PCR-Based Approach Targeting Mucorales-Specific Gene Family for Diagnosis of Mucormycosis

Abstract

Mucormycosis is an aggressive, life-threatening infection caused by fungi in the order Mucorales. The current diagnosis of mucormycosis relies on mycological cultures, radiology and histopathology. These methods lack sensitivity and are most definitive later in the course of infection, resulting in the prevention of timely intervention. PCR-based approaches have shown promising potential in rapidly diagnosing mucormycosis. The spore coating protein homolog encoding CotH genes are uniquely and universally present among Mucorales. Thus, CotH genes are potential targets for the rapid diagnosis of mucormycosis. We infected mice with different Mucorales known to cause human mucormycosis and investigated whether CotH could be PCR amplified from biological fluids. Uninfected mice and those with aspergillosis were used to determine the specificity of the assay. CotH was detected as early as 24 h postinfection in plasma, urine, and bronchoalveolar lavage (BAL) samples from mice infected intratracheally with Rhizopus delemar, Rhizopus oryzae, Mucor circinelloides, Lichtheimia corymbifera, or Cunninghamella bertholletiae but not from samples taken from uninfected mice or mice infected with Aspergillus fumigatus Detection of CotH from urine samples was more reliable than from plasma or BAL fluid. Using the receiver operating characteristic method, the sensitivity and the specificity of the assay were found to be 90 and 100%, respectively. Finally, CotH was PCR amplified from urine samples of patients with proven mucormycosis. Thus, PCR amplification of CotH is a promising target for the development of a reliable, sensitive, and simple method of early diagnosis of mucormycosis.

Keywords: CotH; Cunninghamella; Lichtheimia; Mucor; Mucorales; Rhizopus; diagnosis.

FIG 1

Design of CotH-specific primers. (A) Different Mucorales harbor one to seven copies of CotH genes, which would allow amplification of a PCR signal. (B) A phylogenetic tree was constructed using each species' CotH sequence that presented the highest similarity among all others Mucorales. (C) Multialignment analyses were performed with available online tools. The result showed many regions of high similarity or identity, especially in the 5′ region, that were used to design candidate primers.

FIG 2

Determination of the specificity and sensitivity of the designed primers. PCR amplification of genomic DNA (gDNA) of R. delemar (Rd), R. oryzae (Ro), L. corymbifera (Lc), C. bertholletiae (Cb), and M. circinelloides (Mc) was performed using CotH-specific primer sets #2, #4, and #6. gDNA from A. fumigatus (Af) was included as a negative control. (A) Best candidate primers specifically amplified the desired band from all tested Mucorales but not from A. fumigatus. A fast, inexpensive, and reliable method for the extraction of DNA from low-volume/low concentration samples was optimized in our laboratory. (B) The method used for panel A was compared to commercially available kits in extracting gDNA from R. delemar prior to conducting PCR using primer set #6. Note the amplification of the CotH fragment from cultures spiked with even 1 spore/ml, showing a much higher sensitivity with the optimized DNA extraction method compared to the two commercially available kits.

FIG 3

Detection and sensitivity and specificity analysis of CotH in urine samples of DKA mice infected with different Mucorales. Urine samples were collected from mice infected with different Mucorales species on different days postinfection. The samples were extracted for DNA and PCR conducted with primer set #6. A larger volume of starting samples (>100 μl) increased the probability of CotH detection with an efficiency between 88 and 92% (A). *, DNA extracted from urine samples of <100 μl. Spaces between gels indicate the assembled pictures form more than one gel. The ROC was utilized to determine the sensitivity and specificity of the assay by including the results for urine samples collected from mice infected with Mucorales species or those infected with A. fumigatus or with (B) or without (C) including urine samples collected from Mucorales-infected mice that had been treated with antifungal therapy.

FIG 4

Detection of CotH in urine samples from patients with proven mucormycosis. PCR amplification of a 433-bp CotH fragment using gDNA extracted from urine samples of four different patients with proven mucormycosis. The space in the gel indicates assembled picture from more than one gel.