Fungal Deoxyribonucleic Acid Research Articles

Fast and reliable method to estimate global DNA methylation in plants and fungi with high-pressure liquid chromatography (HPLC)-ultraviolet detection and even more sensitive one with HPLC-mass spectrometry.

DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2µM for HPLC-UV and 0.02µM for HPLC-MS of methylated cytosine.

Molecular Identification of Fungi Causing Tissue Mycoses From Formalin Fixed Paraffin Embedded (FFPE) Archive Specimens

Introduction: Isolation of fungi from tissue specimens using conventional methods is time consuming. However, in some cases, the histopathological examination (HPE) of tissue alone is unable to provide a definite identity of the fungus. Alternatively, a non-culture method, such as polymerase chain reaction (PCR) detecting the internal transcribed spacer (ITS) rRNA genes of the fungi, is a promising diagnostic tool for rapid and accurate diagnosis of tissue mycoses. Methods: This work investigated the utility of panfungal PCR in identifying agents of tissue mycoses in 87 FFPE archive specimens. Deoxyribonucleic acid (DNA) extraction was performed on FFPE specimens by using QIAamp DNA FFPE Tissue Kit. The ITS2 region was amplified using ITS3/ITS4 primers. The PCR products were sequenced using the same primers and compared to the NCBI nucleotide database for species identification. Results: Fungal DNA was successfully amplified in 52 (59.8%) specimens, from which only 23 (44.0%) fungi were consistent with clinical/HPE findings. The identified fungi were Aspergillus spp., Candida spp., Penicillium spp., Cryptococcus neoformans, Talaromyces marneffei, and Rhizopus oryzae. A few rare fungi were also identified, such as Diaporthe longicolla and fungus-like oomycete such as Pythium insidiosum that are commonly associated with plant pathogens. Conclusion: Although PCR was able to offer accurate genus/species identification, utilising this method on paraffinised tissue specimens must be evaluated by considering many factors that will reduce its sensitivity and specificity. Therefore, it is important to correlate the PCR results with clinical and HPE findings to obtain a correct diagnosis and adequate treatment for tissue mycoses.

Diabetic ketoacidosis and coronavirus disease 2019-associated mucormycosis: a case report

BackgroundMucormycosis is a rare, life-threatening fungal infection that affects immunocompromised hosts. Diabetes mellitus is a common predisposing condition and most often presents with rhino-orbital-cerebral infection. Association with coronavirus disease 2019 infection was revealed following a resurgence in cases of mucormycosis during the second wave of the pandemic wherein poorly controlled diabetes mellitus was the most significant risk factor in the affected population. Rhino-orbital-cerebral mucormycosis has a high mortality rate, and cerebral involvement is a poor prognostic factor. Herein, we report a case of newly diagnosed diabetes mellitus with concurrent coronavirus disease 2019 infection complicated by diabetic ketoacidosis and rhinocerebral mucormycosis at presentation, describe the diagnostic and therapeutic challenges, and discuss the interventions that ultimately resulted in a favorable clinical response.Case presentationWe describe the case of a previously healthy 13-year-old African American female patient with newly diagnosed diabetes mellitus and concurrent severe acute respiratory syndrome coronavirus 2 infection whose disease course was complicated by rhinocerebral mucormycosis. She presented with fever, altered mental status, and Kussmaul respirations and was diagnosed with diabetic ketoacidosis with concern for cerebral edema. Concern for infectious cerebritis arose due to recurring fevers and persistently altered mental status despite correction of her metabolic derangements. This raised concern for infectious cerebritis and prompted evaluation with serial head imaging, lumbar puncture, and initiation of broad empiric antimicrobial regimen. Head imaging revealed an evolving cerebral abscess, and fungal deoxyribonucleic acid was identified on blood metagenomics testing, which ultimately confirmed the diagnosis of rhinocerebral mucormycosis. Treatment was challenging as she required surgical debridement of the frontal lobe and aggressive antifungal therapy complicated by electrolyte derangements and electrocardiogram changes that necessitated modification of the antimicrobial regimen. Despite these challenges and high mortality rate, the patient was discharged from the hospital in stable condition to inpatient rehabilitation service for reconditioning after prolonged hospitalization.ConclusionRhinocerebral mucormycosis mortality is associated with delays in therapeutic interventions, thus a high index of suspicion and early recognition were essential for timely initiation of antifungal therapy and surgical debridement.

Swiss needle cast tolerance in British Columbia’s coastal Douglas-fir breeding population

Abstract Substantial growth losses in Douglas-fir can be the result of Swiss needle cast, a foliar disease caused by the fungus Nothophaeocryptopus gaeumannii. Although the disease is native to western North America, it is expected to become increasingly problematic in regions where climates become warmer in winter and wetter in spring, such as in coastal British Columbia (BC), Canada. Previous research suggests that tolerance to this disease is under partial genetic control. We therefore aim to screen for tolerance and resistance to the disease in the tree breeding population of coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) in BC. We evaluated if early screening for resistance or tolerance to this disease is possible. We worked with 32 families grown for 18-years on two full-sibling genetic field trial sites representing different climates. We assessed >900 trees for disease signs and symptoms from 2017 to 2019. Needle retention was assessed in the field. In the laboratory, the proportion of stomata occluded with pseudothecia was measured, deoxyribonucleic acid (DNA) was extracted, and the proportion of fungal DNA in the needles was assessed using quantitative polymerase chain reaction. Strong differences were observed among families for needle retention, stomatal occlusion and fungal load. The milder and wetter site closer to the coast was generally more severely affected. Families showed rank changes between the two sites for all response variables. Higher needle retention was correlated with increased tree volume. No correlation was found between the proportion of stomata occluded with pseudothecia, fungal DNA load and needle retention. These results indicate that a more complex pathology is involved in causing needle loss. We conclude that screening for Swiss needle cast tolerance in the coastal BC Douglas-fir breeding population is possible if needle retention can be assessed and that area-specific deployment strategies may be needed given family rank changes in different environments.

