection from January 2014 to December 2015, in comparison to the ELSO database age and modality specific data. We obtained ethical approval from our institution. We examined the prevalence of infection, the time of the first positive cultures, the site of the positive cultures, and the underlying microorganisms and compared with ELSO data whenever feasible. Results: There is no specific data on the incidence of infection in the ELSO database with open chest/transthoracic cannulation; our incidence was 0.44. The Candida species was the highest offending organism (24% vs. ELSO 12% concerning the entire ECMO population), followed by Klebsiella 20%, E. coli 16%, and Pseudomonas 12%. The first positive culture was taken on the 8th day of ECMO (median). By site, the highest prevalence of infection is as follows: ventilator-associated pneumonia (VAP), 41%, followed by bloodstream infection (BSI), 22%, and then catheter-associated urinary tract infection (CAUTI), 12%. Conclusions: The highest prevalence of Candida infections is most probably due to the combined antibiotic and steroid therapy for patients with capillary leak syndrome. This may prompt that routine antifungal prophylaxis can be added after 1 week of ECMO for this patient group. Alternatively, the early detection with fungal polymerase chain reaction (PCR) assay should be evaluated.5 The high occurrence of VAP may indicate the need of reinforcing enteral feeding, oral decontamination protocol along with VAP bundle, and investigation of alternative source of contamination. As Gram-negative Enterobacteriaceae and Pseudomonas were in second line as typical multidrug-resistant (MDR) organisms, those should be covered whenever a need for empiric antibiotic therapy arises.

A Pilot Study for the Evaluation of PCR as a Diagnostic Tool in Patients with Suspected Dermatophytoses.

Context:The conventional diagnostic tools for dermatophytoses suffer from several limitations including low sensitivity, specificity, and long turn-around-time.Aims:The present study was, therefore, performed to evaluate the performance of a polymerase chain reaction (PCR)-based method for the diagnosis of this condition.Settings and Design:The study was conducted in the Dermatology outpatient department of a tertiary care teaching hospital in central India over a period of 3 months from July to September 2015.Materials and Methods:Forty participants, including 25 cases and 15 controls, were recruited in this observational study. Direct microscopy and fungal culture were performed from skin scrapings and nail clippings collected from the participants. PCR was also performed to amplify the chitin synthase 1 and internal transcribed spacer 2 genes from DNA samples extracted from the same clinical materials, using the method reported by Brillowska-Dabrowska et al. The diagnostic performance of fungal culture and PCR was compared using OpenEpi software.Results:We observed a significant male predominance among patients with dermatophytoses. The sensitivity of fungal culture and dermatophyte PCR to diagnose dermatophytoses was 24% and 48%, respectively, whereas the specificity of the two assays was 100% and 93.3%, respectively. The likelihood ratio of a positive PCR assay was 7.2 and the negative likelihood ratio was 0.5. PCR assay also delivered a significant shortening of the time-to-diagnosis, with the mean turn-around-time being 8 hours and 14 days for PCR and culture, respectively.Conclusion:This study, thus, highlights the potential merits of the dermatophyte PCR assay in achieving a rapid diagnosis of dermatophytoses and underscores its utility as a complementary test to improve the sensitivity of the conventional diagnostic tools for this condition, as well as to reliably differentiate this condition from other similar skin conditions.

Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients.

BackgroundBroad-range fungal inter spacer region (ITS) polymerase chain reaction (PCR) has been evaluated for the detection and identification of fungi in clinical specimens from severely immunocompromised patients, but not in non-selected patients. Thus, the aim of this study was to compare the diagnostic performance of ITS PCR with that of fungal culture and to investigate its clinical impact on the diagnosis of fungal infections in non-immunocompromised patients. The corresponding patients’ data were retrieved by detailed medical chart reviews.ResultsResults from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture. The analytical sensitivity and specificity of ITS PCR considering culture as gold standard were 87.7 and 90.3 %, respectively, the positive and negative predictive value (PPV and NPV) were 76 and 95.5 %, respectively. Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture.ConclusionsOur data show that, in non-selected and non-immunocompromised patients, the performance of ITS PCR is similar to that of culture for detecting fungal infections, not the least because sensitivity of culture in patients under antifungal treatment is surprisingly high. Compared to culture, PCR has the advantage of a rapid time-to-result (approximately two working days), proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0752-1) contains supplementary material, which is available to authorized users.

