Division and Transmission: Malaria Parasite Development in the Mosquito.

The malaria parasite life cycle alternates between two hosts: a vertebrate and the female Anopheles mosquito vector. Cell division, proliferation, and invasion are essential for parasite development, transmission, and survival. Most research has focused on Plasmodium development in the vertebrate, which causes disease; however, knowledge of malaria parasite development in the mosquito (the sexual and transmission stages) is now rapidly accumulating, gathered largely through investigation of the rodent malaria model, with Plasmodium berghei. In this review, we discuss the seminal genome-wide screens that have uncovered key regulators of cell proliferation, invasion, and transmission during Plasmodium sexual development. Our focus is on the roles of transcription factors, reversible protein phosphorylation, and molecular motors. We also emphasize the still-unanswered important questions around key pathways in cell division during the vector transmission stages and how they may be targeted in future studies.

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle.

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

The molecular mechanisms driving Plasmodium cell division.

Malaria, a vector borne disease, is a major global health and socioeconomic problem caused by the apicomplexan protozoan parasite Plasmodium. The parasite alternates between mosquito vector and vertebrate host, with meiosis in the mosquito and proliferative mitotic cell division in both hosts. In the canonical eukaryotic model, cell division is either by open or closed mitosis and karyokinesis is followed by cytokinesis; whereas in Plasmodium closed mitosis is not directly accompanied by concomitant cell division. Key molecular players and regulatory mechanisms of this process have been identified, but the pivotal role of certain protein complexes and the post-translational modifications that modulate their actions are still to be deciphered. Here, we discuss recent evidence for the function of known proteins in Plasmodium cell division and processes that are potential novel targets for therapeutic intervention. We also identify key questions to open new and exciting research to understand divergent Plasmodium cell division.

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Fluconazole-resistant Candida parapsilosis complex candidemia and analysis of mutations in the ERG11 gene from Pakistan.

BACKGROUND: Recent reports of the emergence of fluconazole resistance in Candida parapsilosis species complex poses a challenge, more specifically in settings where echinocandin-based treatment regime is not feasible. OBJECTIVE: This study reported emergence of fluconazole resistance in C. parapsilosis species complex strains isolated from blood cultures. MATERIALS AND METHODS: This retrospective observational study was conducted from 2018 to 2020 at a tertiary care laboratory from Pakistan. Fluconazole-resistant C. parapsilosis species complex fungemia cases were identified from laboratory database and clinical details were collected. Identification of C. parapsilosis species complex was done using API 20C AUX and Cornmeal Tween80 agar morphology. Minimum inhibitory concentrations (MICs) were determined using Sensititre YeastONE and interpretation was done with CLSI M60 ED1:2017. ERG11 gene region was amplified and sequenced by Sanger sequencing and analysed by MEGA 11 Software. RESULTS: A total of 13 (8.5%) fluconazole-resistant isolates were identified from 152 C. parapsilosis species complex candidemia cases. Fluconazole MICs of resistant isolates ranged between 8 and 256 µg/mL. Analysis of ERG11 gene revealed nonsynonymous mutations at position Y132F in 86% of the fluconazole-resistant isolates. Diabetes and hospitalization were important risk factors for candidemia with fluconazole-resistant C. parapsilosis complex. CONCLUSION: This is the first report of the emergence and molecular mechanisms of fluconazole resistance in C. parapsilosis species complex from Pakistan. Y132F mutation in the ERG11 gene was the most common mutation in fluconazole-resistant strains. These findings are concerning and necessitate better diagnostics, newer antifungals, ongoing surveillance and further insights on resistance mechanisms in the country.

Real-time dynamics of Plasmodium NDC80 reveals unusual modes of chromosome segregation during parasite proliferation.

Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. Species of the genus Plasmodium, the causative agents of malaria, display remarkable aspects of nuclear division throughout their life cycle to meet some peculiar and unique challenges to DNA replication and chromosome segregation. The parasite undergoes atypical endomitosis and endoreduplication with an intact nuclear membrane and intranuclear mitotic spindle. To understand these diverse modes of Plasmodium cell division, we have studied the behaviour and composition of the outer kinetochore NDC80 complex, a key part of the mitotic apparatus that attaches the centromere of chromosomes to microtubules of the mitotic spindle. Using NDC80-GFP live-cell imaging in Plasmodium berghei, we observe dynamic spatiotemporal changes during proliferation, including highly unusual kinetochore arrangements during sexual stages. We identify a very divergent candidate for the SPC24 subunit of the NDC80 complex, previously thought to be missing in Plasmodium, which completes a canonical, albeit unusual, NDC80 complex structure. Altogether, our studies reveal the kinetochore to be an ideal tool to investigate the non-canonical modes of chromosome segregation and cell division in Plasmodium.