Identification of Colletotrichum spp. associated with fruit rot of Capsicum annuum in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.

Microscopic and molecular detection of Leptosphaeria maculans and L. biglobosa ascospore content in air samples

Airborne sexual spores of Leptosphaeria maculans and L. biglobosa are primary inoculum of fungi causing phoma stem canker of oilseed rape. In this study ascospore release of these two species was compared between Lithuania (of autumn 2010 and 2011) and Poland (autumns of 2009 till 2012), using identical equipment and methods. Dynamics of L. maculans and L. biglobosa ascospore dispersal was investigated using volumetric samplers followed by microscopic and molecular methods of detection. In Lithuania, the earliest detection of Leptosphaeria ascospores, using microscopy was on 1 st September (2010) and on 2 nd September (2011), whereas in Poland the earliest detection was on 5 th September (2009 and 2010) and the latest on 11 th October (2011), which demonstrates that differences between the seasons may exceed a month (36 days). In the case of molecular detection performed for the Polish samples, the dates of the earliest and the latest release of the first ascospores ranged from 5 th September to 15 th October (40 days). The number of days with ascospores detected in the air samples ranged from 6 (2012) to 25 (2009). At earliest, the detection of the highest concentration of ascospores was on 6 th September (2010) and the latest – on 28 th October (2009). These results demonstrate big differences between the results of spore monitoring between the years and the importance of aerobiological studies to follow the development of the pathogen in natural infection. The fluctuations in quantities of spores and fungal deoxyribonucleic acid (DNA) strongly depended on the weather conditions, mainly rainfall, air temperature and relative humidity. Ascospore release was observed immediately after the rainfall events, but not later than two days after the rainfall. On days without rain, spores were present in the air samples when average relative air humidity exceeded 90% and day temperature was below 15°C. In 2009, molecular detection revealed the presence of both fungal species in the air samples, whereas in 2010–2012 only L. biglobosa DNA was found. Molecular detection with a quantitative real-time polymerase chain reaction (qPCR) was identified as a fast and accurate method of pathogen detection and identification from air samples. The highest coefficient of correlation between microscopic and molecular detection of Leptosphaeria species was 0.828 (2009) and the lowest was 0.483 (2011); in all years both techniques of detection were statistically correlated. We have demonstrated that the correlation coefficient highly depended on the number of ascospores in air samples. Based on the studies performed in Lithuania and Poland, we have shown that climatic conditions in the northern part of Central Europe are favourable for the spread of Leptosphaeria spp. by ascospores; however, the patterns of ascospore release greatly differ between the both countries.

Evaluation of Microbial Bacterial and Fungal Diversity in Cerebrospinal Fluid Shunt Infection

BackgroundCerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described.ObjectivesTo identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture.MethodsThis descriptive study included eight children ≤18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis.ResultsThe microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment.ConclusionsNumerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection.

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.

The fungal DNA examination is useful as a sensitive parameter for the initiation and the quit of antifungal therapy in immunocompromised pediatric patients after surgery.

The purpose of this study is whether the fungal deoxyribonucleic acid (DNA) examination is useful as a sensitive parameter for pediatric surgical patients with mycosis. The eleven episodes of five cases (4 cases; progressive liver disease after biliary atresia operation, 1 case; short bowel syndrome and long term total parenteral nutrition with megacystis microcolon intestinal hypoperistalsis syndrome) with mycosis were divided into two groups according to the difference of therapeutic protocols. The sensitivity of fungal DNA examination, serum Candida antigen level, plasma beta-D glucan level, and blood culture were evaluated at the onset of infection and at the quit of antifungal medication under the protocols respectively. The duration of medication and the medication free interval in two groups were compared. The 6 episodes (3 cases) were diagnosed and treated under the protocol not including fungal DNA examination, while the 5 episodes (2 cases) under the protocol including fungal DNA examination. The occurrence rate was not significant. The sensitivity of fungal DNA examination was complete, but others were not. Using the fungal DNA examination, the duration of medication became significantly short. We conclude that the fungal DNA examination could be a sensitive parameter not only to start but to quit antifungal medication in pediatric patients with mycosis.

Polymerase chain reaction used to detect candidemia in patients with candiduria

Objectives. Candiduria has been shown to be an early marker of disseminated fungal infection in critically ill patients who have undergone surgery. The management of candidemia and disseminated candidiasis depends on rapid and definitive identification of Candida. Routine or fungus-specific blood cultures are unreliable and require a large quantity of blood for incubation. We describe the importance of the polymerase chain reaction (PCR) procedure in the early detection of candidemia in critically ill patients who develop candiduria and the favorable outcome in treating these patients with systemic antifungal therapy. Methods. We compared the results of cultures and PCR to detect the presence of C. albicans in the blood of two critically ill patients with clinical candidiasis and candiduria. Results. PCR detected the presence of C. albicans deoxyribonucleic acid (DNA) in urine and blood specimens of both patients in spite of negative blood cultures and did not detect fungal DNA after systemic antifungal therapy. Conclusions. Candiduria manifests as an early sign of candidemia, and systemic antifungal therapy timed appropriately based on the clinical condition and onset of candiduria will improve outcome. Detection of fungal DNA in blood by PCR is of value in establishing the diagnosis. Additional studies with a larger sample size are required to evaluate the specificity and sensitivity of PCR as a routine diagnostic test for candidemia.