Cloning and Sequence Analysis of Recombinant Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-142 kDa) In pTZ57R/T Vector

Background:

Carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 is considered as an important antigen in blood stage. Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.

Methods:

In the present study, the genetic diversity of cloned PvMSP-1₄₂ kDa gene from an Iranian patient is analyzed. Parasite DNA was extracted from a P. vivax - infected patient in Iran. The region of PvMSP-1₄₂ kDa was amplified by PCR, cloned into pTZ₅₇R/T vector and then sequenced.

Results:

Sequencing of cloned PvMSP-1₄₂ kDa gene clearly has a high degree of homology (95%) with reference Sal-I sequence and also with the homogeneous sequences from some studied countries (97%). Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-1₄₂ kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-1₃₃ kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-1₁₉ kDa). 2 out of 38 mutations were found as to be novel haplotypes.

Conclusion:

High similarity of cloned PvMSP-1₄₂ kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.     

Genetic Variation of MSP-1 Gene in Plasmodium vivax Isolated from Patients in Hormozgan Province,Iran using SSCP-PCR

Background

The main goal of present study was to detect polymorphism in MSP-1 gene which is a major blood stage candidate for vaccine in Plasmodium vivax by Single Strand Conformational Polymorphism-Polymerase Chain Reaction (SSCP-PCR).

Methods

During 2008 to 2010 fifty samples were collected from Iranian patients with P. vivax in Hormozgan Province, southern Iran. All of the samples were detected by microscopical examination. Amplification of MSP-1 gene was done by PCR after DNA extraction. Single strand DNAs due to using in SSCP, was electrophoresed on polyacrylamid- Bisacrylamid gel then banding patterns were revealed by silver-staining method. Sequencing as a typing method was performed for some isolates.

Results

All of the 50 isolates were positive microscopically. Totally 12 (24%) isolates showed 440 bp and 38 (76%) showed 500 bp in PCR assay. SSCP analysis revealed four banding patterns. Pattern I (10/50), Pattern II (12/50), Pattern III (27/50), and Pattern IV (1/50). The results sequencing analysis of the MSP-1 gene in 19 isolates revealed diversity in nucleotides and amino acid in Iranian P. vivax isolates.

Conclusion

Our study confirms that the SSCP-PCR is a rapid method for detecting polymorphism in MSP-1 gene in P. vivax. The presence of different haplotypes in MSP-1 gene shows that several P. vivax strains exist in malaria endemic areas of Iran.     

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran

Background

Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran.

Methods

Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced.

Results

A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates.

Conclusion

Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.     

Retinal haemorrhage in vivax malaria

Retinal haemorrhage is often observed in patients with Plasmodium falciparum, especially when combined with cerebral malaria. However, few cases of retinopathy have been reported in P. vivax malaria. Benign tertian malaria has re-emerged among soldiers in the South Korean demilitarized zone since 1993. We report an indigenous case of retinal haemorrhage caused by P. vivax and review the relevant literature.     

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.     

Fatal Case of Plasmodium vivax Malaria in a Splenectomized Patient

Malaria is a major problem in tropical and sub-tropical countries, with high morbidity and mortality. Splenectomy makes patients more susceptible to serious bacterial and parasitic infections. We report for the first time in Iran a fatal case of Plasmodium vivax malaria, confirmed by microscopic and molecular (Semi-nested multiplex PCR) tests in a patient who had undergone splenectomy due to hemolytic anemia.     

In vivo Susceptibility of Plasmodium vivax to Chloroquine in Southeastern Iran

Background

Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province, southeastern Iran.

Methods

A total 270 subjects with symptomatic malaria and confirmed P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonucleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0, 5, 6, 7, and 28.

Results

P. vivax was cleared in 15%, 50%, 95%, and 100% of patients on days 1, 2, 3, 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7, 14 and 28. Mean (±standard deviation) parasite clearance time was 2.41 (±0.8) days.

Conclusion

P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compatible with results of neighboring countries Pakistan and Afghanistan.     