Extraintestinal Seeding of Salmonella enterica Serotype Typhi, Pakistan.

We evaluated Salmonella enterica serotype Typhi strains isolated from all body sites in Pakistan during 2013-2018. Despite an increase in overall number of localized, extensively drug-resistant Salmonella Typhi in organ infections during 2018, there was no increase in the proportion of such isolates in comparison with non-extensively drug-resistant isolates.

Plasmodium kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission.

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.

Postprandial fasting related headache during Ramadan in Saudi Arabia: A cross-sectional study.

BACKGROUND: Headache disorders are classified as primary or secondary; however, among the secondary headaches, those attributed to food ingestion are not well understood. Therefore, we conducted this study to describe and characterize a new headache entity that occurred during the holy month of Ramadan. This headache occurred within 4 h of breaking the fast. METHODS: This is a nationwide descriptive community-based cross-sectional study conducted during the last 10 days of Ramadan, based on a random sample of adults living in Saudi Arabia. The demographic data, headache symptomatology, nature and distribution of the pain, possible triggering and relieving factors, and patient management programs were analyzed. RESULTS: Completed questionnaires were obtained from 16,031 participants. Of those, 3147 (19.6%) reported headaches after breaking the fast in Ramadan. In 84.1% of cases, there was no previous diagnosis of headache or migraine. The characteristics of these postprandial fasting-related headaches mostly was episodic in nature (72%). The nature of the headache was variable, mostly heaviness or tightness (53.9%). Triggering factors included ingestion of fried food in (45%) and coffee (26.3%). Lying down and sleeping was found to be an important relieving factor (61%). CONCLUSION: A new headache entity is being described. Appears to be quite common, occurs less than 2 h following the first meal, and is mostly of the heaviness and tension type.

Prevalence and risk factors associated with multi-drug resistant organisms (MDRO) carriage among pediatric patients at the time of admission in a tertiary care hospital of a developing country. A cross-sectional study.

BACKGROUND: The rise of Multidrug-resistant organisms (MDROs) poses a considerable burden on the healthcare systems, particularly in low-middle income countries like Pakistan. There is a scarcity of data on the carriage of MDRO particularly in the pediatrics population therefore, we aimed to determine MDRO carriage in pediatric patients at the time of admission to a tertiary care hospital in Karachi, Pakistan, and to identify the risk factors associated with it. METHODS: A cross-sectional study conducted at the pediatric department of Aga Khan University Hospital (AKUH) from May to September 2019 on 347 children aged 1-18 years. For identification of MDRO (i.e., Extended Spectrum Beta-Lactamase (ESBL) producers, Carbapenem Resistant Enterobacteriaceae (CRE), Vancomycin Resistant Enterococci (VRE), Methicillin Resistant Staphylococcus aureus (MRSA), Multidrug-resistant (MDR) Acinetobacter species and MDR Pseudomonas aeruginosa), nasal swabs and rectal swabs or stool samples were cultured on specific media within 72 h of hospitalization. Data was collected on a predesigned structured questionnaire on demographics, prior use of antibiotics for > 48 h in the last 6 months, history of vaccination in last 6 months, exposure to health care facility regardless of the time of exposure, ICU stay for > 72 h, and about the prior use of medical devices (urinary catheter, central venous lines etc.) in last 1 year. Statistical analysis was performed by Standard statistical software. RESULTS: Out of 347 participants, 237 (68.3%) were found to be MDRO carriers. Forty nine nasal swabs from 346 children (14.2%) showed growth of MRSA. The majority of the stool/rectal swabs (n = 222 of 322; 69%) collected were positive for MDRO. The most isolated species were ESBL Escherichia coli 174/222 (78.3%) followed by ESBL Enterobacter species 37/222 (16.7%) and ESBL Klebsiella pneumoniae 35/222 (15.8%). On univariate analysis, none of the risk factors showed statistically significant association with MDRO carriage. CONCLUSION: Overall, a high prevalence of MDRO carriage was identified among admitted pediatric patients. Implementation of systematic screening may help to identify true burden of MDROs carriage in the health care settings.

