The PfCRT (K76T) point mutation favours clone multiplicity and disease severity in Plasmodium falciparum infection

In Orissa, a malaria-hyperendemic area of India, we assessed the relationship between the PfCRT (K76T) point mutation of Plasmodium falciparum and the clinical severity of malaria. Forty uncomplicated and 36 severe malaria cases were selected, and parasite species, density and schizontaemia determined by examination of Giemsa-stained thick or thin blood films. The PfCRT point mutation was analysed by PCR-RFLP and genotypes of the parasite isolates investigated by nested PCR using the polymorphic region of the merozoite surface protein-2. We found that (i) the prevalence of the PfCRT point mutation was significantly higher (P < 0.01) in severe malaria cases and that (ii) heavy parasitaemia along with clone multiplicity was statistically more common (P < 0.01) in severe cases. These associations may be due to progression of uncomplicated to severe disease after chloroquine treatment failure and/or increased virulence of chloroquine-resistant parasites. The implications for antimalarial treatment policy are discussed.     

High frequency of Plasmodium falciparum PfCRT K76T and PfpghN86Y in patients clearing infection after chloroquine treatment in the Sudan

The clinical response following treatment with chloroquine, and the prevalence of two Plasmodium falciparum DNA polymorphisms known to associate with drug resistance, namely PfCRT K76T and Pfpgh N86Y were investigated in two sites in central and eastern Sudan. Patient's sensitivity to chloroquine was determined according to the standard in vivo test as recommended by the WHO protocol in days 0, 3, 7 and 14, respectively. Clinical un-responsiveness was 75.9% in Gadaref in eastern Sudan and 32.1% in Haj Yousif of the Khartoum state. Difference between the two sites in treatment outcome is not tantamount to allele frequency and genotype distribution of neither Pfcrt K76T nor PfpghN86Y. All post treatment samples in the two areas were carrying the mutant allele of Pfcrt K76T. The higher frequency of PfpghN86Y in Haj Yousif (0.86) than Gadaref (0.72), where chloroquine resistance is higher suggests a minor role, if any, for PfpghN86Y in resistance to chloroquine. Age effect on the clearance of parasitemia was evident in both areas, more significantly though in Gedaref (P<0.000) than Haj Yousif (P=0.043) These results add to reports in the literature, pointing to the complexity of factors that may contribute to a clinical outcome following chloroquine treatment, particularly, in this case are elements of the host immunity that are yet to be identified.     

南通市重症疟疾病例特征及诊治情况分析

目的 回顾性分析南通市重症疟疾病例特征和诊治情况,总结重症疟疾的成因、评价诊治效果,为制定切实可行的重症疟疾救治措施提供科学依据。方法 收集2009-2016年南通市所有疟疾病例资料,对重症疟疾病例的发病时间、临床表现、诊断过程、治疗情况进行汇总分析。结果 2009-2016年南通市共报告疟疾病例359例,其中重症病例26例,均为境外输入性恶性疟病例,其中脑型疟12例、急性肾功能衰竭11例、重度贫血3例。26例重症疟疾病例从发病至就诊的平均时间为3.1 d,平均时间最长者在发病第12天才就诊;从发病至确诊的平均时间为5.2 d,其中11例在发病5 d后得到确诊。乡（镇）医院首诊确诊率为25.00%（1/4）,县级医疗机构首诊确诊率为90.91%（10/11）。26例重症疟疾病例通过抗疟治疗和临床对症治疗均治愈,无死亡病例。结论 重症疟疾临床表现复杂、并发症多、危害严重,加大对赴疟疾高度流行区人员的疟疾宣教、提高诊治单位医务人员对疟疾的诊治能力和镜检技能,可有效减少重症恶性疟病例发生、避免疟疾病例死亡。     

安徽省输入性疟疾疫情流行特征及重症病例影响因素分析

目的分析安徽省输入性疟疾疫情流行特征,探讨重症病例发生的影响因素,为进一步提高输入性疟疾防治水平提供科学依据。方法收集2012年1月1日到2017年4月18日安徽省输入性疟疾病例流行病学资料并分析。结果安徽省输入性疟疾发病人群主要为境外务工的青壮年男性。全省79%(83/105)的县、区有输入性病例发生。共收集到686例病例,其中重症62例,占9.04%,死亡4例,病死率0.58%。年龄、学历、初诊单位、初诊结果、发病到就诊时间、就诊到诊断时间是重症发生的影响因素。结论安徽省输入性疟疾病例中重症比例较高,要进一步提高监测敏感性,增强临床医生的诊断意识、加强对外出务工人员的健康教育。     