用间日疟原虫裂殖子表面蛋白1(PvMSP-1)基因分析福建输入性疟疾病例

[目的]探讨福建输入性疟疾的分子流行病学特征,追踪传染源。[方法]病人血样用套式PCR-RFLP检测,扩增PvMSP-1基因ICB5一ICB6片段。测定DNA序列并进行BLAST相似性搜索和进化树分析。[结果]16例镜检确诊的间日疟病人血样经套式PCR—RFLP检测,7份属Sal-1型,1份为Belem型,8份为重组Ⅲ型。对其中7份样本PCR产物进行DNA直接测序,发现没有两个DNA序列完全相同,PvMSP-1基因ICB5-ICB6片段存在高度多态性,DNA序列搜索结合流行病学词查可初步确定感染来源。[结论]PvMSP-1基因用于疟疾基因分型和判断感染来源有一定价值,可作为疟疾分子流行病学调查的方法之一。     

Estimates of short- and long-term incubation periods of Plasmodium vivax malaria in the Republic of Korea

With the current epidemic of vivax malaria closely associated with the demilitarised zone along the border between North and South Korea, it has been suggested that the incubation period tends, in part, to be prolonged. Based on the detailed travel history of cases from 2000 to 2003 who reside in non-malarious areas, statistical estimates of the incubation periods were obtained. The data suggest that cases fall into two categories with short- and long-term incubation periods, respectively. Of 416 cases with available information, 72 and 79 successfully met our criteria for inferring the durations of short- and long-term incubation periods. The mean short- and long-term incubation periods were estimated to be 26.6 days (95% CI 21.0-32.2) and 48.2 weeks (95% CI 46.8-49.5), respectively. The maximum likelihood method was used to fit gamma and normal distributions to the short- and long-term incubation periods, assisting prediction of the frequency distribution of the overall incubation period, which exhibited a bimodal pattern. We postulate that the observed distribution reflects adaptation of the parasite to the seasonal population dynamics of the vector, Anopheles sinensis, ensuring continued transmission of vivax malaria in this temperate zone.     

Detection of Asymptomatic Carriers of Plasmodium vivax among Treated Patients by Nested PCR Method in Minab,Rudan and Bashagard,Iran

Background

Plasmodium vivax is the most widespread species of Plasmodium in humans and causing about 80 million clinical cases annually. This study was undertaken to detect P. vivax in asymptomatic treated vivax malaria patients to trace latent/sub-patent malaria infection.

Method

The venous blood of all detected cases with P. vivax in Bashagard, Minab and Roodan Districts in Hormozgan Province from 2009 to 2010 was examined by microscopic and nested PCR methods for presence of the parasite.

Results

In microscopic examination of peripheral blood smears, all samples were negative for the presence of the parasites. But, we detected two P. vivax related bands in the electrophoresis of the nested PCR products (120 bp).

Conclusion

Following up the malaria cases after treatment by a combination of methods, or new diagnostics such as RDTs can be included in the priorities of malaria elimination program in Iran.     

Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran

Background

Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far.

Methods

P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42–488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing.

Results

Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador.

Conclusions

We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.     

Serum cytokine profiles in patients with Plasmodium vivax malaria: a comparison between those who presented with and without hepatic dysfunction

The aim of this study was to compare the serum cytokine profiles of Plasmodium vivax malaria patients who presented with and without hepatic dysfunction. This is a retrospective analysis of 74 consecutive cases of P. vivax malaria seen at 3 military hospitals near the Demilitarized Zone in South Korea from 1999 to 2000. All patients studied were adult active duty servicemen. On admission, the mean (+/- SEM) age of the patients who presented with (n = 36) and without hepatic dysfunction (n = 38) was 21.6 +/- 0.24 and 22.5 +/- 0.44 years, respectively (P = 0.72). On admission, there was no significant difference between the 2 patient populations in terms of mean temperature, haemoglobin level, haematocrit, total white blood cell count, platelet count, parasite index, and serum concentration of transforming growth factor-beta. Plasmodium vivax malaria patients who presented with hepatic dysfunction had significantly higher mean serum concentrations of soluble Fas ligand, interleukin (IL)-l, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma than those without hepatic dysfunction, suggesting the involvement of these cytokines in the development of hepatic dysfunction. The mean serum concentration of IL-12 was significantly lower in patients with hepatic dysfunction. The mean body temperature was not significantly different between the 2 patient populations.     