Specificity and selectivity of arbuscular mycorrhizal fungal polymerase chain reaction primers in soil samples by clone library analyses

ABSTRACTAn open question with regard to the community ecology of arbuscular mycorrhizal fungi (AMF) concerns how to best amplify AMF in the soil, which contains a large proportion of DNA from AM extra-radical mycelium and spores. However, to date, a direct comparison of AMF primers for soil samples, which would systematically assess their amplification efficiency, is still missing. In our present study, we compared and characterized four widely used primer sets targeting AMF 18S rDNA or SSU-ITS-LSU rDNA from three soil samples as follows: (1) SSUmAf/LSUmAr + SSUmCf/LSUmBr, (2) GeoA2/Geo11 + NS31/AM1, (3) AML1/AML2 + NS31/AM1 and (4) AMV4.5NF/AMDGR. These primer sets were compared in terms of the proportion of Glomeromycota detected, AMF diversity and community composition. Our data revealed that the newly combined primer set 3 was the most suitable one for amplifying AMF from soil samples. It yielded the highest AMF alpha diversity, and was very specific to Glomeromycota. Primer set 2 was unable to amplify Claroideoglomus from soil 1, which was the dominant AMF clade as proved by other three primer sets. Primer set 4 demonstrated its instability among different soil samples, since the proportion of AMF in total sequences varied from 5% to 83%. Although primer set 1 showed the highest proportion of AMF (95–100%) in the soil samples, it captured the lowest AMF diversity, and the operational taxonomic units obtained by this primer set were only 36.4% of that by primer set 4. Taken together, our data suggested that AMF diversity in soil samples could be underestimated by primer set 1, 2 and 4. Our result confirmed the important role of the choice of AMF primers for analyzing AMF communities in soil and explored the most suitable one for amplifying AMF from soil samples.

Adult Living-Donor Liver Transplantation for a Recipient With a High Preoperative 1,3-Beta-d-Glucan Level and Positive Test Result for Aspergillus Antigen

The patient was a 45-year-old man with underlying alcoholic liver cirrhosis. Two years prior, he was repeatedly hospitalized for liver failure symptoms and requested a living-donor liver transplantation (LDLT) because of end-stage cirrhosis. A pretransplantation blood test revealed a high 1,3-beta-d-glucan (BDG) value of 102.0 pg/mL (reference value <20.0 pg/mL) and a high blood Aspergillus antigen (AsAg) value of 1.6 cutoff index (COI; reference value <0.5 COI). Contrast-enhanced thoracoabdominal-pelvic computed tomography (CT) and cranial magnetic resonance imaging revealed no fungal infection. However, latent fungal infection could not be ruled out, hence preoperative antifungal agent treatment was administered. BDG and AsAg levels showed a decreasing trend after treatment initiation. However, normalization did not occur; the BDG and AsAg levels were 25.8 pg/mL and 1.0 COI, respectively. Although the possibility of latent fungal infection was judged low, we prophylactically administered antifungal agents after LDLT. The BDG level consistently increased at 35–39 pg/mL until postoperative day 5 but subsequently normalized. The AsAg level was higher than the limit of detection at 5.0 COI on postoperative day 3 but normalized to 0.2 COI on postoperative day 5 and did not subsequently increase. The postoperative course was uneventful despite bacterial pneumonia and the patient was discharged on postoperative day 35. A histopathologic examination (Grocott methenamine silver staining) and a fungal polymerase chain reaction assay were performed for the resected liver, but the results of both were negative. At 9 postoperative months, the patient was making ambulatory follow-up visits. Currently, the BDG and AsAg values remain normal and clinical progress is favorable. We found no reports of LDLT for a recipient with a high preoperative BDG level and positive test result for AsAg. Thus, we report on such a case with a discussion of the literature on the causes of high preoperative BDG and AsAg values.

Survey of antifungal prophylaxis and fungal diagnostic tests employed in malignant haematology and haemopoietic stem cell transplantation (HSCT) in Australia and New Zealand.