Marked differences in the prevalence of chloroquine resistance between urban and rural communities in Burkina Faso

BACKGROUND: Chloroquine (CQ) resistance has reached high levels in Africa in recent years. Little is known about variations of resistance between urban and rural areas. OBJECTIVES: To compare the rates of in vivo resistance to CQ and the prevalences of the main molecular marker for CQ resistance among young children from urban and rural areas in Burkina Faso. METHODS: The current analysis used the frame of a randomized controlled trial (ISRCTN27290841) on the combination CQ-methylene blue (MB) (n=177) compared to CQ alone (n=45) in young children with uncomplicated malaria. We examined clinical and parasitological failure rates as well as the prevalence of the Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 mutation. RESULTS: Clinical and parasitological failure rates of CQ-MB differed significantly between urban (70%) and rural areas (29%, p<0.0001). Likewise, CQ failure rates were higher in the urban setting. Matching this pattern, pfcrt T76 was more frequently seen among parasite strains from urban areas (81%) when compared to rural ones (64%, p=0.01). In the presence of parasites exhibiting pfcrt T76, the odds of overall clinical failure were increased to 2.6-fold ([1.33, 5.16], p(LR)=0.005). CQ was detected at baseline in 21% and 2% of children from the urban and the rural study area, respectively (p(Chi)=0.002). CONCLUSION: Even within circumscribed geographical areas, CQ efficacy can vary dramatically. The differences in the prevalence of pfcrt T76 and in CQ failure rates are probably explained by a higher drug pressure in the urban area compared to the rural study area. This finding has important implications for national malaria policies.     

Therapeutic responses to antimalarial and antibacterial drugs in vivax malaria

Plasmodium vivax is the most prevalent malaria infection and is an important cause of morbidity in Central and South America and Asia. P. vivax is generally sensitive to the common antimalarial drugs but high level resistance to chloroquine and/or pyrimethamine has been documented in some geographic locations. In the studies reviewed here, the therapeutic responses to antimalarial and antibacterial drugs in vivax malaria have been assessed in the Bangkok Hospital for Tropical Diseases. The evaluated drugs consisted of the eight most widely used antimalarial drugs and anti-bacterial drugs that possess antimalarial activities (tetracycline, doxycycline, clindamycin or azithromycin). The activities of these drugs in descending order of parasite clearance times were artesunate, artemether, chloroquine, mefloquine, quinine, halofantrine, primaquine, followed by the antibacterial drugs and lastly sulfadoxine-pyrimethamine. Clinical responses to sulfadoxine-pyrimethamine were also poor with evidence of high grade resistance in 42% of the patients. Of the four antibacterial drugs, clindamycin was more effective than azithromycin and can be an alternative to the tetracyclines. Except for chloroquine and mefloquine which have long plasma half lives and may therefore suppress first relapses, the cumulative cure rates for the short acting antimalarial drugs were similar. Double infection with Plasmodium falciparum was common and usually manifested 3-4 weeks following clearance of vivax malaria. The prevalence of cryptic falciparum malaria was 8-15% and was higher in patients treated with less potent antimalarial drugs. Follow-up studies have revealed that the relapse time in Thai patients with vivax malaria is on average only 3 weeks, but can be suppressed by the slowly eliminated antimalarial drugs such as chloroquine and mefloquine. For accurate comparison of relapse/recrudescence rates in vivax malaria, at least 2 month's follow-up is required. It can be concluded that in malarious areas of Thailand, double infection with P. falciparum and P. vivax is common affecting at least 25% of the patients and usually manifests as sequential illnesses. P. vivax in Thailand is sensitive to chloroquine but has acquired high grade resistance to sulfadoxine-pyrimethamine.     

