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ABSTRACT Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex (Plasmodium spp. and Plasmodium falciparum) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human Plasmodium species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving P. falciparum, P. vivax, P. ovale, and P. malariae were positive with duplex and species-specific qPCRs. P. knowlesi qPCR detected DNA from a P. knowlesi culture. Of eight cases of mixed Plasmodium species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log P. falciparum load decrease was observed by qPCR. These new Plasmodium qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of P. falciparum cases, malaria with low parasitaemia, and mixed Plasmodium species infection.IMPORTANCEMalaria is a disease transmitted by a Plasmodium parasite genus. Most cases are caused by Plasmodium falciparum. Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species (Plasmodium spp.) and P. falciparum. We have also developed specific qPCRs for each human plasmodial species.

Background: While invasive fusariosis and lomentosporiosis are known to be associated with fungemia, overall data on mold-related fungemia are limited, hampering early management. This study aimed to describe the epidemiology of mold-positive blood cultures., Methods: Epidemiological and clinical data on mold-positive blood cultures from 2012 to 2022 were obtained from the RESSIF database. Pseudofungemia was excluded using modified Duthie and Denning criteria. Univariable and multivariable Firth logistical regression was used to study factors associated with 90-day mortality., Results: Fusarium spp accounted for 67.5% of the 80 events, involving predominantly Fusarium fujikuroi spp complex (FFSC), Neocosmospora spp, and Fusarium oxysporum spp complex (FOSC). Lomentospora prolificans was the second most frequent (10%), followed by Trichoderma spp, Aspergillus spp, and Mucorales (5% each).Most patients had a history of hematological malignancy (HM) (70%). Forty-three percent had undergone allogeneic hematopoietic stem cell transplantation. Cutaneous and pulmonary lesions were common (43% each). Median time to blood culture positivity was 72 hours.HM and neutropenia were commonly reported in patients with FFSC, Neocosmospora spp, and L. prolificans fungemia. Pulmonary lesions were frequent in cases of L. prolificans fungemia. Patients with gastrointestinal conditions were frequently diagnosed with FOSC molds. HM (75%), particularly acute myeloblastic leukemia, was frequent in patients with Aspergillus spp fungemia. All patients with Trichoderma spp fungemia were exposed to corticosteroids.Day 90 mortality was 53%. Independent predictive factors of day 90 mortality included L. prolificans (odds ratio [OR], 33.3), Aspergillus spp fungemia (OR, 14.2), and corticosteroid exposure (OR, 7.85)., Conclusions: Underlying conditions and clinical presentation vary between genera and could be considered to guide early management., Competing Interests: Potential conflicts of interest . F. D. has received funding from Pfizer for attending scientific events. S. C. has received funding from Gilead and Pfizer for attending scientific events. M.-P. L. has received funding from Gilead, AbbVie, Jazz, Grifols, and Servier for attending scientific events; has received payment from Gilead, Pfizer, Takeda, Servier, and Janssen for lectures; and has participated in advisory boards for AbbVie and Jazz. A. P. B. and C. B. have received funding from Gilead for attending scientific events. V. D. has served as expert witness for Pfizer SAS and Patientys. M. G. M. has received funding from the European Society of Clinical Microbiology and Infectious Diseases for scientific events and payment from Gilead for expert lectures. C. C. has served as unpaid speaker with Gilead. C. A. has received funding for attending scientific events by Gilead and from Pfizer for lectures. A. A. has received payment from Pfizer for educational lectures. E. D. has received funding from Gilead and Mundipharma for attending scientic events, and payment for Mundipharma and bioMérieux for expert lectures. F. L. has served on the speakers bureau and an advisory board for F2G. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2025. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Paracoccidioides are dimorphic fungal pathogens and the etiological agents of paracoccidioidomycosis (PCM). This severe systemic mycosis is restricted to Latin America, where it has been historically endemic. Currently, PCM presents the fewest diagnostic tools available when compared to other endemic mycoses. The main PCM diagnostic methods have also limitations. Molecular methods using different protocols have been proposed, but are restricted to few regions. An analytical transversal study was conducted to evaluate a new molecular tool using specimens from patients diagnosed with PCM at a reference center for endemic mycoses in Rio de Janeiro, Brazil. After whole nucleic acid (WNA) extraction, a RT-qPCR was performed in two independent simplex reactions, targeting the mitochondrial small subunit ribosomal RNA genes of Paracoccidioides brasiliensis and Paracoccidioides lutzii. Additionally, WNAs from all PCM-related Paracoccidioides species and from 114 other fungal strains, as well as from samples obtained from patients diagnosed with other endemic mycoses and tuberculosis, were also tested for specificity. The RT-qPCR targeting P. brasiliensis successfully amplified genetic material from all tested Paracoccidioides species but not P. lutzii, reason why a specific RT-qPCR has been designed. The RT-qPCR efficiency was 1.95 (95%) with 100% analytical specificity for both targets. All included PCM clinical samples were positive (100% sensitivity) for P. brasiliensis and all yielded negative for P. lutzii. Additionally, all samples collected from patients with other diseases were negative, reinforcing the assay's specificity. In conclusion, this study proposes a new accurate tool to cover gaps, contributing with the molecular diagnosis of this neglected disease., (© The Author(s) 2025. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex ( Plasmodium spp. and Plasmodium falciparum ) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human Plasmodium species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving P. falciparum , P. vivax , P. ovale, and P. malariae were positive with duplex and species-specific qPCRs. P. knowlesi qPCR detected DNA from a P. knowlesi culture. Of eight cases of mixed Plasmodium species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log P. falciparum load decrease was observed by qPCR. These new Plasmodium qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of P. falciparum cases, malaria with low parasitaemia, and mixed Plasmodium species infection.IMPORTANCEMalaria is a disease transmitted by a Plasmodium parasite genus. Most cases are caused by Plasmodium falciparum . Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species ( Plasmodium spp.) and P. falciparum . We have also developed specific qPCRs for each human plasmodial species., Competing Interests: The authors declare no conflict of interest.