This article reports the findings of a survey developed to assess the current use of antifungal prophylaxis among haematology and infectious disease clinicians across Australia and New Zealand, and their alignment with existing consensus guidelines for the use of antifungal agents in the haematology/oncology setting (published 2008). Surveyed clinicians largely followed the current recommendations for prophylaxis in the setting of induction chemotherapy for acute myeloid leukaemia, as well as autologous and low-risk allogeneic haemopoietic stem cell transplantation (HSCT). In keeping with guideline recommendations, posaconazole was the agent used by most centres for high-risk allogeneic HSCT. However, its routine continuation for 75-100 days post-transplantation without de-escalation suggested use beyond those indications described in the 2008 guidelines, namely pre-engraftment neutropenia and graft-versus-host disease. Variations in practice were observed in other settings, such as acute lymphoblastic leukaemia and myelodysplastic syndrome, reflecting the general lack of evidence for antifungal prophylaxis in these patient populations and changing perceptions of risk. With regard to the availability of testing in cases of suspected breakthrough IFD, 40% of centres did not have access to investigative bronchoscopy within 48 h of referral, and results of Aspergillus galactomannan (GM), fungal polymerase chain reaction and therapeutic drug monitoring (TDM) were not available within 48 h in 83%, 90% and 85% of centres respectively. The survey's findings will influence the recommendations provided in the updated 2014 consensus guidelines for the use of antifungal agents in the haematology/oncology setting.

Heart Valve Culture and Sequencing to Identify the Infective Endocarditis Pathogen in Surgically Treated Patients

Testing excised valves in surgically treated infective endocarditis (IE) patients provides an opportunity to identify the microbial etiology of IE. Microbial sequencing (universal bacterial, mycobacterial, or fungal polymerase chain reaction followed by DNA sequencing) of valves can identify microorganisms accurately, but the value it adds beyond information provided by blood and valve cultures has not been adequately explored. Three hundred fifty-six patients who underwent surgery for active IE from January 1, 2010, to January 1, 2013, were identified from our cardiovascular information registry and outpatient parenteral antibiotic therapy registry. Their records were reviewed to identify 174 patients whose valves were sent for sequencing. The microbial etiology of IE was defined using comprehensive clinical, pathologic, and microbiological criteria. Blood culture, valve culture, and valve sequencing were examined to determine how frequently they identified the definitive cause of IE. Of the 174 patients, 162 (93%) had acute inflammation on histopathologic examination of their valves. Valve sequencing was significantly more sensitive than valve culture in identifying the causative pathogen (90% versus 31%, p < 0.001), and yielded fewer false positive results (3% versus 33%, p <0.001). The pathogen would not have been identified in 25 patients (15%) had it not been for valve sequencing. All the value provided by sequencing was attributable to bacterial DNA sequencing; mycobacterial and fungal sequencing provided no additional information beyond that provided by blood culture, histopathology, and valve culture. Valve sequencing, not valve culture, should be considered the primary test for identifying bacteria in excised cardiac valves.

Real-time PCR assay to detect Verticillium albo-atrum and V. dahliae in hops: development and comparison with a standard PCR method

Verticillium wilt, caused by Verticillium albo-atrum and V. dahliae, is a devastating disease that causes considerable economic crop losses in hop fields. The fungus can survive in soil for several years by producing resting structures, and due to the lack of effective fungicides, the disease is spreading. Thus, a fast and sensitive detection system is urgently needed. In this study we report a novel routine detection method for the identification of V. albo-atrum and V. dahliae on a molecular basis. The standard polymerase chain reaction (PCR) assay includes isolation of the fungus from affected tissue by following DNA extraction from fungal cultures and PCR identification by specific primers. In order to improve this detection assay, DNA was isolated directly from the hop bine using a commercially available DNA isolation kit. A multiplex real-time PCR assay for the simultaneous detection of V. albo-atrum and V. dahliae was established using a specific primer pairs/TaqMan probes combination. Ninety-six plants were collected from different culti-vars and locations in Germany and tested by standard and real-time PCR assay. The new detection system is more accurate, sensitive and time-saving than the standard PCR method and is suitable for routine use. The test provides a valuable tool for rapid and sensitive detection of V. albo-atrum and V. dahliae in plants and gives farmers and plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against Verticillium.

