Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travellers

Real-time PCR diagnosis of malaria has advantages over traditional microscopic methods, especially when parasitaemia is low and when dealing with mixed infections. We have developed a new real-time PCR with specific genes in each Plasmodium species present only in one copy to identify the four pathogenic Plasmodium spp. for humans. The sensitivity was less than 25 parasites/microl. No cross-hybridisation was observed with human DNA or among the four Plasmodium spp. Using LightCycler PCR and conventional microscopy, we compared the diagnosis of malaria in patients from Vietnam and in returned European travellers with suspicion of malaria. In patients from Vietnam with suspicion of malaria, one mixed infection was observed by PCR only; the remaining data (54 of 55 patients) correlated with microscopy. In 79 patients without symptoms, low parasitaemia was detected in 7 samples by microscopy and in 16 samples by PCR. In returned travellers, PCR results were correlated with microscopy for all four species in 48 of 56 samples. The eight discrepant results were resolved in favour of real-time PCR diagnosis. This new real-time PCR is a rapid, accurate and efficient method for malaria diagnosis in returned travellers as well as for epidemiological studies or antimalarial efficiency trials in the field.     

Point-of-care testing for malaria outbreak management

A rapid antigen assay for malaria was performed on blood samples collected during a simultaneous outbreak of falciparum malaria and vivax malaria on a remote island in the Indonesian archipelago. During the outbreak, a total of 89 patients (4.3% of the population) were diagnosed with malaria within a week. Microscopic examination revealed 78 malaria slide-positive cases, of whom 49 (62.8%) were identified as P. falciparum, 7 (9.0%) as P. vivax and 22 (28.2%) as mixed P. falciparum and P. vivax infections. The rapid malaria assay showed excellent correlation with expert-confirmed routine microscopy for P. falciparum and P. vivax monoinfections and mixed infections with a parasite density >50 parasites/microl. Several slide-negative blood samples collected from febrile patients with clinical malaria tested positive in the rapid test. The estimated sensitivity calculated for the rapid test (91.0%) was slightly higher than that of microscopy (87.6%). The result indicates that rapid antigen detection for malaria could be a useful alternative to microscopy to reduce the workload during emergency outbreak situations.     

Demonstration by the polymerase chain reaction of mixed Plasmodium falciparum and P. vivax infections undetected by conventional microscopy.

Mixed malaria infections (Plasmodium falciparum and P. vivax) are suspected to occur at a greater frequency than is detected by conventional light microscopy. To determine this frequency we carried out a prospective 'blinded' comparison of diagnosis by conventional light microscopy and enzymatic amplification of the circumsporozoite gene extracted from dried spotted blood samples. Patients were previously healthy, active duty Thai soldiers assigned to a malaria risk area presenting with malaria. Microscopy (oil immersion objective at 1000 x magnification) involved examination of Giemsa-stained thick and thin blood films by an experienced microscopist. Whole blood samples (25 microliters) dried on filter paper were used for species-specific parasite deoxyribonucleic acid (DNA) amplification by the polymerase chain reaction (PCR) and hybridization with radiolabelled P. falciparum and P. vivax probes. Of 137 consecutive cases of malaria studied, 9% (3/32) of microscopically diagnosed P. falciparum infections and 5% (5/104) of microscopically diagnosed P. vivax infections were found to be mixed by the PCR/DNA probe systems, while 1 case was diagnosed as mixed by both microscopy and PCR. The possibility that malaria patients may have undetected mixed infections should be kept in mind because of the specific therapy required both for P. falciparum and for radical cure of P. vivax.     

A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO Informal Consultation on Recent Advances in Diagnostic Techniques and Vaccines for Malaria.

Recent advances in the diagnosis of Plasmodium falciparum infections have made it possible to consider supplementing light microscopy with a standardized dipstick antigen capture assay based on the detection of a parasite-specific protein, which is secreted by the asexual blood stages and immature gametocytes but not by the other stages. Field trials indicate that this dipstick assay provides consistently reproducible results, with a threshold of detection of P. falciparum parasitaemia similar to that obtained by high quality routine malaria microscopy and a specificity and sensitivity of around 90% compared with standard thick blood film microscopy. The stability, reproducibility, and ease of use of the assay clearly indicate that it has potential for application in the management of malaria, particularly at the peripheral health care level, provided its accuracy can be assured and that it can be made affordable. Consideration should be given to its wider use where operational requirements and resources so justify, and where decisions are based on adequate evaluation of the existing health delivery systems.     

