Apical membrane protein 1‐specific antibody profile and temporal changes in peripheral blood B‐cell populations in Plasmodium vivax malaria

Plasmodium falciparum‐specific antibodies tend to be short‐lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen‐specific antibodies and B‐cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short‐lived antibodies but long‐lived MBC responses to a major blood‐stage P vivax antigen, apical membrane protein 1 (PvAMA‐1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19⁺CD10^?CD21^?CD27^?) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody‐secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B‐cell memory that may affect both naturally acquired and vaccine‐induced immunity.     

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1₁₉) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1₁₉ were evaluated in individuals (n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1₁₉ and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1₁₉, the frequency of antibodies was determined in the infected subjects (n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1₁₉ was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1₁₉. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1₁₉-based vaccine.     

Rapid Dissemination of Newly Introduced Plasmodium vivax Genotypes in South Korea

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996–2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.     

Genetic polymorphism and effect of natural selection at domain I of apical membrane antigen-1 (AMA-1) in Plasmodium vivax isolates from Myanmar

Malaria is endemic or hypoendemic in Myanmar and the country still contributes to the high level of malaria deaths in South-East Asia. Although information on the nature and extent of population diversity within malaria parasites in the country is essential not only for understanding the epidemic situation but also to establish a proper control strategy, very little data is currently available on the extent of genetic polymorphisms of the malaria parasites in Myanmar. In this study, we analyzed the genetic polymorphism and natural selection at domain I of the apical membrane antigen-1 (AMA-1) among Plasmodium vivax Myanmar isolates. A total of 34 distinguishable haplotypes were identified among the 76 isolates sequenced. Comparison with the previously available PvAMA-1 sequences in the GenBank database revealed that 21 of them were new haplotypes that have never been reported till date. The difference between the rate of nonsynonymous (dN) and synonymous (dS) mutations was positive (dN − dS, 0.013 ± 0.005), suggesting the domain I is under positive natural selection. The Tajima's D statistics was found to be −0.74652, suggesting that the gene has evolved under population size expansion and/or positive selection. The minimum recombination events were also high, indicating that recombination may occur within the domain I resulting in allelic diversity of PvAMA-1. Our results collectively suggest that PvAMA-1 displays high genetic polymorphism among Myanmar P. vivax isolates with highly diversifying selection at domain I. These results have significant implications in understanding the nature of P. vivax population circulating in Myanmar as well as providing useful information for malaria vaccine development based on this antigen.     

Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) is a leading malaria vaccine candidate. This protein is processed to give rise to various sized fragments during merozoite maturation. Here, we describe the analysis of genetic diversity in the 42 kDa C-terminal part of this protein among 33 Indian P. vivax isolates. A total of 27 haplotypes with 72 mutations and 0.0212 ± 0.0005S.D. over all π nucleotide diversity were observed among the isolates. Twenty-six of 27 haplotypes reported here were new as they have not been reported so far from any other country. The difference between non-synonymous (dN) and synonymous (dS) mutations was found to be positive (0.0081 ± 0.0051) for the entire 42 kDa region. Further analysis revealed that 33 kDa (MSP-1₃₃) fragment of the MSP-1₄₂ was highly polymorphic with π nucleotide diversity 0.0290 ± 0.0007S.D. The dN-dS for this region of MSP-1 was also positive (0.0114 ± 0.0071S.E.). On the other hand, there was no non-synonymous mutation in the 19 kDa (MSP-1₁₉) fragment of the MSP-1₄₂ and thus it was highly conserved. In conclusion, MSP-1₃₃ fragment was highly polymorphic and appeared to be under diversifying selection whereas there was no selection at MSP-1₁₉ region among the isolates. Present study will be helpful for the development of PvMSP-1 based vaccine against P. vivax malaria.     

Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well‐consolidated settlement of the Amazon Region

Objective  To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) – a leading malaria vaccine candidate – in a well‐consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBP_II) within the local malaria parasite population. Methods  Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBP_II. Results  The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti‐PvDBP IgG antibodies, as detected by enzyme‐linked immunosorbent assay (ELISA) with subject’s age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBP_II diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion  The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBP_II diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.     

