Molecular detection of Histoplasma capsulatum indoors: A public health approach

Amorim-Conselheiro, Juliana; Orico, Lilian Dias; Galvão-Dias, Maria A.; Grigorio, Iraci Martins; Neto, Hildebrando Montenegro; Rosa, Adriana Rückert da; Oliveira, Débora Cardoso; Taborda, Carlos Pelleschi; Reis-Menezes, Adriana Araujo

doi:10.1016/j.riam.2019.01.002

Cited by 3 publications

(9 citation statements)

References 7 publications

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“…Centre 1 and 2 performed different qPCRs targeting regions of the ITS1 12,49 and ITS2 43 of the H. capsulatum rDNA. Centre 3 used a RT‐qPCR assays, targeting the mitochondrial ribosomal small subunit (mtSSU) 28 and Centre 4 three different qPCR assays targeting ITS2, ITS1 36 and the multicopy cytochrome C oxidase 2 ( COX2 ) mitochondrial gene. Centre 5 performed a broad‐range fungal conventional PCR (cPCR) targeting a part of ITS1 (primers ITS1 and ITS2 77 ), and a specific cPCR 78 targeting a part of the ITS1 region with primers derived from Buitrago et al 29 Both assays were followed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Molecular diagnostic testing on clinical samples has shown its usefulness in diagnosing fungal infections in animals 26,27 and humans 12,[28][29][30][31][32][33][34][35] and detecting environmental niches. [36][37][38][39] Besides existing broad-range fungal PCRs that rely upon amplicon identification by sequencing, multiple specific in-house assays were developed during the past years to detect H. capsulatum and Coccidioides spp. fast (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…They were applied on various sample types, such as formalin-fixed paraffin-embedded and fresh biopsies, 4,32,40,41 blood, 28,[42][43][44][45] serum, 29,33,43,[45][46][47] bronchoalveolar lavage fluid (BAL), 28,45,46,48 cerebrospinal fluid, 28,45,47,49 bone marrow, 28,43,48,49 pleural fluid 28,48 and urine. 50 They target multicopy ribosomal DNAs 28,29,33,36,39,[43][44][45]47,49,[51][52][53][54][55][56][57][58][59] (rDNAs) or species-specific genes, 27,…”

Section: Introductionmentioning

confidence: 99%

“…They were applied on various sample types, such as formalin‐fixed paraffin‐embedded and fresh biopsies, 4,32,40,41 blood, 28,42–45 serum, 29,33,43,45–47 bronchoalveolar lavage fluid (BAL), 28,45,46,48 cerebrospinal fluid, 28,45,47,49 bone marrow, 28,43,48,49 pleural fluid 28,48 and urine 50 . They target multicopy ribosomal DNAs 28,29,33,36,39,43–45,47,49,51–59 (rDNAs) or species‐specific genes, 27,41,42,46,48,60–73 mostly single copy, except for the region identified in the NCBI database as a ‘copia‐like retrotransposon’ 1,37,38,74,75 with approximately 60 copies per Coccidioides genome 74 …”

Section: Introductionmentioning

confidence: 99%

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A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

Wilmes

Hagen

Veríssimo

et al. 2023

Mycoses

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Background: In-house real-time PCR (qPCR) is increasingly used to diagnose the socalled endemic mycoses as commercial assays are not widely available.Objectives: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories.Methods: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing.Results: qPCR assays were the most sensitive for the detection of H. capsulatum DNA.The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

Wilmes

Hagen

Veríssimo

et al. 2023

Mycoses

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“…The target sequence, i.e., ITS1 of H. capsulatum ribosomal DNA, was chosen as suggested in other studies [ 9 , 11 ]. An in-silico analysis of a primer/probe set previously used [ 9 ] was performed to verify: a) the complementarity of primers on the ITS1 region of H. capsulatum ribosomal DNA consensus sequence, obtained from various gene sequences of H. capsulatum , and b) the size of the fragment to be amplified by qPCR.…”

Section: Methodsmentioning

confidence: 99%

Design and optimization of an improved qPCR assay for the detection of Histoplasma capsulatum

da Silva,

Alves Cortez,

Oliveira

et al. 2023

EuJMI

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BackgroundThis study aimed at improving a real-time polymerase-chain-reaction (qPCR) assay for the detection of Histoplasma capsulatum, a fungal pathogen that can cause severe respiratory infections in humans, in clinical and soil samples.MethodsPrimer and probes were in-silico designed, in-silico and in-vitro evaluated including clinical biopsy materials and finally subjected to a real-world application with collected soil samples.ResultsApplying the qPCR assay with liver and lung biopsies from 71 patients each, including 59 patients infected with human immunodeficiency virus (HIV), as well as with Sabouraud (SAB) agar culture as the diagnostic reference standard, diagnostic accuracy of the qPCR assay of 100% (5/5) sensitivity and 96% (63/66) specificity for liver samples and 100% (4/4) sensitivity and 94% (63/67) specificity for the lung samples was recorded. When applying the assay with soil samples from caves near of Presidente Figueiredo city, Amazonas, Brazil, one sample from the Maroaga cave was confirmed as positive.ConclusionsThe improved qPCR assessed in this study was successful in detecting H. capsulatum with high efficiency and accuracy in in-vitro evaluation, including the identification of the target pathogen in both clinical and environmental samples.

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