Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.
Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.
复制标题
DOI：
10.1371/journal.pntd.0003469
复制
发表时间：
2015-01
影响因子：
3.8
通讯作者：
Stewart VA
中科院分区：
文献类型：
--
作者：
Miller RH;Obuya CO;Wanja EW;Ogutu B;Waitumbi J;Luckhart S;Stewart VA
卵形疟原虫由两个遗传上不同的亚种组成，卵形疟原虫curtisi和卵形疟原虫wallikeri。尽管卵形种在形态和地理分布上具有相似性，但等位基因差异表明卵形种curtisi和卵形种walllikeri具有遗传差异。此外，卵形疟原虫curtisi和卵形疟原虫wallikeri之间潜在的临床和潜伏期差异表明，有必要调查这种被忽视的疟疾寄生虫对全球疟疾负担的贡献。为了同时检测到所有卵形圆蚧亚种，我们基于卵形圆蚧curtisi和卵形圆蚧walllikeri在网状细胞结合蛋白2 （rbp2）基因中的保守区域建立了一种具有包容性的卵形圆蚧特异性实时PCR检测方法。此外，我们还利用多位点基因分型技术对22例无症状疟疾感染中卵形疟原虫亚种的流行情况进行了分析，以区分卵形疟原虫curtisi和卵形疟原虫wallikeri。我们的卵形P. ovale rbp2 qPCR验证实验显示，rbp2质粒拷贝数在6.25 ~ 100,000 rbp2质粒拷贝数/微升线性动态范围内，检测限为1.5 rbp2质粒拷贝数/微升。特异性实验表明，rbp2 qPCR检测方法能够在存在其他疟疾寄生虫（包括恶性疟原虫、间日疟原虫和疟疾疟原虫）的情况下检测出低水平的卵形疟原虫。我们通过对富含色氨酸抗原基因、小亚基核糖体RNA基因和rbp2基因的DNA测序，在肯尼亚西部鉴定出了卵形P. curtisi和卵形P. walllikeri。我们的新方法rbp2 qPCR可同时检测出卵形疟原虫curtisi和卵形疟原虫wallikeri，可用于表征疟疾流行地区卵形疟原虫的流行、分布和负担。利用多位点基因分型，我们还首次描述了在疟疾传播全地方性的肯尼亚西部，卵形疟原虫curtisi和卵形疟原虫wallikeri的流行情况。人类可感染五种疟疾寄生虫：恶性疟原虫、间日疟原虫、疟疾疟原虫、诺氏疟原虫和卵形疟原虫。虽然全世界绝大多数疟疾发病率和死亡率可归因于恶性疟原虫，但非恶性疟原虫也可引起临床疾病。研究人员使用基于核酸的检测方法，如聚合酶链反应（PCR），来检测可以逃避显微镜检测的低密度疟疾寄生虫。卵形假种最近被鉴定为两个亚种，卵形假种（P. ovale curtisi）和卵形假种（P. ovale wallikeri），它们看起来相同，但遗传上不同。在这项研究中，我们基于P. ovale curtisi和P. ovale wallikeri之间的保守基因，建立了一种新的实时荧光定量PCR （qPCR）检测所有卵形疟原虫的方法。我们还使用DNA测序从肯尼亚西部的一个小样本的无症状卵形疟原虫感染中区分卵形疟原虫curtisi和卵形疟原虫wallikeri。通过使用我们的新型rbp2 qPCR检测，我们的目标是在未来的流行病学研究中表征卵形疟原虫的患病率，以便更好地了解这种被忽视的疟疾寄生虫物种。
Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden. In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri. Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene. Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission. Humans can be infected with five malaria parasite species: Plasmodium falciparum, P. vivax, P. malariae, P. knowlesi, and P. ovale. Although the vast majority of malaria morbidity and mortality worldwide can be attributed to P. falciparum, non-falciparum malaria parasites can also cause clinical disease. Researchers use nucleic acid based detection methods, such a polymerase chain reaction (PCR), to detect low-density malaria parasitemias that can evade microscopic detection. P. ovale was recently identified to exist as two subspecies, P. ovale curtisi and P. ovale wallikeri, that look identical but differ genetically. In this study, we developed a novel real-time PCR (qPCR) assay to detect all P. ovale parasites, based on a conserved gene between P. ovale curtisi and P. ovale wallikeri. We also used DNA sequencing to differentiate between P. ovale curtisi and P. ovale wallikeri from a small sample of P. ovale asymptomatic infections in Western Kenya. Through the use of our novel rbp2 qPCR assay, we aim to characterize the prevalence of P. ovale in future epidemiological studies in order to better understand this neglected malaria parasite species.
登录
查看更多内容
影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
Lopez R
影响因子：
9.4
作者：
Fuehrer, Hans-Peter;Stadler, Marie-Therese;Noedl, Harald
通讯作者：
Noedl, Harald
影响因子：
3
作者：
Alemu, Abebe;Fuehrer, Hans-Peter;Noedl, Harald
通讯作者：
Noedl, Harald
影响因子：
3.7
作者：
Kamau, Edwin;Alemayehu, Saba;Ockenhouse, Christian F.
通讯作者：
Ockenhouse, Christian F.
影响因子：
3.3
作者：
Cohen, Robert;Feghali, Karla;Zapor, Michael
通讯作者：
Zapor, Michael