Departament of Medicine, Discipline of infectious Diseases, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
PLoS Negl Trop Dis. 2020 Jul 1;14(7):e0008330. doi: 10.1371/journal.pntd.0008330. eCollection 2020 Jul.
Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC = 0.2552-0.3113), marker index (MI = 0.002-0.0039), effective multiplex ratio (E = 17.8519-35.2222), resolving power (Rp = 33.6296-63.1852), discriminating power (D = 0.9291-0.9662), expected heterozygosity (H = 0.3003-0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis.
申克孢子丝菌病是一种由申克孢子丝菌引起的慢性皮下真菌病，主要的病原体包括巴西申克孢子丝菌、申克孢子丝菌和球形孢子丝菌。感染是在哺乳动物皮肤创伤性接种孢子丝菌繁殖体后发生的，可能遵循经典的植物碎片创伤性接种途径（例如，申克孢子丝菌和球形孢子丝菌）或通过动物的人畜共患病传播的替代途径（例如，巴西申克孢子丝菌）。在巴西，曾发生过动物传染病，随后出现了人畜共患病，而里约热内卢是最近猫传播的传染病的中心。需要基于 DNA 的标记物来使用分子方法探索这些申克孢子丝菌扩张的流行病学。本文报告了使用扩增片段长度多态性（AFLP）来评估种内变异程度的方法。我们使用了来自申克孢子丝菌的全基因组序列来生成 2304 个虚拟 AFLP 指纹。在计算机筛选中，突出了 6 对引物组合进行体外测试。该方案用于对 27 种具有医学意义的申克孢子丝菌进行基因分型。根据总体评分的 AFLP 标记（97-137 个片段），多态信息含量（PIC=0.2552-0.3113）、标记指数（MI=0.002-0.0039）、有效多重比（E=17.8519-35.2222）、分辨率（Rp=33.6296-63.1852）、区分能力（D=0.9291-0.9662）、预期杂合度（H=0.3003-0.3857）和平均杂合度（Havp=0.0001）表明这些引物组合在区分申克孢子丝菌方面具有实用性。AFLP 标记揭示了先前认为是最普遍的克隆类型的物种中的隐型多样性，例如负责猫传播孢子丝菌病的巴西申克孢子丝菌，以及负责亚洲大 sapronosis 暴发的球形孢子丝菌。三种组合（#3 EcoRI-FAM-GA/MseI-TT、#5 EcoRI-FAM-GA/MseI-AG 和 #6 EcoRI-FAM-TA/MseI-AA）提供了最佳的多样性指数和最低的错误率。这些方法使在动物传染病和人畜共患病期间更容易追踪疾病传播途径，我们的 DNA 指纹分析可以在实验室之间进一步转移，以深入了解致病性申克孢子丝菌的生态学和进化，并为管理和缓解策略提供信息，以应对孢子丝菌病的蔓延。
