Malaria Journal

Pyronaridine, a Mannich base anti-malarial with high efficacy against drug resistant Plasmodium falciparum, is currently evaluated as a fixed dose combination with artesunate for the treatment of uncomplicated malaria. In this study, the in vitro activity of pyronaridine against clinical isolates of P. falciparum from Lambaréné, Gabon, was assessed in order to obtain baseline data on its activity prior to its future use in routine therapy. Moreover, follow-up assessment on the in vitro activity of chloroquine, artesunate and quinine was performed. In vitro response of field isolates of P. falciparum to pyronaridine, chloroquine, artesunate and quinine was assessed using the traditional WHO microtest. In addition, the histidine-rich protein 2 (HRP-2) assay was performed and evaluated for its future implementation for follow-up of drug susceptibility testing. Pyronaridine exhibited a high in vitro activity against P. falciparum, with a geometric mean cut-off concentration of 9.3 nmol/l. Fifty percent effective concentrations were 1.9 nmol/l and 2.0 nmol/l in the WHO microtest and HRP-2 assay, respectively. Results matched closely in vivo findings from a recent clinical trial on pyronaridine-artesunate treatment. One isolate showed diminished sensitivity to artesunate. For chloroquine and quinine resistance levels were comparable to prior studies from Lambaréné. Results from the novel HRP-2 assay corresponded well to those obtained by the WHO microtest. Pyronaridine is highly active in chloroquine-resistant parasites and seems a promising partner drug for artemisinin-based combination therapy in Africa.

Tại tiểu vùng Mê Kông mở rộng, Plasmodium vivax đã trở thành loài chiếm ưu thế và đặt ra thách thức lớn cho việc loại trừ sốt rét trong khu vực. Nghiên cứu này nhằm điều tra sự biến đổi trong các gen có khả năng liên quan đến kháng thuốc trong các quần thể P. vivax từ khu vực biên giới Trung Quốc–Myanmar. Ngoài ra, nghiên cứu cũng muốn xác định liệu có sự khác biệt nào tồn tại giữa các quần thể ký sinh trùng liên quan đến nhiễm không triệu chứng và sốt cấp tính hay không. Tổng cộng có 66 mẫu P. vivax được thu thập từ bệnh nhân mắc sốt rét cấp tính đến khám tại các phòng khám ở khu vực Laiza, bang Kachin, Myanmar vào năm 2015. Thêm vào đó, 102 mẫu P. vivax có liên quan đến nhiễm không triệu chứng được xác định qua việc sàng lọc các tình nguyện viên không có dấu hiệu hoặc triệu chứng từ các ngôi làng xung quanh. Các mẫu xét nghiệm dương tính trên lam được xác nhận bằng PCR nhúng phát hiện gen 18S rRNA. Các nhiễm trùng đa dòng đã được loại trừ thêm bằng phương pháp genotyping tại các gen msp-3α và msp-3β. DNA ký sinh trùng từ 60 trường hợp có triệu chứng và 81 trường hợp nhiễm không triệu chứng đã được sử dụng để khuếch đại và giải trình tự các gen có thể liên quan đến kháng thuốc, bao gồm pvmdr1, pvcrt-o, pvdhfr, pvdhps, và pvk12. Các đột biến pvmdr1 Y976F và F1076L xuất hiện trong 3/113 (2.7%) và 97/113 (85.5%) mẫu P. vivax, tương ứng. Sự chèn K10 trong gen pvcrt-o được tìm thấy ở 28.2% ký sinh trùng. Bốn đột biến trong hai gen kháng chống axit folic đạt tỷ lệ tương đối cao: pvdhfr S58R (53.4%), S117N/T (50.8%), pvdhps A383G (75.0%) và A553G (36.3%). Các kiểu gen với pvmdr1 kiểu hoang dã (976Y/997K/1076F) và bốn đột biến trong pvdhfr (13I/57L/58R/61M/99H/117T/173I) xuất hiện phổ biến hơn ở các trường hợp có triệu chứng so với không triệu chứng, trong khi kiểu gen đột biến pvmdr1 976Y/997K/1076L lại phổ biến hơn ở các trường hợp không triệu chứng so với có triệu chứng. Ngoài ra, bốn đột biến tại các codon 57, 58, 61 và 117 của pvdhfr cùng với hai đột biến tại các codon 383 và 553 của pvdhps được tìm thấy cả trong nhiễm không triệu chứng và có triệu chứng với tần suất tương tự. Không phát hiện đột biến nào trong gen pvk12. Các đột biến trong pvdhfr và pvdhps phổ biến ở cả nhiễm P. vivax có triệu chứng và không triệu chứng, cho thấy khả năng kháng thuốc nhóm chống axit folic. Những người mang ký sinh trùng không triệu chứng có thể đóng vai trò như một hồ chứa âm thầm duy trì sự lây truyền ký sinh trùng kháng thuốc, điều này cần một chiến lược hợp lý để loại trừ sốt rét trong khu vực này.

Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies. Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis. The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity. The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.

Many countries across sub-Saharan Africa are rapidly increasing insecticide-treated net (ITN) coverage to combat malaria, but systematic data on the use of those ITNs and the factors affecting this use are scarce. A household survey was conducted during malaria season in 23 communities of Amhara and Oromia Regional States, Ethiopia, stratified by degree of urbanization (rural, peri-urban, or urban), whether or not they received indoor residual spraying (IRS), and whether or not free nets had been distributed. Descriptive statistics as well as univariate and multivariate logistic regression analyses were used to describe household net ownership and identify factors associated with use or non-use of nets already in the household. A qualitative component consisting of observations of ITNs in households and several open-ended questions provided further understanding of the reasons for ITN use and non-use. Of 857 surveyed households, 91% owned at least one ITN, but only 65% of ITNs owned had been used the prior night. The multivariate analysis found that the factors significantly associated with an ITN being used were regional state (Amhara) (Odds Ratio [OR] = 0.61; 95% Confidence Interval [C.I.] 0.43 - 0.86]; p < 0.01), residing in a house sprayed with IRS (OR = 1.89; 95% C.I. 1.36 - 2.63; p < 0.001), age of ITN (<12 months) (OR = 0.55; 95% C.I. 0.37 - 0.82; p < 0.01), shape (conical) (OR = 2.27; 95% C.I. 1.10 - 4.68; p < 0.05), and paying for the ITN rather than receiving it free (OR = 2.16; 95% C.I. 1.32 - 3.53; p < 0.01). The most common reasons for ITN non-use identified through the qualitative component of the study were: there are few mosquitoes around or malaria is not a serious problem; the ITN is no longer effective; ITN is in poor condition; the ITN is being saved. Observations showed many ITNs hanging incorrectly, and some being used for purposes other than as a bed net. The very high ITN ownership in the study areas suggests that a strategy targeting free nets to rural and poor households combined with support for the commercial sector is an effective means of achieving high coverage. The data suggests that use of ITNs owned could be increased by distribution of conical ITNs, continued development of the commercial sector, replacement schemes for worn-out ITNs, assistance with hanging of ITNs, and communication addressing misperceptions about ITNs.

Plasmodium falciparum resistance to artemisinin has been reported in South-East Asia. Long half-life drugs are increasingly being used for malaria prevention. The potential spread of parasite resistance to these regimens is real and makes regular efficacy surveillance a priority. From August to December 2004 and July to December 2005, a randomized open label trial of sulphadoxine-pyrimethamine (SP) + artesunate (AS) versus SP + amodiaquine (AQ), and SP alone, was conducted in two villages of Mali. PCR was used to distinguish new infections from recrudescent P. falciparum infections. Patients were followed for 28 days to assess treatment efficacy. Overall 912 children aged between six to 59 months, with uncomplicated P. falciparum malaria were recruited. Baseline characteristics were similar in the three treatment arms. Crude ACPRs were 94.9%; 98.6% and 93.5% for SP + AS; SP + AQ and SP alone arms respectively (SP + AS versus SP + AQ, p = 0.01; SP + AS versus SP, p = 0.5; SP + AQ versus SP, p = 0.001). After PCR adjustment, cACPRs were 99%; 100% and 97.2% for SP + AS; SP + AQ and SP alone arms, respectively (SP + AS versus SP + AQ, p = 0.25; SP + AS versus SP, p = 0.12; SP + AQ versus SP, p = 0.007). Sulphadoxine-pyrimethamine + amodiaquine therapy was as efficacious as sulphadoxine-pyrimethamine + artesunate, but more efficacious than sulphadoxine-pyrimethamine alone in the treatment of uncomplicated P. falciparum malaria in Mali.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IE) is a parasite adhesion phenotype that has been associated with severe malaria in some, but not all, field isolate studies. A variety of experimental conditions have been used to study clumping in vitro, with substantial differences in parasitaemia (Pt), haematocrit (Ht), and time of reaction between studies. It is unknown whether these experimental variables affect the outcome of parasite clumping assays. The effects of Pt (1, 4 and 12%), Ht (2, 5 and 10%) and time (15 min, 30 min, 1 h, 2 h) on the clumping of P. falciparum clone HB3 were examined. The effects of platelet freshness and parasite maturity were also studied. At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p = 0.0004). Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p = 0.0004). Combinations of high Ht and high Pt were impractical because of the difficulty assessing clumping in densely packed IE and the rapid formation of enormous clumps that could not be counted accurately. There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4°C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites. The Pt and Ht at which in vitro clumping assays are set up have a profound effect on the outcome. All previous field isolate studies on clumping and malaria severity suffer from potential problems in experimental design and methodology. Future studies of clumping should use standardized conditions and control for Pt, and should take into account the limitations and variability inherent in the assay.