Study of the early molecular diagnosis methods in invasive fungal infection of clinical humoral specimens

Objective The aim of this study was to investigate the diagnostic impact of polymerase chain reaction (PCR) assays for fungal pathogens in fluid samples,and to evaluate the feasibility of fast PCR diagnostic method for the invasive fungal infection.Methods The sterility body fluid samples from 60 cases hospitalized immunocompromised patients with clinical underlying diseases and suspect of invasive infections with fungi between January 2008 and December 2011 were processed for microscopy and cultures.Applying fungal ITS4 and ITS86 universal primers to amplify pathogenic fungi genes from the sterility body fluid with method of rapid PCR.The results were compared between the standard and PCR methods.Results Humoral direct clinical specimens by PCR amplification of DNA fragments with the scan results were similar.Positive rate of PCR test with clinical body fluid samples and the traditional fungal cultivation was similar.There was no significant statistical difference [38.3％ (23/60) vs 33.3％ (20/60),P ＞ 0.05].Conclusions PCR test is feasibility with clinical fungal diagnosis from directly humoral specimens.To amplify the clinical sterility body fluid samples with ITS fungal universal primers and PCR method might provide an accurate and rapid approach to detect the pathogenic fungi.Its methods on early diagnosis and prognosis of invasive fungal infections are of guiding significance. Key words: SUPERFICIALITY & ORIGIN; Body fluids/microbiology; Mycoses/diagnosis; Polymerase chain reaction; Early diagnosis

Fungal polymerase chain reaction testing in equine ulcerative keratitis

To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. The expected fungus was identified by nested PCR and qPCR in all control fungal cultures. In all fungal culture-positive affected eyes (10/43), one or more fungi were identified by nested PCR and 4/10 were positive by qPCR. In 6/10 animals, the same fungus was identified by nested PCR and culture. Of these 6, only three were positive by qPCR. Fungal agents were identified by morphology in 8/10 horses. Diagnosis of fungal keratitis was reserved for only those cases in which the same fungus could be identified by PCR, culture, and morphology (5 horses). In 33/43 culture-negative affected eyes and in 6/9 unaffected eyes, one or more fungi were identified by nested PCR in 26 samples and by qPCR in 2 samples. Apart from Aspergillus spp, similar fungi were identified in affected and control eyes. Most eyes harbored mixed bacterial and fungal agents. Nested PCR results confirmed all cytologically positive cases of fungal keratitis. Nested PCR identified a greater spectrum of agents than either culture or qPCR.

Two Cases of Cokeromyces recurvatus in Liquid-Based Papanicolaou Tests and a Review of the Literature

We present 2 cases of Cokeromyces recurvatus in routine, liquid-based Papanicolaou tests (ThinPrep). Patient 1 is a healthy, asymptomatic, 26-year-old woman with no pertinent past medical history. Patient 2 is a healthy, asymptomatic, 47-year-old woman with no pertinent past medical history. The Papanicolaou tests from both patients showed many fungal-like elements as globose, yeastlike forms measuring 10 to 30µm in diameter with multiple, narrowly attached apparent "daughter" buds. This morphology was consistent with Paracoccidioides brasiliensis. However, broad-range fungal polymerase chain reaction and deoxyribonucleic acid sequence analysis performed with GenBank Basic Local Alignment Search Tool showed an exact match for C recurvatus. Our cases highlight the importance of molecular techniques to prevent misdiagnosis of C recurvatus as P brasiliensis, based on morphology alone. There have been 8 previously published cases of C recurvatus infection in humans, 3 of which were reported in the female genital tract.

Invasive fungal diseases in children with hematological malignancies and stem cell transplant recipients