Sensitive detection and accurate monitoring of Plasmodium vivax parasites on routine complete blood count using automatic blood cell analyzer (DxH800(TM))

Introduction: Plasmodium vivax malaria is one of the most important infectious diseases plaguing humanity and causes significant mortality and morbidity worldwide. The gold standard of P. vivax malaria diagnosis is the microscopy of blood smears. Although microscopy is a rapid, cost‐effective, and readily applicable method, it has many disadvantages, including low sensitivity, specificity, and precision. Therefore, there is a clear need for an effective screening test for P. vivax malaria detection both in high‐prevalence areas and developed countries. Methods: A total of 1761 complete blood count (CBC) samples generated by the automated hematology analyzer (DxH 800?; Beckman Coulter Inc., Miami, FL, USA) were retrospectively analyzed. The sample pool contained 123 samples from 52 P. vivax malaria patients and 1504 nonmalarial samples including 509 patients with leukopenia (white blood cell <2000/μL) and 134 normal subjects. Results: The P. vivax malaria samples exhibited easily recognizable typical malaria signals on the nucleated red blood cell (nRBC) plots (sensitivity 100%) in DxH 800?. All 1504 samples without P. vivax infection were negative for malaria signal (specificity 100%). The size of P. vivax malaria signals correlated roughly with the parasite burden. Conclusion: DxH800? provides very sensitive and specific, easily recognizable P. vivax malaria signals on routine CBC without need for the additional reagents or special procedures.     

Stronger host response per parasitized erythrocyte in Plasmodium vivax or ovale than in Plasmodium falciparum malaria

Objective and methods Fever tends to start at a lower level of parasitemia in Plasmodium vivax or ovale than in P. falciparum malaria, but hyperparasitemia and complications are more likely to occur in P. falciparum malaria. Therefore, we compared the relationship between parasitemia and host response parameters before therapy in 97 patients with P. faciparum malaria (18 with complications), and 28 with P. vivax or ovale malaria. Results In both types of malaria, parasitemia correlated with blood levels of tumour necrosis factor alpha (TNF‐alpha), lactate dehydrogenase (LDH), Thrombin–antithrombin III (TAT) and elastase, and these parameters were higher in P. falciparum malaria than in P. vivax or ovale malaria. In contrast, the ratios of TNF‐alpha, TAT, elastase, and LDH per parasitized erythrocyte were higher in P. vivax or ovale malaria than in uncomplicated P. falciparum malaria. They were lowest in complicated disease. Multivariate regression analysis confirmed that parasitemia did not affect these differences. Conclusion The host response may reach full strength at lower parasitemia in Plasmodium vivax or ovale, than in P. falciparum malaria. With hyperparasitemia in P. falciparum malaria, the host response seems to be unable to control parasite multiplication.     

Microgeographical Differences of Plasmodium vivax Relapse and Re-Infection in the Peruvian Amazon

To determine the magnitude of Plasmodium vivax relapsing malaria in rural Amazonia, we carried out a study in four sites in northeastern Peru. Polymerase chain reaction-restriction fragment length polymorphism of PvMSP-3α and tandem repeat (TR) markers were compared for their ability to distinguish relapse versus reinfection. Of 1,507 subjects with P. vivax malaria, 354 developed > 1 episode during the study; 97 of 354 (27.5%) were defined as relapse using Pvmsp-3α alone. The addition of TR polymorphism analysis significantly reduced the number of definitively defined relapses to 26 of 354 (7.4%) (P < 0.05). Multivariate logistic regression modeling showed that the probability of having > 1 infection was associated with the following: subjects in Mazan (odds ratio [OR] = 2.56; 95% confidence interval [CI] 1.87, 3.51), 15–44 years of age (OR = 1.49; 95% CI 1.03, 2.15), traveling for job purposes (OR = 1.45; 95%CI 1.03, 2.06), and travel within past month (OR = 1.46; 95% CI 1.0, 2.14). The high discriminatory capacity of the molecular tools shown here is useful for understanding the micro-geography of malaria transmission.     

Development of PCR-RFLP assay for the discrimination of Plasmodium species and variants of P. vivax (VK210, VK247 and P. vivax-like) in Anopheles mosquitoes

The identification of Plasmodium species in Anopheles mosquitoes is an integral component of malaria control programs. We developed a new assay to identify Plasmodium falciparum, Plasmodium malariae, and Plasmodium vivax variants. Specific primers were designed to hybridize to CS gene-specific regions. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to distinguish the P. vivax variants VK210, VK247, and P. vivax-like. The new PCR–RFLP assay revealed good agreement when compared with a nested PCR using artificially infected Anopheles mosquitoes. This sensitive PCR–RFLP method can be useful when detection of Plasmodium species and P. vivax variants is required and may be employed to improve the understanding of malaria transmission dynamics by Anopheles species.     

Pulmonary Edema Due to <Emphasis Type="Italic">Plasmodium vivax</Emphasis> Malaria in an American Missionary

Abstract Pulmonary edema is a recognized complication of Plasmodium falciparum malaria but is uncommon with Plasmodium vivax infection. We report the case of a non-immune adult with imported P. vivax malaria who developed pulmonary edema during treatment. The case was further complicated by a recurrent malaria episode after failure of acute quinine and doxycycline treatment followed by terminal primaquine therapy. Prompt recognition and appropriate management of pulmonary edema is needed for optimal outcomes of P. vivax infection, as well as awareness of the potential failure of terminal therapy for liver hypnozoites.     

