Real-Time PCR Method for Detection of Zygomycetes

Abstract

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/l, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.Zygomycetes are common environmental fungi capable of producing serious disease in immunocompromised hosts. The vast majority of infections occur in individuals who are neutropenic, those receiving cytotoxic therapies, or those who have underlying metabolic acidosis. Although previously considered a relatively rare cause of disease, the incidence of opportunistic infections due to zygomycetes has increased over the last decade. Studies from multiple centers have indicated infection rates from 2.5% in bone marrow transplant recipients to 5.7% in solid-organ transplant recipients (10). Depending on the patient population, underlying disease, clinical presentation, and an early, accurate diagnosis, overall mortality rates due to zygomycete infections have ranged from 20% in localized infections to 100% in cases of disseminated disease (5, 13).Members of the order Mucorales include species of the genera Rhizopus, Absidia, Mucor, Rhizomucor, Apophysomyces, Cunninghamella, and Saksenaea, and all have been implicated in human disease. Approximately 90% of all infections are caused by members of the genus Rhizopus, most commonly, R. arrhizus, followed by R. microsporus var. rhizopodiformis.Currently, a diagnosis of zygomycosis is based upon identification of broad, ribbon-like, pauciseptate hyphae by histopathology or the use of macroscopic and microscopic morphology analysis following fungal culture. Histopathology determinations suffer from subjectivity that is dependent upon the experience of the reader. In addition, tissue processing, fixation, and staining may require sever...

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“…Four studies reported the development of PCR assays to diagnose the pathogens causing this disease (229)(230)(231)(232). An early study focused on the identification of Rhizopus spp.…”

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