Rodrigues Anderson Messias, de Hoog G Sybren, de Camargo Zoilo Pires
Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.
PLoS Negl Trop Dis. 2015 Dec 1;9(12):e0004190. doi: 10.1371/journal.pntd.0004190. eCollection 2015 Dec.
Sporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective.
We developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens.
Primer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10-100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals.
This PCR-based method could successfully detect and identify a single species in samples from cultures and from clinical specimens. The method proved to be simple, high throughput, sensitive, and accurate for diagnosing sporotrichosis.
孢子丝菌病是由长喙壳目嗜温双态真菌引起的一种慢性（亚）皮肤感染。这些真菌的特点是在传播途径、宿主偏好、物种毒力和对抗真菌药物的敏感性方面存在重大差异。孢子丝菌属物种以暴发形式出现。在巴西和中国分别持续存在大型人兽共患病和腐生菌病。由于致病和非致病孢子丝菌之间的表型密切相关且成分重叠，目前基于形态学和生理学的诊断方法不准确。迫切需要特异性强、灵敏度高且具有成本效益的新型诊断工具。
我们基于钙调蛋白（CAL）基因序列开发了一组新型标记物，用于孢子丝菌属及其相关属长喙壳属临床相关成员的大规模诊断和流行病学研究。我们鉴定了针对巴西孢子丝菌、申克孢子丝菌、球形孢子丝菌、墨西哥孢子丝菌、苍白孢子丝菌和窄孢长喙壳的基于聚合酶链反应（PCR）的特异性标记物。我们采用播散性孢子丝菌病小鼠模型优化用于检测临床标本中孢子丝菌的PCR检测方法。
引物-基本局部比对搜索工具（Primer-BLAST）搜索揭示了在单个物种内保守的候选序列。物种特异性引物与孢子丝菌属以外的人、小鼠或微生物无明显同源性。在一轮PCR中，用于鉴定巴西孢子丝菌、申克孢子丝菌、球形孢子丝菌、墨西哥孢子丝菌和苍白孢子丝菌的检测限为10 - 100飞克DNA。以分生孢子作为DNA来源的简单直接PCR检测方法对于快速、低成本基因分型有效。来自播散性孢子丝菌病小鼠模型的样本证实了在感染动物的脾脏、肝脏、肺、心脏、脑、肾脏、尾巴和粪便中检测巴西孢子丝菌和申克孢子丝菌DNA的可行性。
这种基于PCR的方法能够成功检测和鉴定培养物样本及临床标本中的单个物种。该方法被证明对于诊断孢子丝菌病简单、高通量、灵敏且准确。
