Calchi Ana Cláudia, Moore Charlotte O, Bartone Lillianne, Kingston Emily, André Marcos Rogério, Breitschwerdt Edward B, Maggi Ricardo G
Vector-Borne Bioagents Laboratory (VBBL), Department of Pathology, Reproduction and One Health, School of Agricultural and Veterinarian Sciences (FCAV), São Paulo State University (UNESP), Jaboticabal CEP 14884-900, Brazil.
Intracellular Pathogens Research Laboratory, Comparative Medicine Institute, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.
Pathogens. 2024 Dec 11;13(12):1094. doi: 10.3390/pathogens13121094.
More than one-hundred species that affect animals and humans have been described, eight of which have been associated with emerging and underdiagnosed zoonoses. Most diagnostic studies in humans have used serology or molecular assays based on the 18S rRNA gene. Because the 18S rRNA gene is highly conserved, obtaining an accurate diagnosis at the species level is difficult, particularly when the amplified DNA fragment is small. Also, due to its low copy number, sequencing of the product is often unsuccessful. In contrast, because the internal transcribed regions (ITS), between 18S rRNA and 5.8S rRNA, and between 5.8S rRNA and 28S rRNA, contain highly variable non-coding regions, the sequences in these regions provide a good option for developing molecular assays that facilitate differentiation at the species level. In this study, the complete ITS1 and ITS2 intergenic regions of different Piroplasmida species were sequenced to add to the existing GenBank database. Subsequently, ITS1 and ITS2 sequences were used to develop species-specific PCR assays and specific single-plex and multiplex conventional (c)PCR, quantitative real-time (q)PCR, and digital (d)PCR assays for four zoonotic species (, , , and ). The efficacy of the assay protocols was confirmed by testing DNA samples extracted from human blood or enrichment blood cultures. Primers were first designed based on the 18S rRNA-5.8S rRNA and 5.8S rRNA-28S rRNA regions to obtain the ITS1 and ITS2 sequences derived from different Piroplasmida species (, , , , , , -like, , , , , , , and ). Subsequently, using these sequences, single-plex or multiplex protocols were optimized targeting the ITS1 region of , , and . Each protocol proved to be sensitive and specific for the four targeted sp., detecting 10 (for and ) and 10 (for and ) DNA copies per microliter. There was no cross-amplification among the species tested. Using 226 DNA extractions from blood or enrichment blood cultures obtained from 82 humans, (seven individuals), (seven individuals), and (two individuals) were detected and identified as a single infection, whereas co-infection with more than one sp. was documented by DNA sequencing in six (7.3%) additional individuals (representing a 26.8% overall prevalence). These newly developed protocols proved to be effective in detecting DNA of four species and facilitated documentation of co-infection with more than one sp. in the same individual.
已描述了一百多种可感染动物和人类的物种，其中八种与新出现的、诊断不足的人畜共患病有关。大多数针对人类的诊断研究使用基于18S rRNA基因的血清学或分子检测方法。由于18S rRNA基因高度保守，在物种水平上进行准确诊断很困难，尤其是当扩增的DNA片段较小时。此外，由于其拷贝数低，产物测序往往不成功。相比之下，由于18S rRNA与5.8S rRNA之间以及5.8S rRNA与28S rRNA之间的内部转录间隔区（ITS）包含高度可变的非编码区，这些区域的序列为开发有助于在物种水平上进行区分的分子检测方法提供了一个很好的选择。在本研究中，对不同梨形虫物种的完整ITS1和ITS2基因间隔区进行了测序，以补充现有的GenBank数据库。随后，利用ITS1和ITS2序列开发了针对四个人畜共患病物种（、、和）的物种特异性PCR检测方法以及特异性单重和多重常规（c）PCR、定量实时（q）PCR和数字（d）PCR检测方法。通过检测从人血或富集血培养物中提取的DNA样本，证实了检测方案的有效性。首先基于18S rRNA - 5.8S rRNA和5.8S rRNA - 28S rRNA区域设计引物，以获得来自不同梨形虫物种（、、、、、、类、、、、、、和）的ITS1和ITS2序列。随后，利用这些序列，针对、和的ITS1区域优化了单重或多重检测方案。每个检测方案对四种目标物种均具有敏感性和特异性，每微升可检测到10个（对于和）和10个（对于和）DNA拷贝。在所检测的物种之间未出现交叉扩增。使用从82名人类获取的血液或富集血培养物中提取的226份DNA样本，检测并鉴定出（7人）、（7人）和（2人）为单一感染，而通过DNA测序在另外6名个体（占总体患病率的26.8%）中记录到感染了不止一种梨形虫物种的共感染情况。这些新开发的检测方案被证明在检测四种梨形虫物种的DNA以及便于记录同一个体中感染不止一种梨形虫物种的共感染情况方面是有效的。