Systematic analysis of Plasmodium myosins reveals differential expression, localisation, and function in invasive and proliferative parasite stages.

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.

Efforts to improve diagnosis of bacteraemia by reducing blood culture contamination in an emergency department: strategies and outcome.

OBJECTIVE: To assess the strategies and outcome for reducing blood culture contamination in order to improve the diagnosis of bacteraemia. METHODS: The interventional study was conducted at a tertiary care hospital in Karachi from January 1, 2013, to December 31, 2016. The blood culture contamination data related to the first year of the study was taken as the baseline pre-intervention data. Strategies were planned as intervention for improvement by consolidating training and education in the form of dedicated lectures, practising on mannequins and developing in-house video, replacing povidone with 2% chlorhexidine preparation spray plus 70% isopropyl alcohol swabs and inducting dedicated phlebotomy team whose only responsibility was blood sample collection and minimising the probability of error. RESULTS: In 2013, there were 8868 samples; 7402 in 2014; 6897 in 2015; and 9756 samples in 2016. The contamination rate in 2013 was 8% which went down to 7.75% in 2014, 4.25% in 2015 and 3.9% in 2016. The decline became statistically significant (p<0.001) after implementing a dedicated phlebotomy team in the emergency department. CONCLUSIONS: Apart from teaching and training, the concept of blood culture collection kit with checklist and dedicated blood collection team was found to be vital in reducing blood culture contamination.

Use of pefloxacin as a surrogate marker to detect ciprofloxacin susceptibility in Salmonella enterica serotypes Typhi and Paratyphi A.

OBJECTIVE: To determine the use of pefloxacin as a surrogate marker to detect fluoroquinolone (ciprofloxacin) susceptibility against Salmonella enterica serotypes Typhi and Paratyphi A. METHODS: The prospective, descriptive cross-sectional study was conducted at the Aga Khan University Hospital, Karachi, from September 2016 to March 2018, and comprised Salmonella Typhi and Paratyphi A isolates of blood cultures. Disk susceptibility tests and broth microdilution to test minimum inhibitory concentration were performed as per standard guidelines. Data was analysed using SPSS 21. RESULTS: Of the 138 isolates, 91(66%) were intermediate resistant to ciprofloxacin but were resistant to pefloxacin, 42(30%) were resistant to both ciprofloxacin and pefloxacin, and 5(4%) were susceptible to both ciprofloxacin and pefloxacin. Of the isolates that were intermediate resistant to ciprofloxacin, 85(93%) had minimum inhibitory concentration range0.12-0.5mg\L, while 6(7%) had MIC>1mg\L (p<0.0001). CONCLUSIONS: Pefloxacin disk diffusion test was found to be reliable in detecting fluoroquinolone resistance among enteric fever causing Salmonella.

Is non-traditional therapy for multiple sclerosis overwhelming in Saudi Arabia.

OBJECTIVE: To describe the prevalence, knowledge and attitudes about complementary and alternative medicine (CAM) use and the proportion that seek advice from their physician about CAM use. METHODS: This cross-sectional observational study was performed in multiple sclerosis (MS) clinic of King Fahd Hospital of Universityin Alkhobar, Kingdom of Saudi Arabia from January-June 2017. A total of 133 patients have completed the survey. RESULTS: The mean age of patients was 32.3+/-7.6 years and 84 (63.2%) were female. Approximately 83.5% of the patients reported the use of CAM. Among all the reported forms of CAM, vitamins were the most prevalent form, followed by cupping, special prayers and meditation. The majority of patients (62%) obtained knowledge of CAM through social media. A significant number of patients (75.6%) did not disclose the use of CAM to their physician. There was a trend for using CAM more in highly educated, older age, and female patients. The most commonly reported rationale to use CAM was overall improvement in health status. CONCLUSION: The use of CAM among Saudi patients with MS is highly prevalent, without disclosure of its use to physicians. These factors should be taken into account in the doctor-patient consultation to avoid adverse events.

