Evaluation of a new Histoplasma spp. reverse transcriptase quantitative PCR assay

Alexandre Alanio (1, 2, 3, 4) , Maud Gits-Muselli (1, 3, 2) , Fanny Lanternier (1, 3, 5, 4) , Aude Sturny-Leclère (1) , Marion Benazra (1) , Samia Hamane (2) , Anderson Messias Rodrigues (6) , Dea Garcia-Hermoso (1, 4) , Olivier Lortholary (1, 3, 5, 4) , Françoise Dromer (1, 4) , Stéphane Bretagne (2, 1, 3) , The French Mycoses Study Group

1 Mycologie moléculaire - Molecular Mycology
2 Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris]
3 UPCité - Université Paris Cité
4 CNRMA - Centre National de Référence Mycoses Invasives et Antifongiques - National Reference Center Invasive Mycoses & Antifungals
5 Centre d'infectiologie Necker-Pasteur [CHU Necker]
6 Unifesp - Federal University of Sao Paulo

Laboratory diagnosis of histoplasmosis is based on various methods including microscopy, culture, antigen and DNA detection of Histoplasma capsulatum var. capsulatum (Hcc) or H. capsulatum var. duboisii (Hcd). To improve sensitivity of existing quantitative PCR assays, we developed a new reverse transcriptase qPCR (RTqPCR) assay allowing amplification of whole nucleic acids of Histoplasma spp.. and validated on suspected cases.The limit of detection was 20 copies and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp.. Whole nucleic acids of 1,319 prospectively collected consecutive samples from 907 patients suspected of histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. 44 had proven histoplasmosis due to Hcc (n=40) or Hcd (n=4) infections. RTqPCR was positive in 43/44 patients (97.7% sensitivity), in at least one specimen. Nine out of 863 cases (99% specificity) were RTqPCR positive and therefore classified as possible cases. RTqPCR was positive in 13/30 (43.3%) blood tested in proven cases. A positive RTqPCR in blood was significantly associated with Hcc progressive disseminated histoplasmosis with a positive RTqPCR in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RTqPCR assay enabling amplification of hcc and hcd is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and BAL.

Mots clés: Histoplasmosis; qPCR; reverse transcriptase; whole nucleic acids; Whole blood; fungal load; quantification cycle

Domaines: Mycologie; Systématique, phylogénie et taxonomie; Maladies infectieuses

Alexandre Alanio, Maud Gits-Muselli, Fanny Lanternier, Aude Sturny-Leclère, Marion Benazra, et al.. Evaluation of a new Histoplasma spp. reverse transcriptase quantitative PCR assay. Journal of Molecular Diagnostics, 2021, 23 (6), pp.698-709. ⟨10.1016/j.jmoldx.2021.02.007⟩. ⟨pasteur-03149041⟩