Fibrinolysis,inhibitors of blood coagulation,and monocyte derived coagulant activity in acute malaria

Different parameters of fibrinolytic systems like t-PA, PAI, D-dimer, and inhibitors of blood coagulation, i.e., protein C (PC), protein S(PS), and antithrombin III (AT-III), have been studied in cases of acute malaria due to Plasmodium falciparum and plasmodium vivax infection, and these patients were followed up. It was observed that the plasma PAI-1 was very high in cases of P. falciparum malaria infection as compared to normal controls and P. vivax infection. The changes in complicated cases of P. falciparum were remarkable as compared to uncomplicated ones. The PC, PS, and AT-III levels were also low in P. falciparum, particularly so in complicated cases, and were normal in P. vivax infection. The factor VIII R:Ag levels were invariably high in acute malaria. On follow-up of some of these cases the values came back to normal after the antiparasite treatment. The monocyte procoagulant activity was found to be significantly higher in P. falciparum infection as compared to that of P. vivax infection. All these findings therefore contribute towards the production of a hypercoagulable state in P. falciparum infection and partly explain the complications of P. falciparum infection like cerebral malaria. Am. J. Hematol. 54:23–29, 1997 © 1997 Wiley-Liss, Inc.     

Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite

Plasmodium vivax is geographically the most widely distributed cause of malaria in people, with up to 2·5 billion people at risk and an estimated 80 million to 300 million clinical cases every year—including severe disease and death. Despite this large burden of disease, P vivax is overlooked and left in the shadow of the enormous problem caused by Plasmodium falciparum in sub-Saharan Africa. The technological advances enabling the sequencing of the P vivax genome and a recent call for worldwide malaria eradication have together placed new emphasis on the importance of addressing P vivax as a major public health problem. However, because of this parasite's biology, it is especially difficult to interrupt the transmission of P vivax, and experts agree that the available methods for preventing and treating infections with P vivax are inadequate. It is thus imperative that the development of new methods and strategies become a priority. Advancing the development of such methods needs renewed emphasis on understanding the biology, pathogenesis, and epidemiology of P vivax. This Review critically examines what is known about P vivax, focusing on identifying the crucial gaps that create obstacles to the elimination of this parasite in human populations.     

Exflagellated Microgametes of <Emphasis Type="Italic">Plasmodium Vivax</Emphasis> in Human Peripheral Blood: An Uncommon Feature of Malaria

In the life cycle of malarial parasite exflagellation of microgametes occur in mosquitoes. Usually this will not occur in the peripheral blood of human beings. However, exflagellation can occur in the collected blood and may create diagnostic dilemma. We report a case of vivax malaria with exflagellated microgametes, which were mistaken for a double infection of vivax malaria and borrelia.     

Retrospective analysis of vivax malaria patients presenting to tertiary referral centre of Uttarakhand

Introduction

Despite the high prevalence of Plasmodium vivax (P vivax) malaria, research into its complications has lagged disproportionately as compared to Plasmodium falciparum (P falciparum) malaria.

Material and methods

The present retrospective observational study was conducted on cases with P vivax mono-infection presenting with severe malaria on the basis of one or more criteria as per the World Health Organization guidelines being used for severe falciparum malaria in children, as well as other manifestations been classified as complicated malaria, during an outbreak of malaria in a single tertiary referral hospital of north India.

Results

Seventy-four patients of acute malaria presented during the outbreak, of which 50 cases with P vivax mono-infection were included for the study. Complicated malaria was diagnosed in 41/50 cases, with thrombocytopenia being the commonest manifestation. Other presentations of severe malaria in our patients were liver dysfunction (with or without jaundice) 31/50 cases, respiratory involvement 14/50 cases, renal impairment 11/50 cases, circulatory collapse (Shock) 8/50 cases, severe anaemia 3/50 cases and central nervous system (CNS) involvement 2/50 cases.

Conclusion

The term “benign tertian malaria” no longer holds true for P vivax mono-infection. The authors wish to open a new front for researches on the possible genotypic abnormalities that the parasite or its carrier might have acquired over decades and has transformed into a species with the malignant potential of P falciparum.     

