Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Carvalho-Pereira J

PROVIDER: S-EPMC7712097 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Carvalho-Pereira Joana J Fernandes Filipa F Araújo Ricardo R Springer Jan J Loeffler Juergen J Buitrago María José MJ Pais Célia C Sampaio Paula P

Journal of fungi (Basel, Switzerland) 20201123 4

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 identificatio ...[more]

PMID: 33238439

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Project description:The Magicplex Sepsis Real-time Test (MST) is a commercial multiplex PCR that can detect more than 90 different pathogens in blood, with an analysis time of six hours. The aim of the present study was to evaluate this method for the detection of bloodstream infection (BSI). An EDTA whole blood sample for MST was collected together with blood cultures (BC) from patients with suspected sepsis at the Emergency Department of a university hospital. Among 696 study patients, 322 (46%) patients were positive with at least one method; 128 (18%) were BC positive and 268 (38%) were MST positive. Considering BC to be the gold standard, MST had an overall sensitivity of 47%, specificity of 66%, positive predictive value (PPV) of 23%, and a negative predictive value of 87%. Among the MST positive samples with a negative BC, coagulase-negative staphylococci (CoNS) and species that rarely cause community-acquired BSI were frequently noted. However, the quantification cycle (Cq) values of the MST+/BC- results were often high. We thus hypothesized that the performance of the MST test could be improved if the Cq cut-off level was adjusted downwards. With a lower Cq cut-off value, i.e. 6.0 for Staphylococcus species and 9.0 for all other species, the number of MST positive cases decreased to 83 (12%) and the overall sensitivity decreased to 38%. However, the PPV increased to 59% and the specificity increased to 96%, as many MST positive results for CoNS and bacteria that rarely cause community-acquired BSI turned MST negative. In conclusion, our study shows that with a lower Cq cut-off value, the MST will detect less contaminants and findings with unclear relevance, but to the cost of a lower sensitivity. Consequently, we consider that a positive MST results with a Cq value above the adjusted cut-off should be interpreted with caution, as the result might be clinically irrelevant. In a correspondent way, quantitative results could probably be useful in the interpretation of positive results from other molecular assays for the detection of BSI.

Project description:Echinocandins, such as anidulafungin, are the first-line treatment for candidemia or invasive candidiasis in critically ill patients. There are conflicting data on the pharmacokinetic properties of anidulafungin in intensive care unit (ICU) patients. Adult ICU patients (from 3 hospitals) receiving anidulafungin for suspected or proven fungal infections were included in the present study. Patients were considered evaluable if a pharmacokinetic curve for day 3 could be completed. Twenty-three of 36 patients (7 female and 16 male) were evaluable. The median (range) age and body weight were 66 (28 to 88) years and 76 (50 to 115) kg, respectively. Pharmacokinetic sampling on day 3 (n = 23) resulted in a median anidulafungin area under the concentration-time curve from 0 to 24 h (AUC0-24) of 72.1 (interquartile range [IQR], 61.3 to 94.0) mg · h · liter-1, a median daily trough concentration (C24) of 2.2 (IQR, 1.9 to 2.9) mg/liter, a median maximum concentration of drug in serum (Cmax) of 5.3 (IQR, 4.1 to 6.0) mg/liter, a median volume of distribution (V) of 46.0 (IQR, 32.2 to 60.2) liters, and a median clearance (CL) of 1.4 (IQR, 1.1 to 1.6) liters · h-1 Pharmacokinetic sampling on day 7 (n = 13) resulted in a median AUC0-24 of 82.7 (IQR, 73.0 to 129.5) mg · h · liter-1, a median minimum concentration of drug in serum (Cmin) of 2.8 (IQR, 2.2 to 4.2) mg/liter, a median Cmax of 5.9 (IQR, 4.6 to 8.0) mg/liter, a median V of 39.7 (IQR, 32.2 to 54.4) liters, and a median CL of 1.2 (IQR, 0.8 to 1.4) liters · h-1 The geometric mean ratio for the AUCday7/AUCday3 term was 1.13 (90% confidence interval [CI], 1.03 to 1.25). The exposure in the ICU patient population was in accordance with previous reports on anidulafungin pharmacokinetics in ICU patients but was lower than that for healthy volunteers or other patient populations. Larger cohorts of patients or pooled data analyses are necessary to retrieve relevant covariates. (This study has been registered at ClinicalTrials.gov under identifier NCT01438216.).

Dataset Information

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

Publications

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

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