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.     

Sulfadoxine-pyrimethamine plus artesunate compared with chloroquine for the treatment of vivax malaria in areas co-endemic for Plasmodium falciparum and P. vivax: a randomised non-inferiority trial in eastern Afghanistan

Chloroquine (CQ) is an effective treatment of choice for vivax malaria in most settings, but with the spread of CQ-resistant Plasmodium falciparum, many countries now use artemisinin-based combination therapy for treatment of falciparum malaria. In areas co-endemic for falciparum and vivax malaria incorrect differential diagnosis is always a risk. In Afghanistan the adoption of sulfadoxine-pyrimethamine plus artesunate (SP+AS) as first-line falciparum treatment raises the prospect of a significant proportion of vivax malaria being misdiagnosed and treated with the combination. SP is considered to have limited efficacy against vivax malaria, and the efficacy of SP+AS against Plasmodium vivax has not been established in areas that are using SP+AS. A randomised, non-inferiority trial comparing SP+AS with CQ monotherapy was undertaken on 190 vivax malaria patients in eastern Afghanistan. Standard WHO procedures for in vivo evaluation of antimalarial drugs were followed. A total of 180 individuals completed the trial to day 42. Using a per protocol analysis, both regimens resulted in > or =96% treatment success at 28 d, but significantly more cases failed in the CQ arm (46%) than in the SP+AS arm (24%) by day 42. In areas where vivax infections might be misdiagnosed as falciparum infections and treated with SP+AS, patient management would be as good, or better than, with the standard CQ treatment.     

Effectiveness of primaquine terminal prophylaxis against late primary attacks of Plasmodium vivax malaria: a case-control study among troops of the Republic of Korea army

Plasmodium vivax malaria is an important cause of morbidity among troops operating in endemic areas near the Demilitarized Zone in the Republic of Korea (ROK). The ROK Army has been administering antimalarial chemoprophylaxis to those troops at greatest risk of malaria since 1997. The number of recipients increased from 15000 in 1997 to 90000 in 2001. We undertook a case-control study to estimate the effectiveness of primaquine prophylaxis against late primary attacks of P. vivax malaria in ROK Army troops. Microscopically confirmed cases of P. vivax malaria were identified through hospital-based surveillance. Controls were matched by unit. Between 1 November 2001 and 31 May 2002, 68 cases and 137 matched controls with confirmed chemoprophylaxis status were enrolled. The estimated effectiveness of primaquine prophylaxis was 32% (95% CI 23-63%). Our results suggest that the effectiveness of primaquine prophylaxis against late primary attacks of P. vivax malaria may be insufficient for soldiers of the ROK Army.     

Compliance to recommendations for the management of curative treatment of Plasmodium vivax/ovale infections

Objectives

We assessed the compliance to recommendations for the routine management of Plasmodium vivax/ovale malaria, and analyzed the impact of discrepancies on the quality of care.

Patients and methods

We reviewed the cases of P. ovale and P. vivax malaria treated at the Besançon University Hospital, France (2008–2013).

Results

Chloroquine was prescribed in 44% of the 18 cases (4 due to P. ovale, 14 to P. vivax). Radical cure with primaquine was prescribed after the first bout of malaria for 6 patients. The primaquine dose prescribed was inferior to the recommended one for 4 patients. The mean delay between schizonticide treatment and primaquine cure was 43 days.

Conclusions

The delay before access to primaquine radical cure was the only parameter, likely to alter treatment effectiveness, but also difficult to shorten. Future national guidelines should take into account that not all patients have access to primaquine treatment immediately after schizonticide treatment.