采用混合样本和新一代测序技术对坦桑尼亚湖泊区和南区耐周效磺胺-乙胺嘧啶的疟原虫进行监测。
Surveillance for sulfadoxine-pyrimethamine resistant malaria parasites in the Lake and Southern Zones, Tanzania, using pooling and next-generation sequencing.
作者信息
Ngondi Jeremiah M, Ishengoma Deus S, Doctor Stephanie M, Thwai Kyaw L, Keeler Corinna, Mkude Sigsbert, Munishi Oresto M, Willilo Ritha A, Lalji Shabbir, Kaspar Naomi, Kitojo Chonge, Paxton Lynn A, Hathaway Nicholas J, Bailey Jeffrey A, Juliano Jonathan J, Meshnick Steven R, Gutman Julie
机构信息
RTI International, Dar es Salaam, Tanzania.
National Institute for Medical Research, Tanga, Tanzania.
出版信息
Malar J. 2017 Jun 5;16(1):236. doi: 10.1186/s12936-017-1886-9.
BACKGROUND
Malaria in pregnancy (MiP) remains a major public health challenge in areas of high malaria transmission. Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended to prevent the adverse consequences of MiP. The effectiveness of SP for IPTp may be reduced in areas where the dhps581 mutation (a key marker of high level SP resistance) is found; this mutation was previously reported to be common in the Tanga Region of northern Tanzania, but there are limited data from other areas. The frequency of molecular markers of SP resistance was investigated in malaria parasites from febrile patients at health centres (HC) in seven regions comprising the Lake and Southern Zones of mainland Tanzania as part of the ongoing efforts to generate national-wide data of SP resistance.
METHODS
A cross-sectional survey was conducted in the outpatient departments of 14 HCs in seven regions from April to June, 2015. 1750 dried blood spot (DBS) samples were collected (117 to 160 per facility) from consenting patients with positive rapid diagnostic tests for malaria, and no recent (within past 2 months) exposure to SP or related drugs. DNA was extracted from the DBS, pooled by HC, and underwent pooled targeted amplicon deep sequencing to yield estimates of mutated parasite allele frequency at each locus of interest.
RESULTS
The dhps540 mutation was common across all 14 sites, ranging from 55 to 98.4% of sequences obtained. Frequency of the dhps581 mutation ranged from 0 to 2.4%, except at Kayanga HC (Kagera Region, Lake Zone) where 24.9% of sequences obtained were mutated. The dhfr164 mutation was detected only at Kanyanga HC (0.06%).
CONCLUSION
By pooling DNA extracts, the allele frequency of mutations in 14 sites could be directly determined on a single deep-sequencing run. The dhps540 mutant was very common at all locations. Surprisingly, the dhps581 was common at one health center, but rare in all the others, suggesting that there is geographic micro-heterogeneity in mutant distribution and that accurate surveillance requires inclusion of multiple sites. A better understanding of the effect of the dhps581 mutant on the efficacy of IPTp-SP is needed.
背景
在疟疾高传播地区，妊娠疟疾（MiP）仍然是一项重大的公共卫生挑战。建议采用磺胺多辛-乙胺嘧啶（SP）进行孕期间歇性预防治疗（IPTp），以预防MiP的不良后果。在发现dhps581突变（高水平SP耐药的关键标志物）的地区，SP用于IPTp的有效性可能会降低；此前有报道称该突变在坦桑尼亚北部的坦噶地区很常见，但其他地区的数据有限。作为生成全国范围内SP耐药数据的持续努力的一部分，对坦桑尼亚大陆湖泊和南部地区七个地区的卫生中心（HC）发热患者的疟原虫中SP耐药分子标志物的频率进行了调查。
方法
2015年4月至6月，在七个地区的14个卫生中心的门诊部进行了一项横断面调查。从同意参与的疟疾快速诊断检测呈阳性、且近期（过去2个月内）未接触过SP或相关药物的患者中收集了1750份干血斑（DBS）样本（每个机构117至160份）。从DBS中提取DNA，按卫生中心进行汇集，并进行汇集靶向扩增子深度测序，以得出每个感兴趣位点的突变寄生虫等位基因频率估计值。
结果
dhps540突变在所有14个位点都很常见，在所获得序列中的比例为55%至98.4%。dhps581突变的频率在0至2.4%之间，但在卡扬加卫生中心（卡盖拉地区，湖泊区）除外，在该中心所获得序列中有24.9%发生了突变。仅在卡尼扬加卫生中心检测到dhfr164突变（0.06%）。
结论
通过汇集DNA提取物，一次深度测序运行就能直接确定14个位点的突变等位基因频率。dhps540突变体在所有地点都非常常见。令人惊讶的是，dhps581在一个卫生中心很常见，但在其他所有中心都很罕见，这表明突变分布存在地理微观异质性，准确的监测需要纳入多个地点。需要更好地了解dhps581突变体对IPTp-SP疗效的影响。
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