Identification of Histoplasma capsulatum from Culture Extracts by Real-Time PCR

Abstract

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.Histoplasma capsulatum is a slow-growing, dimorphic fungus that causes disease that ranges from focal and self-limited to disseminated and rapidly fatal (15,19). Immunocompromised individuals, particularly those with advanced AIDS, are at risk for disseminated histoplasmosis (19,20). A variety of tests are used in the laboratory for the diagnosis of histoplasmosis, but all have limitations. Histoplasma antigen detection in urine and/or serum has a variable range of sensitivity, depending on the clinical pattern, the chronicity of the affliction, and the underlying condition of the patient (6). The sensitivity of the Histoplasma urinary antigen test is as high as 97% in AIDS patients with disseminated histoplasmosis but ranges from 20 to 81% in nonimmunosuppressed patients with acute pulmonary histoplasmosis (6,18,22,23). Serologic testing by immunodiffusion and complement fixation also has utility, but both false-positive and false-negative results may occur. False-positive serologic tests may be seen in patients with other disseminated mycoses, while false-negative results may occur in immunocompromised individuals who are unable to produce an antibody response (20,21). Histopathologic analysis of tissue is also useful but is dependent upon adequate sampling, the experience of the observer, and the histochemical stains used (9, 15). Unfortunately, H. capsulatum may be misidentified in histologic sections because of the variety of morphologically similar small yeast forms, such as Candida glabrata and Sporothrix schenckii. Culture, which is often considered the "gold standard," is limited by the slow growth of the organism, which may take more than 20 days to grow. In addition, confirmatory tests are needed for organisms suspected to be H. capsulatum because saprophytic morphological mimics of the mold phase of the organism exist (3, 7, 15). Confirmatory assays include the H. capsulatum AccuProbe (Gen-Probe, Inc., San Diego, Calif.), exoantigen testing, and temperature-induced mycelium-toyeast conversion. The development of a real-time rapid PCR method that could be used as an alternate method of culture confirmation and potentially to test clinical specimens directly is warranted.The LightCycler PCR system (Roche Molecular Biochemicals, Indianapolis, Ind.) affords nucleic acid amplification and detection in a closed system in a real-time format. We designed a real-time PCR assay for the detection of H. capsulatum that targeted the internal transcribed spacer region of the rRNA gene complex (GenBank accession number AB055231) and used hybridization probes and fluorescent resonance energy transfer technology. We used Hcap-F (5Ј-TTGTCTACCGGA CCTG-3Ј) as the forward primer and Hcap-R (5Ј-TTCTTCA TCGATGTCGGAAC-3Ј) as th...

TBHEB 2015-3 Vol 72 Full Printed Journal

“…İnsan sağlığına etkilerini bilenler %17-98, çevreye ve diğer canlılara olan etkilerini bilenler %17-87 olarak bildirilmiştir (26,28,(30)(31)(32)(33). Zararlı (23,25,27,29,32,(37)(38)(39). Türkiye'deki çalışmalarda bildirilen kullanım sıklıkları insektisitler için %29 ile %65 arasında, herbisitler %32, fungusitler %18 ile %29 arasında, akarisitler %12 ile %15 arasındadır (20,34,35).…”

“…Dünyadaki çalışmalarda bitki koruma ürünlerinin uygulanması sırasında tarım çalışanlarının yeme-içme davranışında bulunma düzeyi %9-79, bir şeyler yeme düzeyi %1-22, bir şeyler içme düzeyi %2-10, sigara içme düzeyi %4-39 arasında bildirilmiştir (25,26,28,33,38,41,51).…”

“…Dünyadaki çalışmalarda tarım çalışanlarında bitki koruma ürünlerini kullanırken ya da sonrasında herhangi bir şikayetin görülme sıklığı %20 ile %70 arasında bildirilmiştir (23,25,27,29,32,33,37,38,53 (23,25,27,29,32,33,37,38,53). Koruyucu önlem alanlarda ve kişisel koruyucu donanım kullananlarda sağlıkla ilgili şikayetlerin daha az görüldüğü bildirilmiştir (33,38).…”

“…Koruyucu önlem alanlarda ve kişisel koruyucu donanım kullananlarda sağlıkla ilgili şikayetlerin daha az görüldüğü bildirilmiştir (33,38). Ürün kullanımı sırasında/sonrasında sağlık sorunu görülmesinin gelir düzeyinin düşük olması, ürün hazırlama işleminin kapalı alanda yapılması, ürün kullanımı sırasında bir şeyler yeme ve bir şeyler içme ile ilişkili olduğu tespit edilmiştir (20,36).…”

“…This technology was used to develop assays for Aspergillus, Candida, and the zygomycetes (26). Real-time PCR was also used fort he identification of culture isolates of Histoplasma capsulatum (38). The Light Cycler may also be used to identify organisms to the species level using specific primers and probes (5).…”