Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

Simon, Stéphane; Véron, Vincent; Boukhari, Rachida; Blanchet, Denis; Aznar, Christine

doi:10.1016/j.diagmicrobio.2009.10.010

Cited by 62 publications

(47 citation statements)

References 16 publications

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“…Regarding test performance, Simon et al reported on the ability to detect Histoplasma DNA at a concentration to 10 fg/l while testing fresh tissue using a real-time PCR assay (11). In comparing the lower analytical sensitivity of our real-time PCR assay to these results, the difference would not be unexpected since FFPE tissue was used in our study and the PCR target and primers were different between the studies.…”

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confidence: 44%

“…Culture therefore is the gold standard to confirm Histoplasma capsulatum (6,12). Reported molecular methods to detect H. capsulatum in fresh tissues include nested PCR with nucleotide sequencing (2), realtime PCR (1,11), conventional PCR with nucleotide sequencing (4), and digoxigenin-labeled PCR (9). In situ hybridization using formalin-fixed, paraffin-embedded (FFPE) tissue has also been done to confirm the presence of H. capsulatum (5).…”

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confidence: 99%

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Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

2012

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bA real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/l of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue.H istoplasma capsulatum is a thermally dimorphic fungus that is endemic across the Americas. Exposure to H. capsulatum conidia causes disease states ranging from fatal disseminated fungemia in immunosuppressed patients to an asymptomatic infection in immunocompetent patients (6). A minimally symptomatic infection often results in lung or mediastinal nodules or calcifications that may be detected only incidentally years later (10).Granulomatous pulmonary nodules related to histoplasmosis radiographically mimic malignancy, and biopsies are performed to resolve the differential diagnosis (7). Ideally, following biopsy, fresh tissue should be submitted for culture; however, the specimen is often evaluated histologically only. Lesions that consist of granulomatous inflammation are generally stained using Gomori's methenamine silver (GMS) stain or periodic acid-Schiff stain to look for fungal elements. In a patient with an illness consistent with an endemic mycosis where culture was not performed, the EORT/MSG Consensus Group has suggested that histoplasmosis could be diagnosed with the histopathologic demonstration of distinctive 2-to 4-m oval, narrow-based budding intracellular yeast forms in tissue macrophages (3). However, the yeast form of Histoplasma may mimic other fungal agents, such as Cryptococcus neoformans, Candida glabrata, endospores of Coccidioides immitis, or the protozoan Leishmania species, in tissue. Culture therefore is the gold standard to confirm Histoplasma capsulatum (6,12). Reported molecular methods to detect H. capsulatum in fresh tissues include nested PCR with nucleotide sequencing (2), realtime PCR (1, 11), conventional PCR with nucleotide sequencing (4), and digoxigenin-labeled PCR (9). In situ hybridization using formalin-fixed, paraffin-embedded (FFPE) tissue has also been done to confirm the presence of H. capsulatum (5). Like all assays, each method has limitations and advantages. For utility in an anatomic pathology service, a real-time PCR assay to test FFPE biopsy material for the presence of H. capsulatum would be useful. This study evaluates a novel PCR assay for the direct identification of H. capsulatum using FFPE human tissue samples.Real-time PCRs were performed on a LightCycler 1.5 using software version 3.5.3 (Roche, Indianapolis, IN). Primers specific for a 99-bp portion of the H. capsulatum-specific 100-kDa protein gene were generated as previously described (9). A fluorescent probe specific for the same H. capsulatum-specific 100-kDa protein gene amplicon using a 3=-black hole quencher (BHQ) and a 5=-6-carboxyfluorescein (FAM) fluorophore with the sequence 5=-CCAAGCCACCGATACAGTT-3= was purchased (Eurofins, Hunstville, AL). The reaction...

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confidence: 44%

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confidence: 99%

Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

2012

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“…Although culture has typically been considered the gold standard for diagnosis, molecular methods may in fact be more sensitive. Indeed, in a study comparing real-time PCR to culture for the detection of H. capsulatum, 10 of 11 patients with culture-negative PCR-positive samples were confirmed to have histoplasmosis based on positive cultures from other specimens or positive histopathology (33). Fewer studies have examined molecular methods in comparison to antigen and antibody detection.…”

Section: Molecular Methodsmentioning

confidence: 99%

Laboratory Diagnostics for Histoplasmosis

2017

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The diagnosis of histoplasmosis is based on a multifaceted approach that includes clinical, radiographic, and laboratory evidence of disease. The gold standards for laboratory diagnosis include demonstration of yeast on pathological examination of tissue and isolation of the mold in the culture of clinical specimens; however, antigen detection has provided a rapid, noninvasive, and highly sensitive method for diagnosis and is a useful marker of treatment response. Molecular methods with improved sensitivity on clinical specimens are being developed but are not yet ready for widespread clinical use. This review synthesizes currently available laboratory diagnostics for histoplasmosis, with an emphasis on complexities of testing and performance in various clinical contexts.

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“…The AccuProbe H. capsulatum culture identification test (Gen-Probe) can be used for rapid molecular identification of H. capsulatum in cultures, but this test is expensive and is not readily available in developing countries. Several additional molecular assays for detection of H. capsulatum have been developed (1,4,5), but none has been subjected to large-scale interlaboratory evaluation. These assays rely on PCR methodology and require expensive reagents and equipment, which may be unsustainable in laboratories with limited funding.…”

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Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

et al. 2014

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c Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n ‫؍‬ 6) collected from HIV-positive individuals with culture-and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n ‫؍‬ 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was <6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.

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Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

Cited by 62 publications

References 16 publications

Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

Laboratory Diagnostics for Histoplasmosis

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

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