Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

BACKGROUND & OBJECTIVES: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases. METHODS: The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/µl. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India. RESULTS: Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/µl of blood with a very high sensitivity (96-100%) and specificity (100%). INTERPRETATION & CONCLUSIONS: The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera.

Induction of cell-mediated immune responses to peptide antigens of P. vivax in microparticles using intranasal immunization.

T-cells play a critical role in resistance to malaria, not only because they function as helper cells for an antibody response, but also because they serve as effector cells. Such cellular immunity is directly implicated in protection from sporozoites as well as from blood stage parasites. The aim of this study was to induce cell mediated immune responses to peptide antigens of Plasmodium vivax co-encapsulated with CpG oligodeoxynucleotide (ODN) in microparticles. In the present study, we have investigated the immunomodulatory effects of two CpG adjuvants, CpG 1826 and CpG 2006 to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen (Pvs24) in microparticle delivery. The T-cell proliferation response study of the cells collected from spleen, lamina propria and peyer's patches showed significantly high (p<0.001) stimulation index when primed with peptide antigens in microparticles co-encapsulating CpG ODN adjuvant as compared to peptide alone primed mice. The cytokine measurement profile of IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-10 in culture supernatants of cells primed with peptide antigens in microparticles co-encapsulating CpG ODN showed higher levels of IFN- gamma followed by TNF-alpha and IL-2, with relatively low levels of IL-4 and IL-10.

A preliminary study on pro- and anti-inflammatory cytokine profiles in Plasmodium vivax malaria patients from central zone of India.

The aim of this preliminary study was to investigate the plasma cytokine profiles in a group of patients suffering from Plasmodium vivax malaria during the peak of its transmission season. Plasma samples of 173 P. vivax patients and 34 healthy individuals were analyzed for IFN-gamma, TNF-alpha, IL-10 and IP-10 levels by ELISA. Levels of both pro- and anti-inflammatory cytokines were significantly higher in P. vivax patients compared to controls. Children with P. vivax infection had significantly higher levels of IFN-gamma than adults (P=0.017). Asexual parasitaemia versus TNF-alpha (r=-0.31, P=0.01), IL-10 (r=-0.30, P=0.015) and gametocytaemia versus IFN-gamma (r=-0.26; P=0.034) levels showed significant negative correlation in children compared to adults. The median concentrations of IFN-gamma (P=0.001), IL-10 (P=0.032) and IP-10 (P

Acquired immune response to defined Plasmodium vivax antigens in individuals residing in northern India.

In this investigation, we evaluated the naturally acquired immune response to Plasmodium vivax stage-specific antigens in individuals of different age groups belonging to malaria endemic areas of northern India. Four synthetic peptides containing both B- and T-cell epitopes from P. vivax circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen-1 were used to determine both humoral and cellular immune responses. Immunity, in terms of antibody response and T-cell proliferation against these stage-specific peptides, has been observed in the study subjects. The results demonstrated age-dependent antibody response in this population. Forty two patients were diagnosed with P. vivax. There was a significant association (P=0.013) between number of antibody responders and recognition of stage-specific epitopes by antibodies. The antibody response to B-epitopes of P. vivax CSP, MSP1, AMA1 and GAM1 was associated with age; adults responded more frequently to these antigens than did younger children. In this population, 66% (201/304) cases showed seropositivity to all peptides and 13% (41/304) showed negative response. Peripheral blood mononuclear cells of more than 75% of individuals proliferated in response to stimulation by all four epitopes. In conclusion, the results demonstrated immunogenicity of the epitopes to P. vivax in population of this endemic zone.

Induction of mucosal and systemic humoral immune responses in murine system by intranasal immunization with peptide antigens of P. vivax and CpG oligodeoxynucleotide (ODN) in microparticle delivery.