Analysis of circulating populations of Plasmodium falciparum in mild and severe malaria in two different epidemiological patterns in Madagascar

Objective To investigate whether the severity of Plasmodium falciparum attack in endemic areas was associated with the multiplicity of infection (MOI) and/or with a particular genotype(s). Method In two areas of different malaria transmission pattern in Madagascar (Sainte‐Marie – mesoendemic and Tsiroanomandidy – hypoendemic) the number and the proportions of msp‐2 genotypes within isolates were determined for each patient using a capillary electrophoresis genotyping method. DNA sequencing was performed to identify the msp‐2 allelic family of dominant clones. Results Eighty six uncomplicated and 33 severe cases were included in Sainte‐Marie and 48 uncomplicated and 69 severe cases were included in Tsiroanomandidy. We found no association between the MOI and severity of malaria as the same mean number of msp‐2 genotypes was found in isolates from uncomplicated and from severe malaria cases (3.72 and 3.73, respectively, P>0.05). The study of the association of dominant clones with clinical status showed no particular genotype or allelic family associated with malaria severity. Conclusions Severity of malaria was not associated with higher MOI in our study. Severity did not appear restricted to some particular genotypes either. On the contrary, severe malaria appeared to be caused by very common genotypes in the studied areas. More comprehensive explorations including immunity and genetic factors of the host are needed to acquire new information about this complex condition.     

Socioeconomic and environment determinants as predictors of severe malaria in children under 5 years of age admitted in two hospitals in Koudougou district,Burkina Faso: A cross sectional study

Burkina Faso has a high incidence and death rate of severe malaria, especially for children under 5 years of age. Although the malaria elimination program is a high-priority public health project, finding an effective strategy for managing the problem is a major challenge. Understanding the various factors that contribute to the severity of malaria is essential in designing an effective strategy. In this study, parental and environmental factors associated with severe malaria in Burkinabè children were investigated in two hospitals in Koudougou Health District, Burkina Faso. Between July and September 2012, a cross-sectional study was used to test 510 children under 5 years of age (mean age: 23.5 months) admitted with suspected malaria. Each child was screened using a blood smear to identify whether he or she had severe malaria based on the criteria established by the World Health Organization (WHO). When a child was diagnosed with malaria, either severe or not severe, the parents were interviewed by a trained interviewer using a structured questionnaire. A logistic regression was used to identify the determinants of severe malaria and associated deaths. Of the 510 children having malaria, 201 (39.4%) had severe malaria. Most of the patients (54.9%) lived in rural areas. The main factors associated with severe malaria were low education level of the father, low socioeconomic status [odds ratio (OR) = 4.11, 95% confidence interval (CI) = 1.44–11.75], delayed treatment [OR = 4.53, 95% CI = 1.76–11.65], treating children at home as a typical practice when the child has a fever [OR = 3.24, 95% CI = 1.40–7.51], living in rural area [OR = 6.66, 95% CI = 3.36–13.22], and living beside a water gathering pond (OR = 1.67, 95% CI = 1.02–2.74]. Parental and environmental context associated with severe malaria for children under 5 years of age remains a serious public health problem that affects malaria outcomes in resource-limited areas. Promotion of early care is urgently required. Parents should be given information on the risks of not consulting a health facility when children exhibit symptoms of malaria.     

CTLA-4 positive T cells in contrast to procalcitonin plasma levels discriminate between severe and uncomplicated Plasmodium falciparum malaria in Ghanaian children

Procalcitonin (PCT) plasma levels and the fraction of CTLA-4-positive T cells are both elevated in acute Plasmodium falciparum malaria in human adults and the degree of elevation is positively correlated with other markers of disease severity, for example with parasitaemia. However, the clinical manifestations of malaria are strongly age-dependent and children from endemic areas carry the main disease burden. Therefore, we measured PCT plasma levels and CTLA-4 expression by T cells in four groups of children from the Ashanti Region in Ghana: asymptomatic children with or without parasitaemia, children with uncomplicated P. falciparum malaria and children with severe disease. PCT levels were highly elevated in both groups with acute malaria but they did not discriminate between uncomplicated and severe disease. In contrast, CTLA-4-expression by T cells was increased only in severe malaria. The fraction of CTLA-4 positive T cells in the blood of children with severe disease differed significantly from that in uncomplicated malaria, which was not elevated in spite of the high parasite loads observed in these children. This was unexpected, as in adults uncomplicated malaria is associated with a dramatic sixfold increase of the fraction of CTLA-4-positive T cells. The data from this study support the hypothesis that strong T cell activation as measured here by CTLA-4 expression is not just the by-product of a high parasite burden, but that it contributes to the pathogenesis of P. falciparum malaria.     

Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat,Central India: implications for the diagnosis of malaria and its control

Objective During an epidemiological study (January–July 2012) on malaria in forest villages of Central India, Plasmodium malariae‐like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species‐specific nested polymerase chain reaction (PCR) and DNA sequencing. Methods All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae. Results Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3‐day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum. Discussions Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re‐affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.     

