scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

scholarly journals New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

2016 ◽  
Vol 54 (12) ◽  
pp. 2910-2918 ◽  
Author(s):  
Clara Valero ◽  
Laura de la Cruz-Villar ◽  
Óscar Zaragoza ◽  
María José Buitrago
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Infections ◽  
Melting Curve ◽  
Fungal Species ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Pcr Assay ◽  
Suitable Alternative

The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these “probe-positive” results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.

Molecular Diagnosis of Invasive Fungal Infections: Species Identification and Quantitative Analysis by Broad-Spectrum PCR Assays.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5339-5339
Author(s):  
Lenka Baskova ◽  
Sandra Preuner ◽  
Thomas Lion
Keyword(s):  
Quantitative Analysis ◽  
Dna Sequences ◽  
Fungal Pathogens ◽  
Fungal Infections ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Invasive Fungal Infections ◽  
Fungal Genome ◽  
Quantitative Detection ◽  
Correct Identification

Abstract Invasive fungal infections (IFI) play an increasingly important role as life-threatening complications in immunocompromised patients. Early application of antimycotic agents is an essential prerequisite for successful therapy. However, standardized diagnostic techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically relevant fungi have been lacking. We have developed two different real-time PCR systems for quantitative analysis of pathogenic fungi. The Pan-AC assay* permits in a single reaction the detection of all important Aspergillus and Candida species, which are responsible for the great majority of IFI in immunosuppressed individuals. In view of the increasing incidence of invasive infections caused by hitherto uncommon fungal species, new diagnostic tests with very broad specificity are required. We have therefore developed an additional two-reaction Pan-fungus assay*, which facilitates quantitative detection of a wide spectrum of fungal species (n>50), including also the newly emerging pathogenic fungi. The assays display high sensitivity and show no cross-reactivity with non-fungal pathogens or human DNA sequences. We have established an additional rapid molecular assay based on PCR fragment length analysis of a variable region in the fungal genome permitting rapid identification of the fungal species present, in order to facilitate selection of the most appropriate antifungal treatment. Correct identification of specific fungal pathogens including different Aspergillus, Candida and Fusarium species detected by the above assays in patients with febrile neutropenia has been confirmed by sequence analysis. The new assays are readily applicable to routine clinical diagnosis and provide a rapid and economic approach to the screening and monitoring of invasive fungal infections.

scholarly journals Molecular and morphological approaches for species delimitation and hybridization investigations of two Cichla species

2017 ◽  
Vol 107 (0) ◽  
Author(s):  
Andrea A. F. Mourão ◽  
Diogo Freitas-Souza ◽  
Diogo T. Hashimoto ◽  
Daniela C. Ferreira ◽  
Fernanda D. do Prado ◽  
...  
Keyword(s):  
Species Delimitation ◽  
Parental Species ◽  
Future Research ◽  
Correct Identification ◽  
Pcr Multiplex ◽  
Pcr Rflp ◽  
Genetic Pattern ◽  
Morphometric Methods ◽  
Species Specific ◽  
Specific Nucleotide

ABSTRACT The hybridization is a widely-discussed issue in several studies with fish species. For some authors, hybridization may be related with diversification and speciation of several groups, or also with the extinction of populations or species. Difficulties to differentiate species and hybrids may be a problem to correctly apply a management of wild species, because hybrid lineages, especially the advanced ones, may resemble the parental species. The genus Cichla Bloch & Schneider, 1801 constitutes an interesting experimental model, considering that hybridization and taxonomic uncertainties hinder a correct identification. Considering these problems, in this study, we developed genetic methodologies and applied meristic and morphometric approaches in wild samples in order to identify species and for test a possible hybridization between Cichla kelberi Kullander & Ferreira, 2006 and Cichla piquiti Kullander & Ferreira, 2006. For this, C. kelberi, C. piquiti and potential hybrid ( carijó) individuals were collected in Paraná and Tietê rivers (SP, Brazil). For meristic and morphometric methods, the individuals were analyzed using the statistical software Pcord 5:31, while for molecular methods, primers for PCR-multiplex were designed and enzyme for PCR-RFLP were selected, under the species-specific nucleotide. All results indicated that the carijó is not an interspecific hybrid, because it presented identical genetic pattern and morphology closed to C. piquiti. Thus, we propose that carijó is a C. piquiti morphotype. In addition, this study promotes a new molecular tool that could be used in future research, monitoring and management programs of the genus Cichla.