Risk factors for imported fatal Plasmodium falciparum malaria, France, 1996-2003

Plasmodium falciparum malaria is a serious health hazard for travelers to malaria-endemic areas and is often diagnosed on return to the country of residence. We conducted a retrospective study of imported falciparum malaria among travelers returning to France from malaria-endemic areas from 1996 through 2003. Epidemiologic, clinical, and parasitologic data were collected by a network of 120 laboratories. Factors associated with fatal malaria were identified by logistic regression analysis. During the study period, 21,888 falciparum malaria cases were reported. There were 96 deaths, for a case-fatality rate of 4.4 per 1,000 cases of falciparum malaria. In multivariate analysis, risk factors independently associated with death from imported malaria were older age, European origin, travel to East Africa, and absence of chemoprophylaxis. Fatal imported malaria remains rare and preventable. Pretravel advice and malaria management should take into account these risk factors, particularly for senior travelers.     

Malaria incidence and prevention among European and North American travellers to Kenya

A longitudinal survey was conducted among travellers departing from Nairobi airport to determine the use of malaria prevention measures and assess the risk for malaria while travelling in Kenya. Among 5489 European and North American travellers, 68 different drug regimens were used for prophylaxis, and 48% of travellers used both regular chemoprophylaxis and more than 1 antimosquito measure during travel; 52% of 3469 travellers who used chemoprophylaxis did so without interruption during their travel and for 4 weeks after departure. Compliance was lowest among travellers who visited friends and relatives, who were young, or who stayed more than 3 weeks. Sixty-seven (1%) travellers experienced symptoms of malaria, but the diagnosis could be verified for only 16 of these. Long-stay travellers appeared to be at higher risk for malaria than short-stay travellers, and health information needs to be targeted especially to the former. Similar investigations are needed among international travellers to other malaria-endemic countries. With comparable data available, consistent and effective malaria prevention guidelines can be developed.     

多重聚合酶链反应技术诊断疟疾及混合感染的研究

目的建立一种特异、敏感、简便的疟疾聚合酶链反应（PCR）快速诊断方法。方法针对疟原虫SSU rRNA基因序列设计3条分别具有属、种特异性的引物,通过多重PCR反应扩增间日疟原虫和恶性疟原虫不同大小的DNA片段。结果 19份恶性疟原虫血样均扩增出长度为400 bp的特定基因片段,16份间日疟原虫扩增出长度为300 bp的特定基因片段,20份健康人群对照血样经PCR扩增均为阴性。结论该多重PCR检测系统具有高效、敏感、特异、稳定、简便等特点,适宜于大批量样品同时检测,对疟疾的快速诊断、鉴别混合感染有较高的应用价值。     

Delayed recognition of falciparum malaria from a non-endemic region in the Dominican Republic

A 28-year-old patient had suffered from fever, headache, abdominal pains and vomiting for the past three weeks. She had visited a region in the Dominican Republic where the risk of malaria is considered to be low. The complaints were initially regarded as a viral infection. Later, however, she was found to have severe falciparum malaria. She recovered completely following antibiotic therapy. Since November 2004, 17 cases of falciparum malaria have been reported world-wide among travellers to non-endemic regions in the Dominican Republic. Physicians should always consider the possibility of malaria in travellers because a timely diagnosis of falciparum malaria can be of vital importance.     

快速检测试剂条、镜检和PCR在疟疾诊断中的应用研究

目的采用快速检测试剂条（rapiddiagnostictest,RDT）、镜检及聚合酶链反应（polymerasechainreac-tion,PCR）,3种检测方法进行疟原虫检测,比较3种方法在疟疾诊断中的敏感性、特异性,为科学分析检测结果和探究RDT能否在基层替代疟原虫镜检提供依据。方法收集安徽省2012年1月～2013年3月确诊以及疑似病例抗凝血148份,血片148张,采用RDT进行检测,并与镜检法和PCR的结果对比分析其敏感性与特异性。结果148份血样采用RDT、镜检和PCR3种方法检测结果阳性率分别为43．24％、36．49％、37．84％,经配对x2检验RDT阳性率高于镜检和PCR;镜检和PCR均检出2例卵形疟,而RDT结果为阴性;以镜检为标准,对间日疟和恶性疟进行分析得出,RDT的敏感度为100％,特异度87％,阳性预测值81％,阴性预测值100％;以PCR为标准,对间日疟和恶性疟进行分析得出,RDT的灵敏度为100％,特异度89％,阳性预测值84％,阴性预测值100％。3种方法均检出间日疟16例,而对恶性疟分别为RDT检出48例,镜检检出36例,PCR检出38例。结论RDT对间日疟和恶性疟敏感性较高,可以覆盖全部阳性病例,在今后疟疾低流行时期RDT有望取代基层镜检对疟疾进行诊断。     

