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.     

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.     

Cryptic Plasmodium falciparum parasites in clinical P. vivax blood samples from Thailand

Polymerase chain reaction detection revealed cryptic Plasmodium falciparum infections in 21 of 160 samples collected from Thai patients diagnosed (by microscopy) with vivax malaria. The clinical and biological significance of these mixed infections is discussed in the context of chloroquine resistance and the low inoculation rates which characterize malaria epidemiology in Thailand.     

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.     

一种简易疟原虫PCR模板制备方法

目的:寻找一种高效、简便、快速疟原虫PCR模板制备方法。方法:收集2010年-2011年自非洲、东南亚等疟疾流行区回国人员中疟疾现症患者(显微镜检查阳性),采滤纸血,用蒸馏水直接溶血法和Chel-ex-100法分别制备疟原虫PCR模板,采用巢式PCR技术检测疟原虫ss RNA基因,并对两法结果进行比较。结果:分别用蒸馏水直接溶血法和Chelex-100法制备疟原虫PCR模板32个,其中恶性疟原虫26个,间日疟原虫6个,直接溶血法巢式PCR检测全部出现特异性扩增带,而Chelex-100法均未出现扩增带。结论:蒸馏水直接溶血法是一种较为理想制备疟原虫PCR模板的方法。     

Concurrent dengue and malaria due to Plasmodium falciparum and P. vivax

Concurrent infections of dengue and malaria are rare. We report a case of dengue fever with acute malaria due to Plasmodium falciparum and P. vivax in which the presence of mixed infection with P. vivax was overlooked and confirmed later on during recurrence of the fever that had initially responded to conventional antimalarial treatment and symptomatic treatment for dengue fever. We suggest that in concurrent infections of dengue and malaria, possibility of mixed infection with various Plasmodium species should be excluded to ensure a better treatment outcome.     

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.     

Imported malaria in Kuwait

The number of imported malaria cases in Kuwait rose from 87 in 1980 to 504 in 1983, an increase of 579%. The continued resurgence of malaria in endemic zones, improved diagnostic techniques and a heightened awareness of imported malaria have contributed to the increase in the number of microscopically proved cases. Thick blood films fixed in acetone and stained in Giemsa proved a rapid method of diagnosis; species identification on the basis of a thin film on the same slide was performed with ease. Malaria was acquired in 38 countries. Most patients were young male adults. Most of the cases were due to Plasmodium vivax originating from India, although an increasing number of P. falciparum cases are also now being diagnosed from there. P. falciparum infections were evenly distributed throughout the year and most cases presented within 14 days of their arrival in the country. The highest number of P. vivax cases were diagnosed between May and October, when heat stress might have been a factor in precipitating a clinical attack of an infection previously acquired in the endemic zone. Attention is drawn to the importance of delayed attacks of P. vivax and, in semi-immunes, of P. falciparum. The time interval involved in establishing a history of "recent" travel in clinically suspected cases of malaria needs to be more clearly defined in each geographical area. Cases of induced malaria due to transfusion, accidental and congenital infections were identified. The fatality rate due to P. falciparum infections was low. In terms of the risk of renewed transmission, Kuwait may be considered a vulnerable area.     

Malaria relapse and recrudescence among travellers to the tropics

This study describes 14 cases of relapse and recrudescence of malaria, treated between 1991 and 2003. In that period, 146 patients were hospitalized in the Clinic of the Institute in Gdynia: 20 women and 126 men. In 103 cases the disease was caused by Plasmodium falciparum, in 31 cases by Plasmodium vivax, in 5 cases by Plasmodium malariae, and in 2 cases by Plasmodium ovale. Five patients were found to have mixed infections, with either P. falciparum and P. vivax or P. falciparum and P. ovale. Relapses in patients previously treated in the country or abroad accounted for 9.6% of all the treated cases of malaria. Recrudescences and relapses were diagnosed of both the tropical malaria (6 cases), and the tertian malaria caused by P. vivax (4 cases). Moreover, in 4 patients diagnosis was made of secondary malaria due to P. vivax infection, while the primary attack was caused by invasion of P. falciparum. Also discussed was the issue of drug-resistance of plasmodia and the resulting problems with the treatment.     

Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region

The circumsporozoite protein (CSP) of the Plasmodium vivax infective sporozoite is considered to be a major target for the development of recombinant malaria vaccines. The Duffy blood group molecule acts as the red blood cell receptor for P. vivax. We review the frequency of P. vivax CSP variants and report their association with the Duffy blood group genotypes from Brazilian Amazon patients carrying P. vivax malaria. Peripheral blood samples were collected from 155 P. vivax-infected individuals from five Brazilian malaria-endemic areas. The P. vivax CSP variants and the Duffy blood group genotypes were assessed using PCR/RFLP. In single infections, the VK210 variant was the commonest followed by the P. vivax-like variant. The typing of P. vivax indicated that the frequency of variants among the study areas was significantly different from one to another. This is the first detection of the VK247 and P. vivax-like variant in single infections in endemic areas of Brazil. Association of the CSP P. vivax variants with the heterozygous Duffy blood group system genotype was significant for VK210 single infection. These observations provide additional data on the Plasmodium-host interactions concerning the Duffy blood group and P. vivax capability of causing human malaria.     