In the present study we have investigated the immunomodulatory effects of two adjuvants, CpG 1826 (two copies of CpG motifs) and CpG 2006 (three copies of CpG motifs) to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein (CSP), merozoite surface protein-1 (MSP1#1, MSP1#23), apical membrane antigen-1 (AMA-1) and gametocyte surface antigen (Pvs24) in alum and microparticle formulations, using intramuscular and intranasal routes of immunization. Alum formulation without CpG ODN generated low serum IgG and IgA antibody titers and the predominant IgG isotypes were IgG1 but with the addition of CpG ODN (1826 or 2006), the antibody titers were increased by four fold with the predominance of IgG2a/2b isotypes. The SIgA peak titers in lung and intestinal washes were significantly increased with the intranasal mode of administration. Specific activity measurement was done to calculate for the accurate amounts of total serum IgG, IgA and SIgA in washes and showed direct correlation between antibody titer and its concentration. High titer anti-Pvs24 antibodies have significant inhibitory effects on parasite development in the mosquito midgut when tested in membrane feeding assays. The immunofluorescence results show that the peptide specific antisera reacted with the air-dried parasite antigens isolated from P. vivax patients. The present study demonstrates that intranasal route of immunization appears to be an alternate mode of inducing protective immunity in P. vivax malaria.

Synthesis of highly substituted 2-perfluoroalkyl quinolines by electrophilic iodocyclization of perfluoroalkyl propargyl imines/amines.

A series of highly substituted 2-perfluoroalkyl-3-iodoquinolines are prepared by two different methods in good to excellent yields under mild reaction conditions. The first method involves iodocyclization of perfluoroalkyl propargyl imines with I(2)-CAN. The second method involves iodocyclization of perfluoroalkyl propargyl amines using I(2) and ICl. The perfluoroalkyl propargyl amines are prepared in excellent yields via Sonogashira coupling of easily accessible imidoyl iodides with alkynes followed by reduction with NaBH(3)CN. The scope of this methodology is extended by using the resulting 2-perfluoroalkyl-3-iodo quinolines in Suzuki, annulation, dehalogenation and carboxylation reactions. Antimalarial activity of the 2-perfluoroalkyl-3-iodoquinolines is discussed.

A longitudinal investigation of Plasmodium falciparum malaria in children in northern India.

A group comprising 27 young children (1-4 y of age) suffering from uncomplicated falciparum malaria were studied to characterize the isolates and to measure humoral immune responses during acute infection and after recovery. Finger prick blood from each individual was collected on d 1. After treatment with chloroquine, a further blood sample was collected from each child on d 7, 30, 90 and 180 for assay of antibody responses to P. falciparum antigens. Isolates from individual patients were incubated in vitro for demonstration of rosette formation, assay of plasmodial growth rate and analysis of Pfcrt gene polymorphism. Out of 27 isolates of P. falciparum, 20 showed formation of rosettes in vitro. The growth rate at 96 h varied widely among the isolates. In Pfcrt gene analysis at 76-codon site, 14 showed wild-type Lys 76, 7 showed mutant type Thr 76 and 6 had mixed type. 14 children, all with anaemia on d 7, showed a positive direct antiglobulin test (DAT). Sera positive by ELISA IgG on d 90 also showed parasite growth inhibitory activity in vitro. Significant levels of IgG, IgG1 and IgG3 subclass antibodies against MSP1 were detected in 14 sera collected on d 90. On d 180, there was a decline in IgG and its subtypes. These findings suggest that a variability in isolates may occur in one and the same seasonal area, making children prone to infection. As a consequence, they develop antibodies during recovery phase from an acute attack, which remain in circulation for a period of 4-5 months. After that, a decline in antibody level may again make them susceptible to the disease. Prevalence of different serotypes in a small area may suggest the complexity of malaria transmission.

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.

BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Congenital malaria with atypical presentation: a case report from low transmission area in India.

BACKGROUND: Malaria during first few months of life may be due to transplacental transfer of parasitized maternal erythrocytes. Although IgG and IgM antimalarial antibodies can be detected in maternal blood, only IgG antibodies are present in the infant's blood. These antibodies can delay and modify the onset of clinical manifestations. CASE PRESENTATION: An infant is described who presented with irritability and feeding problems. Clinical examination and investigations revealed that the infant was afebrile, had jaundice, hepatosplenomegaly and haemolytic anaemia. Peripheral smear demonstrated Plasmodium vivax. While the mother had significant levels of immunoglobulin G (IgG), the infant was found negative for IgG and had low immunoglobulin M (IgM) levels. The mother had a history of febrile illness during pregnancy and her peripheral smear was also positive for P. vivax. Both were successfully treated with chloroquine in the dose of 25 mg/kg/day over three days. CONCLUSION: The case emphasizes the importance of considering the diagnosis of malaria even in infants in low transmission area, who may not present with typical symptoms of malaria, such as fever, but have other clinical manifestations like jaundice and haemolytic anaemia.

