Project description:We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.
Project description:Cryptococcus neoformans/gattii and Histoplasma capsulatum var. capsulatum may present atypical histopathological features inducing diagnostic errors. We aimed to estimate the frequency of these atypical features in formalin-fixed tissue (FT) samples and to assess the relevance of an integrated histomolecular diagnosis using specific H. capsulatum PCR and panfungal PCR followed by Sanger sequencing and/or targeted massive parallel sequencing (MPS). A total of 27 FT from 23 patients with a histopathological diagnosis of cryptococcosis (n = 16 FT from 13 patients) or histoplasmosis (n = 11 FT from 10 patients) were retrospectively included. All FT were consultation cases. Mycological identifications on equivalent fresh tissue were available for 11/23 (47.8%) patients. The expert pathologist review modified the diagnosis suggested by the initial pathologist in 7/27 (25.9%) FT. Fungal morphology and tissue inflammation were compared between both mycoses. The most discriminant atypical criterion was the presence of dented-looking yeasts, observed in 68.75% (11/16) of C. neoformans/gattii and none (0/11) of H. capsulatum var. capsulatum (P = .002). For the 12/23 (52.2%) patients without mycological identification on fresh tissue, an integrated histomolecular diagnosis on FT using specific PCR or panfungal PCR followed by Sanger sequencing and/or MPS led to fungal identification in 9/12 (75%) cases; for cryptococcosis, the targeted MPS sensitivity was higher than that of Sanger sequencing (P = .041). Thus, because atypical histopathological features may be tricky, integrated histomolecular diagnosis is essential for optimal patient care.
Project description:Histoplasma capsulatum var. duboisii (Hcd) infections have been well documented to cause chronic granulomatous disease, mainly involving the skin of baboons and humans in African countries primarily. This retrospective study classified the subspecies of Histoplasma and developed a phylogenetic tree utilizing DNA sequences extracted from formalin-fixed, paraffin embedded (FFPE) tissues from 9 baboons from a research colony in Texas histologically diagnosed with Hcd. Based on sequence analysis of ITS-2, Tub-1, and ARF, Hcd isolated from the archived samples closely aligns with the African clade and has 88% sequence homology with a sample isolated from an individual in Senegal.
Project description:Histoplasmosis is an endemic mycosis in the Americas. However, its diagnosis is challenging due to the complexity and limited availability of conventional laboratory techniques-antigen tests, culture, and staining. Microscopic preparations often confuse with other pathogens, such as Leishmania spp. The genus Histoplasma capsulatum comprises three varieties: var. capsulatum, var. duboissi, and var. farciminosum, which cannot be distinguished using conventional techniques. An infant from a tropical region of Ecuador was hospitalized for fever, bloody diarrhea, and anemia persisting for two months. Upon admission, he received antibiotics and immunosuppressants. Histopathological examination of the lymph nodes, intestines, and bone marrow aspirate reported the presence of Leishmania-like amastigotes, and treatment was initiated with meglumine antimoniate and conventional amphotericin B. However, subsequent analysis of samples using PCR and DNA sequencing identified H. capsulatum var. capsulatum but not Leishmania. Despite fluconazole and amphotericin B, the infant succumbed to the disease. The delay in clinical and laboratory diagnosis of histoplasmosis and the use of nonspecific and ineffective drugs such as fluconazole led to disease dissemination and, ultimately, death. Implementing molecular diagnosis and antigen tests in laboratories located in endemic regions and reference hospitals is crucial.