scholarly journals Efficacy of the new β-D-glucan measurement kit for diagnosing invasive fungal infections, as compared with that of four conventional kits

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255172
Author(s):  
Yuuki Bamba ◽  
Kei Nagano ◽  
Hiroshi Moro ◽  
Hideyuki Ogata ◽  
Mariko Hakamata ◽  
...  
Keyword(s):  
Measurement Method ◽  
Diagnostic Performance ◽  
Fungal Infections ◽  
Pneumocystis Pneumonia ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Clinical Diagnoses ◽  
European Regulations ◽  
Pre Treatment ◽  
Positive Results

Background Each of the currently available (1→3)-β-D-glucan (BDG) measurement kits follows a different measurement method and cut-off value. Comparisons of diagnostic performance for invasive fungal infections (IFIs) are desirable. Additionally, ecological considerations are becoming increasingly important in the development of new measurement kits. Methods The plasma BDG levels in clinical samples were measured using the following currently available kits: the Fungitec G test MKII, the Fungitec G test ES, Fungitell, the β-Glucan test Wako, and the newly developed Wako kit (Wako-Eu). Wako-Eu uses a pre-treatment solution that conforms to European regulations for the registration, evaluation, authorisation, and restriction of chemicals. The values obtained for the samples using each kit were studied and compared. Results Of the 165 patients evaluated, 12 had IFIs, including pneumocystis pneumonia, aspergillosis, and candidiasis. BDG values obtained using the kits were moderately correlated with each other. Clinical diagnoses of the evaluated cases indicated that 21 false positives were diagnosed by at least one kit. The sensitivity of the Fungitell kit was relatively low, but those of the other four were over 90%. The specificity was above 90% for all kits. For positive predictive value, the Wako and the Wako-Eu methods were superior to the others owing to fewer false positive results. Conclusions The newly developed Wako-Eu method, which considers ecological concerns, shows diagnostic performance equivalent to that of its predecessor. To improve the diagnostic accuracy of IFIs, it is necessary to interpret the results carefully, giving due consideration to the characteristics of each measurement kit.

scholarly journals Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Dequan Zhang ◽  
Xuelian Lu ◽  
Yong Liao ◽  
Zhikuan Xia ◽  
Zhuoying Peng ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Intergenic Spacer ◽  
Glass Beads ◽  
Clinical Samples ◽  
Trichosporon Asahii ◽  
Colony Pcr ◽  
Extraction And Purification ◽  
Simple Detection ◽  
Mock Infection ◽  
Species Specific

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

scholarly journals Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease

2018 ◽  
Author(s):  
David K. Hong ◽  
Timothy A. Blauwkamp ◽  
Mickey Kertesz ◽  
Sivan Bercovici ◽  
Cynthia Truong ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Invasive Fungal Disease ◽  
Invasive Fungal Infections ◽  
High Morbidity ◽  
Non Invasive ◽  
Treatment Information ◽  
Life Threatening ◽  
Pathogen Dna ◽  
Microbiologic Diagnosis ◽  
Free Plasma

AbstractDiagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The non-invasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.

scholarly journals An alarming rise of non-albicans Candida species and uncommon yeasts in the clinical samples; a combination of various molecular techniques for identification of etiologic agents

