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.     

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 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.     

Absence of Asymptomatic Malaria Infection in Endemic Area of Bashagard District,Hormozgan Province,Iran

Background

A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.

Methods

Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques.

Results

Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1₁₉-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1₁₉, using ELISA.

Conclusion

This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.     

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.     

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.     

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.     

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.     

DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates

Background:

Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum.

Methods:

Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013.

Results:

Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%–76% nucleotide and 90.4%–90.76% amino acid homology.

Conclusion:

pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8–100% homology with 1–3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.     

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.     

Seasonality of presentation of imported Plasmodium vivax malaria in Birmingham, UK

Residents of the UK returning from northern Pakistan with Plasmodium vivax infection tend to developsymptoms and present to hospital in the summer months, irrespective of the month of return. Thus, infections acquired in the cooler months of November to April appear to have a longer latency before presentation. Experiments suggest that more hypnozoites arise from the liver when ambient temperatures fall, somehow ‘programming’ parasites within biting mosquitoes.     

Seroprevalence of Acanthamoeba Antibodies in Rheumatoid Arthritis Patients by IFAT,Tehran, Iran 2007

Background

This preliminary study was conducted to discriminate the prevalence of Acanthamoeba antibodies in rheumatoid arthritis (RA) patients and healthy controls to analyze the correlation between these two groups.

Methods

From October 2006 to August 2007 a total of 121 serum samples from RA patients attending the Rheumatolgy Department at Shariati Hospital in Tehran were obtained and stored at -20°C until using by indirect fluorescent-antibody test (IFAT). RA was diagnosed according to the American Collage of Rheumatology classification criteria. The organism used in this study was isolated from various water resources in Tehran, Iran cultured axenically and then went on a PCR assay based on 18S rRNA to identify the genus Acanthomoeba. Indirect immunofluorescence antibody (IFA) staining of serum samples was carried out to detect anti Acanthomoeba antibodies.

Results

In culture, out of 22 samples, 13(59%) were grown in xenic but only two in axenic medium. PCR amplified a 904bp fragment, specific for Acanthamoeba. Of examined serum samples, Acanthamoeba antibodies were present in 70 (57.8%) and 52 (41.2%), respectively. The highest titer of antibodies (1:320) was detected in one patient with RA.

Conclusion

Our study supports the hypothesis that some parasitic microorganisms can involve and contribute toward the development of rheumatoid syndromes.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

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.     

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.     

Monitoring of Plasmodium vivax sensitivity to chloroquine in vitro in Thailand

The sensitivity of Plasmodium vivax to chloroquine in vitro was investigated in patients admitted to the Bangkok Hospital for Tropical Diseases, Thailand, between September 2001 and May 2002. Of 42 isolates, 34 were successfully tested for parasite sensitivity to chloroquine in vitro; the results showed a significant decrease in sensitivity compared with data published in 1989 and 1995: the IC50 and IC90 were 187.2 and 1217.9 ng/mL blood, respectively, an approximate 4-fold decrease in sensitivity in comparison with other data from the past 2 decades. A number of in vitro tests were performed simultaneously using both WHO microplates and our own laboratory-prepared pre-dosed microplates under the same conditions and there was no significant difference between the results.     

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.