Malaria-induced thrombocytopenia

Summary Platelet counts were investigated in 26 patients withP. falciparum malaria and 39 patients withP. vivax malaria before and after treatment. Before schizontocidal treatment 22 of 26 (85%) patients withP. falciparum malaria and 30 of 39 (72%) patients withP. vivax malaria had depressed platelet counts below 150,000/l blood. There was a correlation between low platelet counts and high counts of malarial plasmodia (parasitized red blood cells) inP. falciparum andP. vivax infections (p < 0.001). Platelet survival, studied by malonaldehyde formation in three patients during the period of decreasing parasitaemia, revealed a shortened life span to 2–3 days in comparison to 7–10 days in normal controls. In all patients platelet counts rose to threefold the initial values within 5 days after clearance of parasites.The results demonstrate that, first, thrombocytopenia is a common feature in human malaria, second, thrombocytopenia induced by malaria is due to shortened life span in the peripheral blood and, third, some interaction is present between platelets and malaria plasmodia or parasitized red cells.This study was supported by the Deutsche Forschungsgemeinschaft     

Presence of anti-modified protein antibodies in idiopathic pulmonary fibrosis

Background and Objective

Studies of autoimmunity and anti-citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti-cyclic citrullinated peptide (anti-CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti-modified protein antibodies (AMPA) in IPF.

Methods

We included patients with incident and prevalent IPF (N = 120), sex and smoking-matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1–Q3: 1–28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom-made peptide microarray.

Results

AMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit₍₂₀₃₃₎-TNC_2025–2040; Cit₍₂₁₉₇₎-TNC_2177–2200; Cit₍₂₁₉₈₎-TNC_2177–2200)_, fibrinogen (Cit_(38,42)-Fibα_36–50; Cit₍₇₂₎-Fibβ_60–74) and filaggrin (Acet-Fil_307–324, Carb-Fil_307–324). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009).

Conclusion

A significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.     

Vivax infection alters peripheral B‐cell profile and induces persistent serum IgM

B cell–mediated humoral responses are essential for controlling malarial infection. Studies have addressed the effects of Plasmodium falciparum infection on peripheral B‐cell subsets but not much is known for P. vivax infection. Furthermore, majority of the studies investigate changes during acute infection, but not after parasite clearance. In this prospective study, we analysed peripheral B‐cell profiles and antibody responses during acute P. vivax infection and upon recovery (30 days post‐treatment) in a low‐transmission area in India. Dengue patients were included as febrile‐condition controls. Both dengue and malaria patients showed a transient increase in atypical memory B cells during acute infection. However, transient B cell‐activating factor (BAFF)–independent increase in the percentage of total and activated immature B cells was observed in malaria patients. Naïve B cells from malaria patients also showed increased TLR4 expression. Total IgM levels remained unchanged during acute infection but increased significantly at recovery. Serum antibody profiling showed a parasite‐specific IgM response that persisted at recovery. A persistent IgM autoantibody response was also observed in malaria but not dengue patients. Our data suggest that in hypoendemic regions acute P. vivax infection skews peripheral B‐cell subsets and results in a persistent parasite‐specific and autoreactive IgM response.     

Prevalence of over-/misdiagnosis of asthma in patients referred to an allergy clinic

Objective: Increasing asthma incidence may be due to an overall increase in asthma awareness by physicians, potentially resulting in overdiagnosis. One of the unique features of asthma is bronchial hyperresponsiveness, which can be assessed by methacholine bronchial challenge (MBC). Overdiagnosis may result in over- or mistreatment. The aims of this study were to describe the prevalence of the over-/misdiagnosis of asthma and the use of anti-asthmatic drugs in patients with asthma-like symptoms who had not yet undergone a respiratory function assessment to confirm the diagnosis of asthma. Methods: This was a retrospective study analyzing all MBCs performed by our Outpatient Allergy Clinic in a two-year period to confirm/exclude the diagnosis of asthma in patients referred by general practitioners and complaining of asthma-like symptoms. Anti-asthmatic medications used by the patients until the MBC date were recorded. Results: 43.8% of the reviewed MBCs were positive and 37.4% of the patients with a positive MBC were previously taking anti-asthmatic drugs (568.8?±?76.4?mcg mean beclomethasone equivalents), compared to 51.2% of those patients with a negative MBC (464.8?±?57.8?mcg). No differences were found in the daily doses of inhaled corticosteroids or other anti-asthmatic drugs, or in the duration of treatment before the assessment of bronchial hyperresponsiveness. Conclusions: A sizeable percentage of subjects who reported physician-diagnosed asthma had a negative MBC. Nevertheless, a greater proportion of negative MBC patients were taking anti-asthmatic drugs compared to those with a confirmed diagnosis of asthma, illustrating that the overdiagnosis of asthma may lead to over- and mistreatment of respiratory symptoms.     

Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat,Central India: implications for the diagnosis of malaria and its control

Objective During an epidemiological study (January–July 2012) on malaria in forest villages of Central India, Plasmodium malariae‐like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species‐specific nested polymerase chain reaction (PCR) and DNA sequencing. Methods All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae. Results Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3‐day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum. Discussions Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re‐affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.