A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.     

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.     

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.     

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.     

Malaria in Mazandaran,Northern Iran: Passive Case Finding During 1997-2012

Background

Malaria is one of the most important parasitic diseases in tropical and temperate regions. The aim of this study was to determine the trend of malaria in Mazandaran Province, northern Iran during 1997-2012.

Methods

This retrospective study was conducted from 1997 to 2012. The population''s study was individuals who registered at health centers of Mazandaran Province. Peripheral blood smear were prepared for each case, stained with Giemsa and examined by light microscope. In addition to demographic data, other parameters including Slide Positive Rate (SPR), Annual Parasite Incidence (API) and Annual Blood Examination Rate (ABER) were analyzed.

Results

In total, 844 cases of malaria were reported. Plasmodium vivax was predominant species with 821 cases (97.4%). The number of malaria cases increased from 1997 to 2005 and then decreased to 3 cases in 2011. Some cities had not reported any cases during last three years. The highest infection rate, 163(20.07%), was seen in 2001-02. The SPR had the highest value (0.54%) in 2004-05. The maximum API and ABER were observed in 2001-02 and 1997-98. 641(75.9%) of cases were imported from hyperendemic areas such as Afghanistan and South-eastern Iran and 94 (11.1%) malaria patients were recorded as introduced cases. The highest infection rate of malaria (21.3%) was seen in Babolsar.

Conclusion

Extensive malaria control should be continued to Mazandaran to become malaria-free region and in prevention of re-introduction stage.     

The Rate of Plasmodium vivax Infectivity within Gloucose-6-Phosphate Dehydrogenase (G6PD) Deficient Individuals in Hormozgan Province,Iran

Background

One of the most important enzymatic disorders that interact with malaria is deficiency of G6PD (Gloucose-6-phosphate dehydrogenase). This enzyme protects red blood cells from hydrogen peroxide and other oxidative damages. Distribution of this enzyme deficiency usually accompanies with low level distribution of malaria disease in most malarious areas. So this hypothesis may be considered that the G6PD deficiency could be protective against malaria.

Methods

Totally 160 samples were taken from vivax malaria infected and non-infected individuals. Preparing blood smears and quantitative test for G6PD deficiency were employed for all of the samples. To ensure accuracy of the malaria in negative samples besides using microscopical examination, semi-nested multiplex PCR was also performed for the two groups.

Results

In microscopical examination 36 and 124 samples were vivax malaria positive and negative respectively. Out of 36 P.vivax positive cases 3 (8.3%) cases were detected to be G6PD deficient versus 30 (24.2%) cases out of 124 P. vivax negative cases. The results showed a significant differentiation between P. vivax positive and P. vivax negative cases in the rate of G6PD deficiency (3/36 in positive cases versus 30/124 in negative cases) (P<0.05).

Conclusion

vivax malaria positive individuals with G6PD deficiency showed too mild symptoms of Malaria or even asymptomatic.     

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.     

Sequence Diversity of pfmdr1 and Sequence Conserve of pldh in Plasmodium falciparum from Indonesia: Its implications on Designing a Novel Antimalarial Drug with Less Prone to Resistance

Background

pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in Plasmodium falciparum, a parasitic carrier for malaria disease. A novel strategy to treat malaria disease is by disrupting parasite lactate dehydrogenase (pLDH), a crucial enzyme for Plasmodium survival during their erythrocytic stages. This research was aimed to investigate and characterize the pfmdr1 and pldh genes of P. falciparum isolated from Nusa Tenggara Indonesia.

Methods

Genomic DNA of P.falciparum was isolated from malaria patients in Nusa Tenggara Indonesia. pfmdr1 was amplified using nested PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). pldh was amplified, sequenced, and analyzed using NCBI public domain databases and alignment using Clustal W ver. 1.83.

Results

Genotyping of the pfmdr1 revealed that sequence diversity was extremely high among isolates. However, a sequence analysis of pldh indicated that open reading frame of 316 amino acids of the gene showing 100% homology to the P. falciparum 3D7 reference pldh (GeneBank: {"type":"entrez-nucleotide","attrs":{"text":"XM_001349953.1","term_id":"124513265","term_text":"XM_001349953.1"}}XM_001349953.1).

Conclusion

This is the first report which confirms the heterologous of pfmdr1 and the homologous sequences of P.falciparum pldh isolated from Nusa Tenggara Islands of Indonesia, indicating that the chloroquine could not be used effectively as antimalarial target in the region and the pLDH-targeted antimalarial compound would have higher chance to be successful than using chloroquine for curbing malaria worldwide.     