Development of diagnostic reagents: raising antibodies against synthetic peptides of PfHRP-2 and LDH using microsphere delivery.

Malaria causes significant morbidity and mortality worldwide, including countries with mainly imported malaria. In developing nations, scarce resources lead to inadequate diagnostic procedures. Microscopy of Giemsa-stained thick and thin films remains the current gold standard for diagnosis. Although it has good sensitivity and allows species identification, it is time consuming, requires microscopical expertise and maintenance of equipment. Antigen tests are promising tools for the diagnosis of malaria. Two such antigens are Plasmodium falciparum histidine rich protein (pfHRP-2) and lactate dehydrogenase (LDH). The present study was aimed to develop indigenous, rapid and sensitive immunodiagnostic method based on detection of PfHRP-2 and LDH antigen in the blood. Unique peptide sequences of PfHRP-2 (two regions) and LDH (three regions) antigen were synthesized by solid phase technique and purified to homogeneity. The antibodies raised against these sequences were raised in mice as well as rabbit using microspheres (PLGA) to generate high titre and affinity antibodies. The peptide-specific peak titres varied from 25,000 to 50,000 and affinity of the antibodies produced was found to be in order of 0.73-5.3 nM. The antibodies generated using microspheres were able to detect the PfHRP-2 and LDH antigen in the culture supernatant and parasitized RBC lysate of P. falciparum respectively by sandwich ELISA up to 0.002% parasitaemia level. The assay allowed the detection of parasite infections of 0.08-2.68% parasitaemia with a sensitivity of 100% in the whole blood of P. falciparum positive patients. No cross-reactivity was observed with P. vivax infected patients.

Assessment of immunometric parameters in malaria: role of enzyme immunoassay.

Human blood samples collected from local malaria clinics, hospitals, and by cross-sectional surveys in malaria endemic areas were tested by enzyme immunoassay for circulating malarial antigen, antimalarial antibody, and antigen-specific circulating immune complexes. The assays were done in serum and finger-prick blood absorbed on filter paper. The results obtained from the present study suggest their roles as effective immunometric indicators.

Epidemiology of malaria transmission in forest and plain ecotype villages in Sundargarh District, Orissa, India.

A study of the epidemiology of malaria transmission was undertaken in 13 tribal villages located in forest and plain areas of Sundargarh District of Orissa state, India, from January 2001 to December 2003. In forest areas, intense transmission of malaria is attributed to the highly anthropophagic vector Anopheles fluviatilis sibling species S and is complemented by A. culicifacies sibling species C. In plain areas, A. culicifacies sibling species C is responsible for malaria transmission. The entomological inoculation rate in the forest and plain areas was 0.311 and 0.014 infective bites/person/night, respectively, during 2003. Malaria transmission is perennial both in forest and plain areas but is markedly low in the plain area compared with the forest area. Plasmodium falciparum accounted for 85.0% of the total malaria cases during the study period. In forest and plain areas, the number of P. falciparum cases per 1000 population per year was 284.1 and 31.2, respectively, whereas the parasite rate was 14.0% and 1.7%, respectively. In forest areas, clinical malaria occurs more frequently in children aged 0-5 years and declines gradually with increasing age. The study showed that villages in forest and plain areas separated by short geographical distances have distinct epidemiology of malaria transmission.

Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India.