Invasive Fungal Disease (IFD) remains a major cause of morbidity and mortality in patients with hematological malignancies or undergoing stem cell transplantation. New surrogate markers of IFD - galactomannan antigen, β -D-glucan, fungal polymerase chain reaction… - and the availability of modern antifungal drugs have changed the management of this serious complication, but both have limitations in children. There are still some unclear issues, particularly in pediatric patients, regardless of the medical and economic possibilities of hospitals: IFD remains difficult to diagnose in a timely way, stem cell transplant recipients have different risk of IFD, antifungal drugs are not free from adverse effects and prospective studies in children are scarce. In this sense, diagnosis criteria have to be assessed , risk stratification should be redefined and the different types of prophylaxis/treatment -prophylaxis, preemptive, empiric and targeted treatment- must be used correctly. We consider low risk for IFD children undergoing autologous stem cell transplantation and children with acute lymphoblastic leukemia, both should receive fluconazole as primary prophylaxis and empiric therapy with an agent with activity against mold. In those undergoing allogeneic stem cell transplantation or chemotherapy for acute myeloid leukemia, prophylaxis should be performed with a mold active agent -triazoles priority-. We rarely use preemptive therapy because of a high false positive rate of galactomannan. In proven and probable IFD we use targeted therapy, selecting a drug based on the type of infection, sensitivity to the drug in our hospital and prior antifungal prophylaxis/treatment. In selected patients, antifungal combination therapy should be a valid option. Despite great advances, there are still thought provoking questions on the diagnosis and management of IFD in children with hematological diseases.

Application of an automated ribosomal intergenic spacer analysis database for identification of cultured Antarctic fungi

AbstractWe utilized an automated ribosomal intergenic spacer analysis (ARISA) method as a more rapid alternative to classical morphological/nutritional identification and a less expensive alternative to sequencing for identification and grouping of isolates in culture-based fungal abundance studies. This method is well suited for the study of culturable Antarctic soil fungal communities where both abundance and diversity are relatively low. We optimized template concentration and verified the effect of primer selection from eight commonly used fungal polymerase chain reaction primers on ARISA chromatographs for 46 fungal species commonly isolated from south Victoria Land. A database of Antarctic fungal electropherograms was produced containing each of the species and was used as the first step in a tiered system for species identification. In addition, isolates containing more than one species were identified, allowing isolates not in the database to be sequenced for further analysis. This method unambiguously identified 78% of the fungal taxa in this study and we were able to rapidly determine which isolates should be subjected to further analysis by DNA sequencing. Using this approach, the cost of analysis for abundance studies can be greatly reduced compared to DNA sequencing of each isolate.

Application of denaturing gradient gel electrophoresis to microbial diversity analysis in Chinese Douchi

Douchi is a traditional Chinese soybean food which has been consumed for thousands years as an important protein source and flavouring ingredient. Studies have rarely been carried out to investigate its microbial composition and these are urgently required for the commercial labels and safety considerations. Microbial counts were statistically significant different among Douchi samples. Although the maximum diversity indexes of bacterial, bacillus and fungal polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) patterns were only 79%, 70% and 64%, some microorganisms, e.g. Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas sp., Saccharomyces cerevisiae and Pichia farinose, were found to share dominant positions in most Douchi samples. In addition, some pathogens, e.g. Staphylococcus saprophyticus, Pantoea sp., Staphylococcus sciuri, Enterobacter sp. and Staphylococcus sp., were also identified. The PCR-DGGE technique was used for the first time as an effective method to assess the microbial communities in different Chinese Douchi samples. This information may be useful in improving the product quality, reformatting production methods, extending shelf life and scaling up the fermentation process.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 106–10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Assessing Responses to Treatment of Opportunistic Mycoses and Salvage Strategies

Invasive fungal disease (IFD) in immunocompromised patients remains a major cause of morbidity and mortality and there is a pressing need for studies of novel antifungal agents and strategies to improve outcomes. Trial design details often determine not only the appropriate interpretation of the results, but also their translation into clinical practice. However, the conduct of IFD clinical trials remains challenging due to the rarity of IFD, heterogeneity of underlying diseases, and the lack of clear and standardized response criteria. Response assessments are influenced by host, underlying disease and treatment factors as well as eligibility criteria. In addition, the criteria used to assess response, when response is assessed and the type of antifungal therapy under study can impact response evaluations. This article will discuss recent trials of primary, salvage, empiric, and prophylactic antifungal therapy with specific attention to the design of these antifungal therapy trials and how their designs influence their interpretation. The potential role of surrogate markers, such as the galactomannan index, fungal deoxyribonucleic acid polymerase chain reaction assay, and (18F) fluorodeoxyglucose positron emission tomography scans in establishing the early diagnosis of IFD, as well as enhancing the ability to assess outcomes to antifungal therapy, and thereby the optimal duration of antifungal therapy, will be discussed.