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.     

Mini-Column for Cytoadherence: A New Method for Measuring the Relative Size of Binding Subpopulations in Plasmodium falciparum Isolates

Background

Cytoadherence of Plasmodium falciparum- infected red blood cells to endothelial cells is an important mechanism for parasite survival and a major trigger for diseases pathology. Here, we describe a new adhesion assay in which different cell types (CHO, CHO/CD36 and CHO/ICAM-1) are attached to Cytodex beads in a mini-column format to measure the relative sizes of various binding subpopulations as a percentage of the total population.

Methods

Relative size of CD36 and ICAM-1-binding subpopulations of erythrocytes infected with P. falciparum were measured by amount of parasitemia before and after passing the infected erythrocytes through a particular column.

Results

The mini-column adhesion assay was a suitable method as parasitemia always reduced after passing through a particular column in independent experiments. For example, in a typical experiment using P. falciparum ITG line, 75% of the parasites are retained on a CHO/ICAM-1 while 0% of clone 3D7 is retained.

Conclusion

This work introduced and validated a method for measuring the relative size of parasite binding subpopulations and the selection of them. Also, the mini-column method is of value for assessments of cytoadherence and can be used as tool for different applications.     

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.     

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.     

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.     

Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar

Background

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.

Methods

A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.

Results

With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.

Conclusion

The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.     

Plasmodium falciparum malaria and atovaquone-proguanil treatment failure

We noticed overrepresentation of atovaquone-proguanil therapeutic failures among Plasmodium falciparum-infected travelers weighing >100 kg. We report here 1 of these cases, which was not due to resistant parasites or impaired drug bioavailability. The follow-up of such patients should be strengthened.     

Haematinic treatment of anaemia increases the risk of Plasmodium vivax malaria in pregnancy

Nutritional deficiency and malaria are 2 major causes of anaemia during pregnancy in tropical areas. The relationship between anaemia, its treatment with iron and folate, and malaria was studied in a prospective cohort of 2112 pregnant Karen women on the north-western border of Thailand between 1993 and 1997. The development of Plasmodium vivax malaria was associated with a past mean haematocrit > 30% (hazard ratio = 1.5, 95% CI 1.2-2, P = 0.001) and recent (< or = 30 d) iron and folate supplementation (hazard ratio = 1.7, 95% CI 1.1-2.6, P = 0.01). There were no associations with P. falciparum infections. Plasmodium vivax has a predilection for young erythrocytes, and these results suggest that pregnant women with larger numbers of circulating young red cells are at greater risk of developing P. vivax malaria. In P. vivax-endemic areas, systematic iron and folate supplementation confers both benefit and risk in pregnancy.     

In Vivo Antimalarial Effects of Iranian Flora Artemisia khorassanica against Plasmodium berghei and Pharmacochemistry of its Natural Components

Background

The aim of this study was to evaluate the antimalarial effects of Iranian flora Artemisia khorassanica against Plasmodium berghei in vivo and pharmacochemistry of its natural components.

Methods

The aerial parts of Iranian flora A. khorasanica were collected at flowering stage from Khorassan Province, northeastern Iran in 2008. They were air-dried at room temperature; powder was macerated in methanol and the extract defatted in refrigerator, filtered, diluted with water, then eluted with n-hexane and finally non-polar components were identified through Gas Chromatography and Mass Spectroscopy (GC-MS). Toxicity of herbal extracts was assessed on naïve NMRI mice, and its anti-malarial efficacy was investigated on infected Plasmodium berghei animals. This is the first application on A. khorssanica extract for treatment of murine malaria. The significance of differences was determined by Analysis of Variances (ANOVA) and Student''s t-test using Graph Pad Prism Software.

Results

The herbal extract was successfully tested in vivo for its anti-plasmodial activity through artemisin composition, which is widely used as a standard malaria treatment.

Conclusion

Although, this study confirmed less anti-malarial effects of A. khorssanica against murine malaria in vivo, however there are some evidences on reducing pathophysiology by this medication. In complementary assay, major components were detected by GC-MS analysis in herbal extract including chrysanthenone (7.8%), palmitic acid (7.4%) and cis-thujone (5.8%). The most retention indices of the component are given as n-eicosane, palmitic acid and n-octadecane.