2019 ◽  
Author(s):  
Mohireh Taei ◽  
Mostafa Chadeganipour ◽  
Rasoul Mohammadi
Keyword(s):  
Molecular Identification ◽  
Candida Species ◽  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Etiologic Agent ◽  
Molecular Techniques ◽  
University Hospital ◽  
Medical Mycology ◽  
Clinical Samples ◽  
Pcr Multiplex

Abstract Objective: Yeasts are opportunistic microorganisms can cause human fungal infection among immunocompromised patients. This study aimed to identify Candida species and uncommon yeasts obtained from clinical specimens in Kashani university hospital and Shefa Lab as a referral medical mycology laboratory, in Isfahan, Iran, by combination of various molecular techniques. Results: A total of 202 yeast strains were isolated from 341 clinical samples between February 2017 to May 2019. All clinical isolates were identified using phenotypic and molecular tests. PCR-RFLP, duplex-PCR, multiplex-PCR, and PCR-sequencing were applied for molecular identification of yeasts. The most clinical samples were obtained from urine (66.8%), nail (9.4%), bronchoalveolar lavage (5.9%), sore (4.4%), and blood (3.9%). One hundred and twenty-one Candida species were identified as non- albicans against 76 Candida albicans. Trichosporon asahii, and Pichia terricola were uncommon non- Candida yeasts isolated from urine samples. For the first time, we isolated P. terricola as etiologic agent of urinary tract infection in a pregnant female. Since non- albicans Candida species and non- Candida yeasts have various virulence factors and antifungal susceptibility profile, precise molecular identification can help us to reach to the advantageous strategies for treatment of these fungal infections.

Invasive Fungal Infection in Febrile Patients with Hematologic Malignancies Undergoing Chemotherapy in Iran

2019 ◽  
Vol 19 (3) ◽  
pp. 302-307 ◽  
Author(s):  
Saba Sheikhbahaei ◽  
Alireza Mohammadi ◽  
Roya Sherkat ◽  
Alireza Emami Naeini ◽  
Majid Yaran ◽  
...  
Keyword(s):  
Blood Culture ◽  
Antifungal Therapy ◽  
Fungal Infections ◽  
Hematologic Malignancy ◽  
Hematologic Malignancies ◽  
Fungal Species ◽  
Antifungal Prophylaxis ◽  
Invasive Fungal Infections ◽  
Severe Neutropenia ◽  
Candida Spp

Background: Patients with hematological malignancies undergoing cytotoxic chemotherapy are susceptible to develop invasive fungal infections particularly Aspergillus and Candida spp. Early detection of these infections is required to start immediate antifungal therapy and increase the survival of these patients. Method: Our study included consecutive patients of any age with hematologic malignancies who were hospitalized to receive chemotherapy and suffer from persistent fever (rectal temperature >38.5°C) for more than 5 days despite receiving broad-spectrum antibiotics. A whole blood sample was taken and sent for blood culture. PCR was also conducted for Aspergillus and Candida species. Results: One hundred and two patients were investigated according to the inclusion criteria. The most common hematologic malignancy was AML affecting 38 patients (37.2%). Six patients were diagnosed with invasive fungal infections (A. fumigatus n=3, C. albicans n=2, A. flavus n=1) by PCR (5.8%) while blood culture showed fungus only in 1 patient. Three more cases were known as probable IFI since they responded to antifungal therapy but the PCR result was negative for them. AML was the most prevalent malignancy in IFI patients (83.3%) and odds ratio for severing neutropenia was 21.5. Odds for each of the baseline characteristics of patients including gender, age>60, diabetes mellitus, previous IFI, history of using more than 3 antibiotics, antifungal prophylaxis, episodes of chemotherapy> 8 and chemotherapy regimen of daunarubicin+cytarabine were calculated. Conclusion: We found that multiplex real-time PCR assay is more accurate than blood culture in detecting fungal species and the results are prepared sooner. Among all factors, the only type of cancer (AML) and severe neutropenia, were found to be risk factors for the development of fungal infections in all hematologic cancer patients and previous IFI was a risk factor only AML patients.