Plasmodium malariae in Haitian refugees, Jamaica

Since 1963, reported malaria transmission in Haiti has been restricted to Plasmodium falciparum. However, screening of Haitian refugees in Jamaica in 2004, by microscopic examination, identified P. falciparum, P. vivax, and P. malariae. PCR confirmed the P. malariae and P. falciparum but not P. vivax infections. DNA sequencing and rRNA gene sequences showed transmission of P. malariae. This report confirms that P. malariae is still being transmitted in Haiti.     

逆转录聚合酶链反应检测疟原虫混合感染的研究

〔目的〕建立在同一反应体系中能同时检测和鉴定恶性疟、间日疟的一步逆转录聚合酶链反应（RT-PCR）检测方法,并用于国境口岸归国劳务人员的疟疾检测。〔方法〕以疟原虫小亚单位核糖体核糖核酸基因为靶片段,设计恶性疟原虫和间日疟原虫的上游通用引物和各自的下游特异性引物,在同一反应体系中同时扩增恶性疟原虫和间日疟原虫的特异片段,对疟疾患者的PCR产物进行序列测定和分析。〔结果〕用建立的RT-PCR检测方法对广东口岸回国劳务人员中发现的2名疟疾患者的不同采样时间的全血进行检测,2006年10月和2007年1月采集的样本被分别扩增出预期大小为360bp和450bp特异扩增带,推测2名患者为恶性疟原虫和间日疟原虫的混合感染,其在入境时处于恶性疟的发作期和间日疟的潜伏期,均属于典型的集体输入性疟疾案例。〔结论〕RT-PCR检测疟原虫方法具有敏感性高、特异性强的特点,对国境口岸疟疾诊断、镜检质量控制和流行病学研究具有较大的实用价值。     

Therapeutic response of multidrug-resistant Plasmodium falciparum and P. vivax to chloroquine and sulfadoxine-pyrimethamine in southern Papua, Indonesia

To determine the level of antimalarial drug resistance in southern Papua, Indonesia, we assessed the therapeutic efficacy of chloroquine plus sulfadoxine-pyrimethamine (CQ+SP) for Plasmodium falciparum infections as well as CQ monotherapy for P. vivax infections. Patients with P. falciparum failing therapy were re-treated with unsupervised quinine+/-doxycycline therapy and those with P. vivax with either unsupervised quinine+/-doxycycline or amodiaquine. In total, 143 patients were enrolled in the study (103 treated with CQ+SP and 40 with CQ). Early treatment failures occurred in four patients (4%) with P. falciparum and six patients (15%) with P. vivax. The failure rate by Day 28 for P. vivax was 65% (95% CI 49-81). After PCR correction for re-infections, the Day 42 recrudescence rate for P. falciparum infections was 48% (95% CI 31-65). Re-treatment with unsupervised quinine+/-doxycycline resulted in further recurrence of malaria in 48% (95% CI 31-65) of P. falciparum infections and 70% (95% CI 37-100) of P. vivax infections. Eleven patients with recurrent P. vivax were re-treated with amodiaquine; there were no early or late treatment failures. In southern Papua, a high prevalence of drug resistance of P. falciparum and P. vivax exists both to first- and second-line therapies. Preliminary data indicate that amodiaquine retains superior efficacy compared with CQ for CQ-resistant P. vivax.     

A stable, oligosymptomatic malaria focus in Thailand

Blood from most of the 250 residents of a non-migratory farming village in south-eastern Thailand was visually examined for malaria parasites monthly for 2 years. Nearly 97% of the population had at least one (median = 5) patent Plasmodium falciparum infection per year; 72% had one due to P. vivax (median = 1). This contrasted with a slide positivity rate of 17% calculated from 12 months of passive case detection before the study began. Children 1-9 years old had the highest mean monthly prevalence (51%) and highest geometric mean density (10/500 white blood cells) of P. falciparum. Fewer than half the expected number of mixed infections were found but these were more common at high densities of P. falciparum. Individuals over 19 years old comprised 52% of the population but accounted for only 18% of P. vivax and 32% of P. falciparum gametocytaemias. Fever rates were marginally higher in those below 10 years old (8%) but occurred with equal frequency in those with patent infections or negative. The spleen rate (89% stage 1) was 24% in those under 15 years old and 7% in those older. No malaria mortality was seen P. falciparum cases treated for 10 d with quinine+tetracycline (QT) cleared the infection as often as those given one dose of mefloquine+sulfadoxine+pyrimethamine (MSP); both treatments reduced densities in cases not cured. Apparently unsupervised compliance was no better with MSP than with QT. The role played by hyperendemic, cryptic foci in Asian epidemics of malaria may have been underestimated.     