Proteomic Identification and Analysis of Arginine-Methylated Proteins of Plasmodium falciparum at Asexual Blood Stages.

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR, or WxxxR. We identified Plasmodium homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth.

Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

Multiple Plasmodium vivax proteins of Pv-fam-a family interact with human erythrocyte receptor Band 3 and have a role in red cell invasion.

Elucidation of molecular mechanisms of receptor-ligand biology during host-parasite interaction helps in developing therapeutic targets. Several Pv-fam-a family proteins of Plasmodium vivax bind to host erythrocytes but their erythrocyte receptors remains to be explored. Here, we show that three merozoite proteins (PvTRAg36, PvATRAg74, and PvTRAg38) of this family interact with Band 3 on human erythrocytes through its three exofacial loops (loop 1, loop 3, and loop 6). These parasite proteins also interfered with the parasite growth in in-vitro, and the inhibition rate seems to be associated with their binding affinity to Band 3. This redundancy in receptor-ligand interaction could be one of the probable mechanism parasite utilizes to invade the host erythrocyte more efficiently.

Host-parasite interaction: selective Pv-fam-a family proteins of Plasmodium vivax bind to a restricted number of human erythrocyte receptors.

BACKGROUND: Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. METHODS: Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. RESULTS: Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. CONCLUSIONS: This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria.

CD4+ T cell response correlates with naturally acquired antibodies against Plasmodium vivax tryptophan-rich antigens.

Tryptophan-rich proteins play important biological functions for the Plasmodium parasite. Plasmodium vivax contains remarkably large numbers of such proteins belonging to the "Pv-fam-a" family that need to be characterized. Earlier, we reported the presence of memory T cells and naturally acquired antibodies against 15 of these proteins in P. vivax malaria-exposed individuals (M. Zeeshan, H. Bora, and Y. D. Sharma, J Infect Dis 207:175-185, 2013, http://dx.doi.org/10.1093/infdis/jis650). Here, we sought to characterize and ascertain the cross talk between effector responses of T and B cells in malarial patients against all Pv-fam-a family proteins. Therefore, we expressed the remaining 21 of these proteins in Escherichia coli and studied the humoral and cellular immune responses based on the same parameters used in our previous study. Naturally acquired IgG antibodies were detected against all 21 antigens in P. vivax patient sera (37.7 to 94.4% seropositivity). These antigens were able to activate the lymphocytes of P. vivax-exposed individuals, and the activated CD4(+) T lymphocytes produced higher levels of Th1 (interleukin-2 [IL-2] and gamma interferon [IFN-γ]) and Th2 (IL-4 and IL-10) cytokines than the healthy controls, but the response was Th2 biased. The combined results of present and previous studies seem to suggest a striking link between induction of the CD4(+) T cell response and naturally acquired antibodies against all 36 proteins of the Pv-fam-a family, the majority of them having conserved sequences in the parasite population. Further work is required to utilize this information to develop immunotherapeutic treatments for this disease.

Presence of memory T cells and naturally acquired antibodies in Plasmodium vivax malaria-exposed individuals against a group of tryptophan-rich antigens with conserved sequences.

BACKGROUND: Tryptophan-rich antigens of malarial parasites have been proposed to be the potential vaccine candidate antigens. Plasmodium vivax contains the largest number of such antigens, which need to be evaluated for their immune responses. METHODS: Recombinant proteins of 15 P. vivax tryptophan-rich antigens (PvTRAgs) were expressed, purified, and used for the human humoral and cellular immune responses. Genetic polymorphism of these 15 genes was also determined among clinical P. vivax isolates. RESULTS: The T lymphocytes of P. vivax exposed individuals expressed higher level of CD69 against all 15 PvTRAgs. These antigens also activated the large population of CD4(+) T cells and produced higher level of intracellular IL-2, INF-γ and IL-4. Although there was a mixed Th1 and Th2 response against these antigens, this response was biased toward Th2. The majority of P. vivax patients (75.7%-100%, n = 33) produced IgG antibodies against these antigens. Most of these antigens showed conserved T- and B-cell epitopes in the parasite population. CONCLUSIONS: These results suggest the presence of memory T cells in humans against these antigens to generate faster and more specific immune responses to minimize the P. vivax infection. Further characterization of these PvTRAgs may lead to the identification of a potential therapeutic target.