间日疟患者外周血NK γδ T Treg细胞含量的变化

目的 分析间日疟原虫现症感染者外周血中NK、γδ T细胞、CD4^＋CD25^＋T淋巴细胞及其FOXP3的表达情况，以初步了解患者的一些细胞免疫特性。方法用流式细胞仪检测25例间日疟原虫现症感染者（AC）、13例免疫对照者（IC）及14例正常对照者（NC）外周血中NK、γδT细胞和CD4^＋CD25^＋T淋巴细胞的百分含量，同时观察CD4^＋CD25^＋T细胞中表达FOXP3群体的百分含量。结果AC组外周血NK细胞百分含量为8．48％，与NC组（15．53％）及IC组（17．69％）比较，明显降低（P均〈0．05）,AC组外周血γδT细胞、CD4^＋CD25^＋T细胞百分含量分别为4．32％和5．42％，较NC组（2．55％和3．94％）及IC组（2．70％和3．44％）明显升高（P均〈0．05）；AC组外周血CD4^＋CD25^＋T淋巴细胞中表达FOXP3的细胞数量为8．14％，低于NC组的11．09％及IC组的11．32％，但差异无统计学意义（P〉0．05）。结论间日疟原虫急性感染期患者外周血NK细胞数量减少，但γδT淋巴细胞增加；CD4^＋CD25^＋T淋巴细胞中表达FOXP3群体的细胞稍有降低。     

Drug resistance-virulence relationship in Plasmodium falciparum causing severe malaria in an area of seasonal and unstable transmission

The pathogenesis of severe Plasmodium falciparum malaria is still obscure, but is believed to be multi-factorial, and among the important factors are intrinsic parasite-properties. Here we investigated the association between clinical manifestation of P. falciparum malaria (an indicator of virulence) and two parasite properties--drug resistance and gametocyte production. Among 996 P. falciparum infections detected in the out-patient clinic of Gedarif Hospital in eastern Sudan, there was no significant association between the incidence of severe versus mild disease and the presence of resistant alleles at the chloroquine-resistance transporter locus (pfcrt-T76) and the multi-drug-resistance locus (pfmdr1-Y86). However, among severe cases, there was a significantly lower prevalence of parasites carrying resistant alleles among patients that died versus survived. There was a trend towards a higher gametocyte rate among severe malaria patients compared with uncomplicated malaria cases. These results are discussed in relation to the fitness of drug resistant parasites.     

High efficacy of two artemisinin-based combinations (artemether-lumefantrine and artesunate plus amodiaquine) for acute uncomplicated malaria in Ibadan, Nigeria

Objective To test the hypothesis that artesunate plus amodiaquine (ASAQ) is as effective as artemether–lumefantrine (AL) in the treatment of acute uncomplicated malaria in Nigerian children. Methods In an open label, randomized controlled clinical trial, children aged 6 months to 10 years were randomized to receive artesunate (4 mg/kg daily) plus amodiaquine (10 mg/kg daily) or AL (5–14 kg, one tablet; 15–24 kg, two tablets and 25–34 kg, three tablets twice daily). Both drug regimens were given for 3 days and follow‐up was for 28 days. Results A total of 132 children (66 in each group) were randomized to receive either ASAQ or AL. Day 28 cure rates in the per protocol (PP) population were 93% for ASAQ and 95% for AL (OR = 0.71, 95% CI = 0.12–3.99, ρ = 0.66). Using Kaplan–Meier product‐limit estimates of failure, the median survival time for ASAQ was 21 days and for AL 28 days (P = 0.294). PCR corrected day 28 cure rate for PP populations were 98.4% for ASAQ and 100% for AL. Both drugs were well‐tolerated. Conclusion ASAQ is as effective as AL and both combinations were efficacious and safe.     