138例输入性疟疾病例的流行病学分析

目的 分析输入性疟疾病例的流行病学特征,为疟疾防控提供科学依据。方法 通过“中国疾病预防控制信息系统”和 “寄生虫病防治信息管理系统”,收集2016 - 2017年郑州市二七辖区医院网络报告的疟疾病例,进行流行病学个案调查。结果 2016 - 2017年郑州市二七区上报疟疾病例138例,全部是境外输入性病例,无本地感染病例,境外发病6例(占4.3%),国内发病132例(占95.7%)。男性136例(占98.6%),镜检结果以恶性疟为主104例(占75.4%),卵形疟13例(占9.4%),间日疟5例(占3.6%),阴性15例(占10.9 %),未做镜检1例(占0.7%)。127例有省级PCR结果反馈,镜检和PCR结果一致率为80.3%(102/127)。138例病例中,初次就诊诊断为疟疾69例(50.0%),住院治疗率90.6%。2016年和2017年并发症发生率分别为63.5%和10.9%。结论 该辖区报告疟疾病例为输入性,无本地感染病例,以恶性疟为主。初次诊断正确率低。     

The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.     

The detection of Plasmodium falciparum and P. vivax in DNA-extracted blood samples using polymerase chain reaction

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.     

套式PCR检测恶性疟原虫与间日疟原虫方法的建立及其应用研究

目的建立简便、灵敏、低成本的恶性疟原虫与间日疟原虫套式PCR检测方法,并探讨应用于间日疟原虫实验室检测的效果。方法制备抗凝静脉血的干滤纸血滴标本,采用干滤纸5%Chelex-100煮沸法提取疟原虫DNA,并进行套式PCR反应;通过比较PCR检测结果与疟原虫镜检结果,评价套式PCR检测恶性疟原虫与间日疟原虫方法的效果。结果在42份样本中,有34份血样PCR检测结果与镜检结果一致,占81%;5份镜检结果无法判断的血样,PCR检测为阳性或阴性;2份镜检结果为阴性的血样,套式PCR检测为阳性。结论套式PCR检测恶性疟原虫与间日疟原虫结果可靠,比镜检方法灵敏,有望在疟疾的实验室诊断中得到应用。     

Differences in automated depolarization patterns of Plasmodium falciparum and P. vivax malaria infections defined by the Cell-Dyn CD4000 haematology analyser

Of 1014 samples submitted for full blood count analysis and malaria screening, 854 were designated malaria-negative by blood film microscopy, 79 were unequivocally identified as Plasmodium vivax and 81 as P. falciparum. All samples were additionally analysed with the Abbott Cell-Dyn CD4000 haematology instrument, and leucocyte differential plots of 90 degrees polarized vs. 90 degrees depolarized (NEU-EOS plot) and 90 degrees depolarized vs. 0 degree light (EOS I plot) scatter were specifically examined for abnormal depolarization patterns. Depolarization pattern types were correlated with microscopy (species) results, and these correlations were consolidated by polymerase chain reaction analysis. All 854 microscopically-designated malaria-negative samples showed a type 1 (normal) CD4000 depolarization pattern. Abnormal pattern types 2, 3a and 3b were entirely restricted to one of the two malaria categories. Plasmodium falciparum malaria showed two CD4000 pattern types only; a 'normal' type 1 pattern was seen in 36/75 (48%) cases and the remaining 39 cases were all abnormal pattern type 3a. In contrast, most (79/85) P. vivax malaria cases showed a distinctive clustered EOS I population (types 2 and 3b patterns) that was not seen with P. falciparum. Automated depolarization analysis provides an effective means of detecting malaria-associated haemozoin, and the patterns of intracellular haemozoin further appear to provide species differentiation between P. falciparum and P. vivax.     

MalaQuick versus ParaSight F as a diagnostic aid in travellers' malaria.

In this study we assessed whether travellers can perform malaria rapid tests, following the provided information leaflet, and correctly interpret performed and pre-prepared test strips. Two Plasmodium falciparum testing systems, namely MalaQuick (ICT) and ParaSight F were used. Test performance and test interpretation of pre-prepared tests were compared. There was no significant difference in test performance between the 2 tests. Interpretation of prepared test strips in both test systems was very reliable in blood parasite densities between 0.1% and 2%, but major problems were encountered at low parasitaemia (< 0.1% blood parasites) and also in ParaSight F test strips showing high parasitaemia (> 2% blood parasites). Low parasitaemia ParaSight F test strips were correctly interpreted by 52.1% compared with 10.8% correct interpretations with MalaQuick (P < 0.0001). Correct interpretation of highly positive (> 2% blood parasites) pre-prepared test strips was higher with MalaQuick (96.8%) than with ParaSight F (33.8%), P < 0.0001. Both tests were associated with high levels of false-negative interpretations which render them unsuitable as self-diagnostic kits. Efforts must be made to assist lay individuals in test performance by technical test improvement, by equiping the test strips with an additional reading aid for interpretation, and by providing instruction by a skilled person.     