scholarly journals PCR Detection of Mixed and Zoonoses Malaria Using Plasmodium spp Dynein Light Chain (dlc-tctex) Gene

2019 ◽  
pp. 1-12
Author(s):  
Maureen W. Kariuki ◽  
Elijah K. Githui ◽  
Andrew G. McArthur ◽  
Rashid A. Aman ◽  
Nyamu M. Njagi ◽  
...  
Keyword(s):  
Malaria Diagnosis ◽  
Clinical Malaria ◽  
Cross Reactivity ◽  
Pcr Detection ◽  
Clinical Samples ◽  
Dynein Light Chain ◽  
Pcr Assay ◽  
Specific Primers ◽  
Dynein Light Chains ◽  
Species Specific

Novel gene targets are needed in accurate diagnosis of malaria. Previous studies show that the dynein light chains (dlc) in Plasmodium are uniquely conserved within the species, possibly due to their role as the cargo adptor moiety. This study aimed at the development of PCR assay for the detection of Plasmodium based on the (dlc-Tctex) as a genus and species-specific tool in malaria diagnosis. Multiple primers were designed based on Plasmodium spp dlc(Tctex) genes. The primers were applied on PCR to detect malaria on clinical samples and on laboratory maintained isolates of P. falciparum and P. vivax for human infecting species and P. knowlesi and P. cynomolgi for zoonoses infection involving primates. The amplified PCR fragments were gene cleaned and sequenced. BLASTn e-values output from the raw nucleotide queries supports that the genes are uniquely conserved.  Species-specific primers amplified P.  falciparum infections with no cross-reactivity to P. vivax, P. knowlesi or P. cynomolgi species. In this assay only 11 out of the 30 microscope positive malaria positive clinical blood samples were positive for PCR detection of P. falciparum infection. Primers designed for Plasmodium genus amplified the target band in all clinical malaria samples but also had another specific band amplification. This preliminary data demonstrate that a species-specific dlc(Tctex) PCR assay can be used for detection of P. falciparum and optimized genus primers can be applied to differentiate mixed malaria infections.

scholarly journals Understanding Pathogenesis and Care Challenges of Immune Reconstitution Inflammatory Syndrome in Fungal Infections

2018 ◽  
Vol 4 (4) ◽  
pp. 139 ◽  
Author(s):  
Sarah Dellière ◽  
Romain Guery ◽  
Sophie Candon ◽  
Blandine Rammaert ◽  
Claire Aguilar ◽  
...  
Keyword(s):  
Immune Reconstitution Inflammatory Syndrome ◽  
Immune Reconstitution ◽  
Fungal Infections ◽  
Current Knowledge ◽  
Immunosuppressive Agents ◽  
Fungal Species ◽  
Invasive Fungal Infections ◽  
Solid Organ ◽  
Mortality And Morbidity ◽  
Inflammatory Syndrome

Immune deficiency of diverse etiology, including human immunodeficiency virus (HIV), antineoplastic agents, immunosuppressive agents used in solid organ recipients, immunomodulatory therapy, and other biologics, all promote invasive fungal infections. Subsequent voluntary or unintended immune recovery may induce an exaggerated inflammatory response defining immune reconstitution inflammatory syndrome (IRIS), which causes significant mortality and morbidity. Fungal-associated IRIS raises several diagnostic and management issues. Mostly studied with Cryptococcus, it has also been described with other major fungi implicated in human invasive fungal infections, such as Pneumocystis, Aspergillus, Candida, and Histoplasma. Furthermore, the understanding of IRIS pathogenesis remains in its infancy. This review summarizes current knowledge regarding the clinical characteristics of IRIS depending on fungal species and existing strategies to predict, prevent, and treat IRIS in this patient population, and tries to propose a common immunological background to fungal IRIS.

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