Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered P. knowlesi.
Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered P. knowlesi.
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DOI：
10.1371/journal.pntd.0006924
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发表时间：
2018-11
影响因子：
3.8
通讯作者：
Noviyanti R
中科院分区：
文献类型：
--
作者：
Coutrier FN;Tirta YK;Cotter C;Zarlinda I;González IJ;Schwartz A;Maneh C;Marfurt J;Murphy M;Herdiana H;Anstey NM;Greenhouse B;Hsiang MS;Noviyanti R
威胁生命的人畜共患传染病诺氏疟原虫的发现增加了及时和准确诊断和监测疟疾的挑战。在这项研究中，从亚齐省，印度尼西亚，疟疾消除设置P.诺氏疟原虫地方性是以前不知道的，我们报告的实验室调查和遇到的困难时，使用分子检测方法的质量保证显微镜下确定的临床病例。2014年至2015年，通过显微镜从四个哨点卫生机构中鉴定出20种（49%）恶性疟原虫、16种（39%）间日疟原虫、3种（7%）三日疟原虫和2种（5%）不确定物种。在一个省级参考实验室，进行了环介导等温扩增（LAMP），一种现场友好的分子方法，并确认了所有样本中的疟原虫，但由于所用平台无法进行非恶性疟原虫种属特异性检测，进一步的种属鉴定受到限制。在国家参考实验室，需要几种分子方法，包括靶向18个小亚基（18 S）核糖体RNA的巢式PCR（nPCR）、靶向细胞色素-b（cytb）基因的nPCR、诺氏疟原虫特异性nPCR和最后测序，以最终将样本分类为：19例（46%）诺氏疟原虫、8例（20%）恶性疟原虫、14例（34%）间日疟原虫。显微镜检查无法识别或错误分类高达56%的确诊病例，包括所有诺氏疟原虫病例。使用靶向四种仅限人类的种属的nPCR方法，诺氏疟原虫被遗漏（18 S rRNA方法）或显示出与间日疟原虫的交叉反应性（cytb方法）。为了便于诊断和管理可能致命的诺氏疟原虫感染，并监测印度尼西亚和其他受影响环境中的人类疟疾，需要在护理点和当地参考实验室进行新的检测方法。在东南亚，最近发现猕猴的疟原虫诺氏疟原虫感染人类。这一新出现的疾病很重要，因为它有可能造成严重疾病和死亡，并对该区域消除疟疾的努力构成威胁。在这份来自印度尼西亚亚齐省的报告中，P. knowlesi最近才被发现，作者报告了区分该物种与其他人类物种的实验室挑战。使用几种不同的分子方法，他们调查了41例疟疾病例，通过显微镜检查，最初报告为：恶性疟原虫（49%），间日疟原虫（39%），三日疟原虫（7%）和不确定（5%）。只有在使用P. knowlesi特异性nPCR方法和测序后，他们才发现近一半是P. knowlesi。与稀疏的文献一致，现场友好的分子方法（属特异性LAMP）可靠地检测到诺氏疟原虫，而使用更标准的参考实验室分子方法（靶向四种人类物种的18 S rRNA nPCR）错过了感染。另外，另一种参考实验室分子方法（cytb nPCR）由于交叉反应性将诺氏疟原虫感染错误分类为间日疟原虫。为了应对诺氏疟原虫的新威胁，需要新的检测方法进行即时检测和参考检测。
The discovery of the life-threatening zoonotic infection Plasmodium knowlesi has added to the challenges of prompt and accurate malaria diagnosis and surveillance. In this study from Aceh Province, Indonesia, a malaria elimination setting where P. knowlesi endemicity was not previously known, we report the laboratory investigation and difficulties encountered when using molecular detection methods for quality assurance of microscopically identified clinical cases. From 2014 to 2015, 20 (49%) P. falciparum, 16 (39%) P. vivax, 3 (7%) P. malariae, and 2 (5%) indeterminate species were identified by microscopy from four sentinel health facilities. At a provincial-level reference laboratory, loop-mediated isothermal amplification (LAMP), a field-friendly molecular method, was performed and confirmed Plasmodium in all samples though further species-identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. At a national reference laboratory, several molecular methods including nested PCR (nPCR) targeting the 18 small sub-unit (18S) ribosomal RNA, nPCR targeting the cytochrome-b (cytb) gene, a P. knowlesi-specific nPCR, and finally sequencing, were necessary to ultimately classify the samples as: 19 (46%) P. knowlesi, 8 (20%) P. falciparum, 14 (34%) P. vivax. Microscopy was unable to identify or mis-classified up to 56% of confirmed cases, including all cases of P. knowlesi. With the nPCR methods targeting the four human-only species, P. knowlesi was missed (18S rRNA method) or showed cross-reactivity for P. vivax (cytb method). To facilitate diagnosis and management of potentially fatal P. knowlesi infection and surveillance for elimination of human-only malaria in Indonesia and other affected settings, new detection methods are needed for testing at the point-of-care and in local reference laboratories. In Southeast Asia, Plasmodium knowlesi, a malaria parasite of macaques, was recently discovered to infect humans. This emerging disease is important because it has potential for causing severe disease and death, and it is a threat to malaria elimination efforts in the region. In this report from Aceh Province, Indonesia, where P. knowlesi was only recently discovered, the authors report on the laboratory challenges of distinguishing this species from other human species. Using several different molecular methods, they investigated 41 malaria cases which by microscopy, were initially reported as: P. falciparum (49%), P. vivax (39%), P. malariae (7%), and indeterminate (5%). Only after using a P. knowlesi-specific nPCR method and sequencing, did they find that nearly half were P. knowlesi. Consistent with a sparse literature, a field-friendly molecular method (genus-specific LAMP) reliably detected P. knowlesi, while use of a more standard reference laboratory molecular method (18S rRNA nPCR targeting the four human-only species) missed the infections. Also another reference laboratory molecular method (cytb nPCR) mis-classified P. knowlesi infections as P. vivax due to cross-reactivity. To address the emerging threat of P. knowlesi, new detection methods are needed for point-of-care and reference testing.
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