BACKGROUND: Effective malaria control programs require continuous monitoring of drug pressure in the field, using molecular markers. METHODS: We used sequence analysis to investigate the pfcrt and pfmdr1 mutations in Indian Plasmodium falciparum isolates. To evaluate the chloroquine drug pressure in the field, isolates were collected from 5 different areas at 2 time points, with an interval of 2 years. RESULTS: In 265 P. falciparum isolates, pfcrt mutations were observed at codons 72, 74, 75, 76, and 220, resulting in 8 different genotypes: SMNTS (61.89%), CIETS (12.08%), CMNKS (0.38%), CMNTA (2.64%), CMNTS (4.91%), SMNTA (0.38%), CIDTS (2.26%), and wild-type CMNKA (15.47%). During the 2-year period, there was a significant decrease in the number of isolates with the SMNTS genotype and an increase in the number of isolates with the highly chloroquine-resistant pfcrt genotype CIETS (P < .05). The N86Y mutation was less prevalent (30.13%) than the Y184F mutation (99.16%) in the pfmdr1 gene in 239 isolates, but the number of isolates with the N86Y mutation increased significantly during the 2-year period (P < .05). The number of isolates with higher total numbers of pfcrt and pfmdr1 2-loci mutations, therefore, increased significantly during this period. There was a regional bias in the mutation rate of these genes, because isolates from areas where chloroquine resistance was high had higher numbers of 2-loci mutations, and areas where chloroquine resistance was low had isolates with lower numbers of 2-loci mutations. CONCLUSION: There was a temporal increase in the number of pfcrt and pfmdr1 2-loci mutations, and this led to the higher level of chloroquine resistance. This is a cause for concern for the antimalarial drug policy in India.

Anti-malarial activity of Baylis-Hillman adducts from substituted 2-chloronicotinaldehydes.

New Baylis-Hillman adducts are synthesized based on substituted 2-chloronicotinaldehydes and screened for their in vitro anti-malarial activity against chloroquine sensitive and chloroquine resistant Plasmodium falciparum. Out of the six new compounds synthesized and screened, 2b, 2c and 2d compounds showed substantial anti-malarial activity.

Stage-specific cytosolic protein kinase C-like activity in human malarial parasite Plasmodium falciparum.

Protein kinase C (PKC)-like activity was characterized in malarial parasite Plasmodium falciparum and its involvement in growth, maturation and differentiation functions, during the asexual stages (ring, trophozoite and schizont) of development was studied. PKC-like activity was found distributed in all the stages of the parasite maturation. The activity was predominantly cytosolic, however it was also present in the membrane fraction. The activation of cytosolic PKC required Ca2+, phosphatidyl serine (PS), and either diacylglycerol or phorbol myristate acetate (PMA). The 9-fold increase in the activity was observed in the presence of the co-factors (Ca2+, PS and PMA) in the late trophozoite stage, as compared to the ring stage. The activation of trophozoites with PMA resulted in redistribution of PKC-like activity from cytosol to membrane fractions. An antimalarial drug, chloroquine (CQ) inhibited directly the PKC-like activity in a dose-dependent manner (IC50 of 45 nM) in trophozoites of chloroquine-sensitive CQ(S) strains, however, the activity remained unaltered in the chloroquine-resistant CQ(R) strains. Kinetic studies showed that the inhibition of cytosolic PKC-like activity by CQ was non-competitive with respect to ATP, histone and PS. The results suggest that the PKC-like activity is developmentally expressed during the parasitic survival and development.

Inter-test comparison between filter paper absorbed blood eluate and serum for malaria serology by enzyme immunoassay: an operational feasibility.

Antimalarial IgG and IgM were detected by enzyme immunoassay in finger-stick blood samples collected in capillary tubes and also spotted onto Whatman filter paper. Assay was performed in 92 blood samples obtained from 53 falciparum malaria patients, representing 23 fever cases (malaria negative) and 16 healthy individuals. A simple indirect ELISA was done using Plasmodium falciparum lysate and MSP1(19) peptide as antigens. Total IgG and IgM contents were also estimated in individual sera and filter paper eluate by single radial immunodiffusion (SRID). Assay results of both serum and filter paper eluates were compared. The sensitivity and specificity of the assays for IgG measurement were comparable between serum and filter paper eluates (P < 0.001), whereas, in case of IgM, detection level was poor in filter paper eluates as observed by ELISA and SRID. The filter paper eluates may serve the purpose of antigen-specific IgG detection in seroepidemiological surveys.