Resistance of falciparum malaria to chloroquine and sulfadoxine-pyrimethamine in Afghan refugee settlements in western Pakistan: surveys by the general health services using a simplified in vivo test

Surveys of drug resistant falciparum malaria were conducted in several Afghan refugee settlements, distributed over a 700 km range in western Pakistan, during the transmission seasons of 1994 and 1995. Symptomatic malaria patients were recruited by a process of passive case detection at the refugees' basic health units. To facilitate follow-up by local health workers, a modified version of the WHO extended in vivo test was adopted in which blood smears were taken from each subject, and clinical symptoms recorded, at weekly intervals. Resistance to chloroquine and sulfadoxine-pyrimethamine was identified in every settlement. The frequency of chloroquine resistance ranged from 18% to 62%. Resistance occurred mostly as RI, with RII resistance never exceeding 11%. Resistance to sulfadoxine-pyrimethamine occurred at much lower frequencies, ranging from 4% to 25%. There was a resumption of clinical symptoms at the onset of parasite recrudescence in over 90% of cases. The policy of using chloroquine as first-line treatment might be changed in favour of sulfadoxine-pyrimethamine in most camps and areas of western Pakistan. The modified in vivo test was almost as accurate as the normal WHO in vivo test in identifying the grade of resistance, and should prove a useful tool for the monitoring of resistance to common antimalarials by district health services.     

江苏省输入性疟疾初诊时间及影响因素分析

目的　了解江苏省输入性疟疾病例回国后就医行为特征,分析病例初诊时间影响因素,为输入性疟疾病例早期发现、防止重症病例发生及继发传播提供科学依据。方法　在中国疾病预防控制中心传染病报告信息管理系统和寄生虫病防治信息管理系统中,收集2019年江苏省报告的输入性疟疾病例个案信息以及发病及初诊时间信息。对输入性疟疾病例回国后就医行为及流行病学特征进行描述性分析,采用多因素logistic回归分析探索病例回国后初诊时间影响因素。结果　2019年江苏省累计报告输入性疟疾病例244例,病例初诊时间在1～12 d,平均初诊时间（1.53 ± 1.65） d、中位初诊时间1 d。出现首发症状当天就医的病例数最多（76例,31.1%）,68例（27.9%）第2天就医、46例（18.9%）第3天就医、54例（22.1%）3 d后就医,其中3例初诊时间在1周以上。归国时间在1月（14例,5.7%）和12月（13例,5.3%）、年龄在41～50岁（32例,13.1%）的外出务工人员中初诊时间延误者比例较高。多因素logistic回归分析发现,归国时间在3月[比值比（OR）= 0.16, P = 0.03, 95%可信区间（CI）：（0.03,0.85）]及有境外疟原虫感染史者[OR = 0.36, P = 0.001, 95% CI :（0.19, 0.67）]的病例初诊时间相对较短。结论　江苏省输入性疟疾患者主动就医及时性有待提高,有疟原虫感染史者就医更及时。     

2015—2016年山东省由赤道几内亚输入的恶性疟原虫抗药性基因多态性分析

目的　了解山东省由赤道几内亚输入的恶性疟原虫抗性基因多态性情况。方法　采集2015—2016年山东省由赤道几内亚务工返乡的输入性恶性疟患者血样,提取疟原虫基因组DNA,对恶性疟原虫抗性基因Pfcrt、Pfmdr1、Pfdhfr、Pfdhps、K13进行套式PCR扩增、DNA测序和序列对比分析。结果　全部样本5种抗性基因目的片段均成功扩增和测序。Pfcrt野生型、突变型、混合型分别占72.8%、18.6%、8.6%,突变型全部为CVIET（下划线表示突变位点,下同）;Pfmdr1野生型、突变型、混合型分别占20.0%、61.4%、18.6%,突变型主要为YF和NF;Pfdhfr野生型、突变型、混合型分别占1.4%、98.6%、0,突变型主要为AIRNI;Pfdhps野生型、突变型、混合型分别占1.4%、94.3%、4.3%,突变型主要为SGKAA;Pfdhfr和Pfdhps完全抗性基因型IRNGE占8.6%;1.4%的样本K13基因发生A578S突变。结论　山东省由赤道几内亚输入的恶性疟原虫Pfcrt、Pfmdr1、Pfdhfr、Pfdhps、K13基因均发生不同程度突变;Pfcrt突变型比例较低,Pfmdr1、Pfdhfr、Pfdhps突变型比例较高;检测到K13基因发生A578S突变。     

Modulation by malaria infection of the induction of T lymphocyte-dependent delayed-type hypersensitivity and antibody formation to sheep erythrocytes in mice

A profound alteration of the inductive phase of delayed-type hypersensitivity and antibody formation to SRBC was found in malaria infected mice when sensitization with this antigen was performed intravenously at a critical time of the disease, but not after subcutaneous immunization, suggesting a major role for the spleen in the mechanism of immunodepression.