Development of a single tube hemi-nested PCR for genus-specific detection of Plasmodium in oligoparasitemic patients

Primers targeting the Plasmodium small-subunit (SSU) rDNA were designed to amplify DNA from P. vivax, P. falciparum, P. malariae, and P. ovale, using conventional PCR, two-step nested PCR (HNPCR), and single tube hemi-nested PCR (STHNPCR). The limit of detection of parasite DNA for the conventional PCR, HNPCR, and STHNPCR were 10 pg, 0.01 pg, and 0.1 pg, respectively, indicating that the STHNPCR is 100-fold more sensitive than conventional PCR, and only 10 times less sensitive than HNPCR. In addition, the detection limit was also defined using blood from a patient infected with P. falciparum. Using the saponin method, the detection limit of the conventional PCR, HNPCR, and STHNPCR were 70, 0.7, and 0.07 parasites/microl, respectively. Finally, the three techniques were evaluated using blood from 30 patients receiving antimalarial treatment, and negative by microscopy and conventional PCR. The HNPCR could still detect specific DNA in 16/30 patients, whereas STHNPCR detected parasite DNA in 10/30 patients, but the difference was not statistically significant. No significant correlation was found between presence of clinical manifestations and presence of parasite DNA, detected by either HNPCR or STHNPCR. We conclude that these sensitive molecular diagnostic systems can be used for the diagnosis of asymptomatic oligoparasitemic patients.     

Induced malaria and antibody titres in acute infections and in blood donors in Kuwait

Concurrent with the increase in the number of imported cases of malaria into non-endemic Kuwait during the past 5 years, induced infections have been identified for the first time. We report 10 such cases over a 4-year-period. Of 8 transfusion-induced infections, 4 were due to Plasmodium falciparum and 4 to P. vivax. The mean incubation period for P. falciparum patients was 13 d and for P. vivax, 17 d. An accidental syringe-needle transmission and a congenital infection were due to P. falciparum and P. vivax respectively. Malarial antibody levels were assayed on commercially-available cultured P. falciparum schizonts by the indirect fluorescent antibody (IFA) test. To establish a base line, the sera of patients with blood film-confirmed P. falciparum and P. vivax were assayed. 96% of the P. falciparum sera were positive, the geometric mean titre (GMT) being 10,280. However, all sera from P. vivax patients were reactive but the GMT was lower at 505. 28% of sera from Kuwaitis and 45% of sera of a consecutive group of blood donors were also reactive, the respective GMTs being 38 and 51. The risk of transfusion malaria was calculated as 79 per million units drawn, an unacceptably high figure for a non-endemic country. We suggest a revised blood donor policy.     

Susceptibility to vivax malaria in Ethiopia.

Plasmodium vivax prevalence rates for Nilotic and Hamitic-Semitic residents of an Ethiopian town were compared. Over a ten-year period, 8,316 blood films from Nilotes were examined and 59 P. vivax infections (0.7%) were diagnosed. In 1,630 films from Hamito-Semites, 75 probable P. vivax infections (4.6%) were found. The problem of morphological differentiation between P. vivax and P. ovale was evaded by combining the two diagnoses. P. vivax/ovale infection rates for Hamitic-Semitic subjects were higher than for Nilotic subjects in all age groups. It was concluded that the two populations are inately different in susceptibility to patient infection with vivax malaria.     

Haemozoin as a marker of placental parasitization

Both Plasmodium vivax and P. falciparum malaria can cause the delivery of low birthweight babies. In this report, we have quantitated haemozoin levels in placentas from women living on the Thai-Burmese border in a region of low transmission for both P. falciparum and P. vivax malaria from June 1995 to January 2000. P. falciparum malaria infections during pregnancy lead to the accumulation of haemozoin (malaria pigment) in the placenta, especially in infections near term and in primigravid pregnancies. Haemozoin concentration was not associated with adverse birth outcomes. Women with P. vivax infections during pregnancy do not have measurable levels of placental haemozoin suggesting that P. vivax-infected erythrocytes do not accumulate in the placenta as much as P. falciparum-infected ones.     

Distribution of Plasmodium vivax variants (VK210, VK247 and P. vivax-like) in three endemic areas of the Amazon region of Brazil and their correlation with chloroquine treatment

The present study evaluated the glass fibre membrane (GFM)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique for genotyping the Plasmodium vivax variants, to verify the distribution of P. vivax variants (VK210, VK247 and P. vivax-like) in parts of Brazil and their correlation with levels of parasitaemia, previous malaria experience and clearance of parasitaemia linked to different treatment schedules. The samples were taken from individuals living in Macapá, Porto Velho and Belém, all of which are endemic areas of vivax malaria in the Amazon region of Brazil. Blood samples were collected on GFMs. The gene that codes for the circumsporozoite proteins of P. vivax variants was amplified by PCR and the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by ELISA. The GFM-PCR-ELISA technique was shown to be accurate for epidemiological surveys of the vivax complex. All variants were detected in all 3 areas, but only P. vivax VK210 was found as a single agent of infection, while the other 2 occurred as mixed infections. The P. vivax-like variant was found to be associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the P. vivax variants was linked with a previous malaria experience. In all cases parasitaemia clearance was identical regarding the type of treatment and consequently it is not possible to confirm the previously reported correlation between P. vivax genotype and response to chloroquine.     

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.