Plasmodium falciparum isolates in India exhibit a progressive increase in mutations associated with sulfadoxine-pyrimethamine resistance.

The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.

Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India.

Chloroquine-resistant Plasmodium falciparum needs to be monitored in the field for effective malaria control strategies. A point mutation K76T in the P. falciparum chloroquine resistance transporter (Pfcrt) protein has recently been proposed as a molecular marker for the faster detection of chloroquine-resistant falciparum malaria in field. We describe here the evaluation of this marker in Indian P. falciparum isolates. A total of 274 Indian P. falciparum isolates were analyzed for the K76T mutation. This mutation was detected in all the clinical isolates obtained from the in vivo chloroquine non-responders. But majority of the clinical isolates from chloroquine responders (71 of 74 patients, i.e. 96%) also harbored this mutation. The K76T mutation was indeed highly prevalent (91%) among 213 clinical isolates. There was a significant association between K76T mutation and the in vitro chloroquine response (P<0.05) but six isolates showed discordant results. In conclusion, the K76T mutation fails to differentiate majority of the chloroquine responders from that of the non-responders and thus will be of limited use in the field in India.

Assessment of therapeutic efficacy of chloroquine and sulphadoxine-pyrimethamine in uncomplicated falciparum malaria.

A standardised protocol has been developed by World Health Organization (CDS/RBM/2002) to assess the efficacy of common antimalarials in the treatment of clinically manifested infection with uncomplicated P. falciparum malaria for areas with low to moderate transmission. The therapeutic efficacy protocol is based on clinical and parasitological responses of the patients and it has the purpose of determining the practical efficacy of the drug regimen in study areas with the ultimate objective of ascertaining its continued usefulness or the necessity for replacing it in the routine treatment. Present study has been conducted at seven sites--Kathiatali and Simonabasti of District Nowgaon, Assam; Sonapur and Boko of District Kamrup, Assam; Keonjhar Town, Padampur and Basudebpur of District Keonjhar, Orissa. In order to reduce the patient recruitment time, health centre close to well-defined community was identified to conduct the activities at peak malaria season by selecting local pockets and organising mobile clinics. Microscopically confirmed cases of P. falciparum were enrolled according to the criteria for inclusion and exclusion. Treatment with recommended drug was given under supervision and a follow-up schedule at various intervals for 28 days was maintained. In chloroquine (CQ) study areas, wherever patients showed treatment failure, they were treated with second line drug--sulphadoxine-pyrimethamine (SP) combination and then followed-up as per study protocol. It was observed that 30% cases showed treatment failure to CQ in District Nowgaon, where revised drug policy has already been introduced. In Kamrup district, treatment failure with CQ was found to be less than 25%, which denotes the said regimen is still effective. Almost all the patients from Padampur and Basudebpur of District Keonjhar responded to CQ, treatment failure was noticed only in two patients (3%). The antifolate combination found to be fully effective as second line and also as first line wherever revised drug policy has been introduced.

A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas--XVI. Asembo Bay Cohort Project.

We have investigated the genetic diversity of the gene encoding the CS protein. A total of 75 complete and 96 partial sequences are studied. We find high levels of genetic polymorphisms as evidenced by 50 and 24 alleles at the Th2R and Th3R epitopes, respectively. Overall, we find that African isolates are more polymorphic as compared with parasites from other geographic regions. We conclude that the uneven geographic polymorphism may have an adverse impact on the effectiveness of vaccines based on this antigen alone. We find extensive polymorphism in the repeat allotypes, or RATs. In order to explore how the protein structure may impose restrictions in the number of repeats, we have simulated the stability of the structure of the tandem repeat region. Our analysis suggests that the protein structure may play an important role in the observed polymorphism in the number of CS repeats in Plasmodium falciparum. We explored the linkage and recombination events among the polymorphic sites. We found that putative recombination events overlap with linked sites. We discuss how this pattern is explained by the action of positive natural selection, where the recombination events detected are convergent mutations. We conclude that it is inappropriate to use linkage-recombination patterns on genes under positive selection for assessing the structure of parasite populations.