Imported malaria in Bordeaux: evaluation of the risk of infection with Plasmodium falciparum as a function of country visited

This study of imported cases of malaria, which was carried out in Bordeaux (France) in 1987-89, emphasizes the major part played by Plasmodium falciparum, especially in areas lying south of the Sahara in Africa, from where falciparum malaria is mainly imported to other countries. The study of these imported cases is strengthening our understanding of the epidemiology of malaria in relation to the country or area, whether the transmission occurs without interruptions or seasonally. The number of cases of P. falciparum per 1000 travellers (seen for vaccination against yellow fever at Bordeaux) gives an index for evaluating the risk of malaria. This risk changes with the epidemiological profile of falciparum malaria in the three major African ecosystems (rain forest, savannah, and sahelian belts), and is related to the progression of chloroquine resistance in Africa and influenced by the type of chemoprophylaxis proposed to travellers. The use of mefloquine for stays shorter than one month in Central Africa reduced the risk of malaria in 1988 and 1989, compared to 1987. [Editorial note. Recent data indicate some undesirable side-effects of mefloquine, e.g., its use during early pregnancy could lead to congenital defects.] Appropriate chemoprophylaxis and advice to travellers to areas lying south of the Sahara are therefore more and more necessary in order to arrest the increase in the number of imported falciparum malaria cases and reduce the number of serious cases, which are costly in terms of public health.     

Review of the prevalence of malaria in Zimbabwe with specific reference to parasite drug resistance (1984-96)

The response of Plasmodium falciparum malaria to antimalarial drugs, mainly chloroquine, the first-line drug of choice for the treatment of malaria in Zimbabwe is reported here for the period 1984-96. Earlier studies (1982-83) had shown that Zimbabwe was free from drug-resistant falciparum malaria. The first chloroquine-resistant cases of malaria were reported in 1984 in the Zambezi Valley in the north-east of Zimbabwe. Following this report several cases of chloroquine resistance have been reported throughout the malaria-endemic regions of the country thus prompting the Ministry of Health to develop a sustainable national surveillance strategy to monitor, on an annual basis, the spread and extent of P. falciparum resistance to antimalarial drugs available to the National Malaria Control Programme (NMCP). Of all the antimalarial drugs assessed in vivo, only chloroquine and halofantrine have shown resistance, while no resistance in vivo was observed for sulfadoxine-pyrimethamine (Fansidar), quinine and mefloquine. The study shows the need to replace chloroquine with alternative antimalarial drugs in areas where chloroquine resistance is high, and an increase in the drug pool against malaria is also recommended considering that all the alternative antimalarial drugs available to the NMCP have faced resistance in various parts of the world.     

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.     

输入性三日疟原虫的实验室诊断

目的 分析输入性三日疟疾的实验室诊断,减少三日疟的误诊与漏诊。 方法 采用厚薄血膜涂片形态学检查、巢氏PCR对疟原虫18S rRNA基因进行扩增等方法确诊输入性三日疟疾病例。 结果 经过形态学和巢氏PCR检测发现首诊结果为未检出、检出未分型和诊断为恶性疟的7例患者感染三日疟原虫,有一例为间日疟原虫和三日疟原虫混合感染。 结论 在消除疟疾的地区,应当持续的进行疟原虫显微镜镜检技术的培训和分子生物学技术的推广应用,以满足现阶段输入性疟疾病例正确诊断和分型要求,尤其是罕见和少见虫种鉴定要求。     

2005-2008年我国疟疾病例诊断和报告情况分析

目的分析我国不同类型疟疾病例诊断和报告中存在的问题,为制订我国疟疾控制和消除策略和措施提供参考。方法以中国疾病预防控制中心疾病监测信息管理系统报告的疟疾病例为基础,描述和分析我国2005-2008年报告的不同类型疟疾病例的构成,以及不同类型疟疾病例和不同单位报告病例的诊断方式、病例的发病-诊断时间等情况。结果 2005-2008年我国报告病例数总体呈下降趋势,降幅为71.77%,未分型疟疾仍占有一定比例;恶性疟和间日疟均以实验室诊断为主,实验室诊断构成比分别为91.44%和71.14%,28.22%的间日疟为临床诊断病例;22.45%未分型疟疾为实验室诊断病例;在各类型病例报告单位中,恶性疟、间日疟来自乡镇医院报告的病例分别占37.54%和71.79%,其中恶性疟报告病例33.41%来自于综合医院;恶性疟、间日疟和未分型疟疾的发病-诊断时间分别为72、96和72h。结论尽管我国疟疾病例诊断方式以实验室诊断为主,但存在诊断条件缺乏、诊断能力不足的情况,应进一步加强基层医疗单位的疟疾实验室诊断能力,实现我国疟疾消除的目标。