Malaria diagnostics and surveillance in the post-genomic era.

Genome sequences are available for 3 human-infecting malaria parasites, Plasmodium falciparum, P. vivax and P. knowlesi, and population genomics data are available for many endemic regions. This review summarizes how genomic data have been used to develop new, species-specific molecular targets for better malaria diagnosis. The combination of bioinformatics and genomics has been used to identify new sequence targets suitable for diagnostic applications and assess their viability within the context of global Plasmodium sequence variation. The selection criteria maximized the sensitivity and specificity of the novel targets. At least one target from each species was found to be suitable for molecular diagnosis of malaria with some advantages over existing molecular methods. The promise of using genome sequence data to develop sensitive, genus- or species-specific diagnostic methods for other pathogens of public health interest is strong. This undertaking together with what we envision as the future of malaria diagnosis in the 'omic' era is discussed.

Malaria prevalence among pregnant women in two districts with differing endemicity in Chhattisgarh, India.

BACKGROUND: In India, malaria is not uniformly distributed. Chhattisgarh is a highly malarious state where both Plasmodium falciparum and Plasmodium vivax are prevalent with a preponderance of P. falciparum. Malaria in pregnancy (MIP), especially when caused by P. falciparum, poses substantial risk to the mother and foetus by increasing the risk of foetal death, prematurity, low birth weight (LBW), and maternal anaemia. These risks vary between areas with stable and unstable transmission. The specific objectives of this study were to determine the prevalence of malaria, its association with maternal and birth outcomes, and use of anti-malarial preventive measures for development of evidence based interventions to reduce the burden of MIP. METHODS: A cross-sectional study of pregnant women presenting to antenatal clinics (ANC) or delivery units (DU), or hospitalized for non-obstetric illness was conducted over 12 months in high (Bastar) and low (Rajnandgaon) transmission districts in Chhattisgarh state. Intensity of transmission was defined on the basis of slide positivity rates with a high proportion due to P. falciparum. In each district, a rural and an urban health facility was selected. RESULTS: Prevalence of peripheral parasitaemia was low: 1.3% (35/2696) among women at ANCs and 1.9% at DUs (19/1025). Peripheral parasitaemia was significantly more common in Bastar (2.8%) than in Rajnandgaon (0.1%) (p < 0.0001). On multivariate analysis of ANC participants, residence in Bastar district (stable malaria transmission) was strongly associated with peripheral parasitaemia (adjusted OR [aOR] 43.4; 95% CI, 5.6-335.2). Additional covariates associated with parasitaemia were moderate anaemia (aOR 3.7; 95% CI 1.8-7.7), fever within the past week (aOR 3.2; 95% CI 1.2-8.6), and lack of formal education (aOR 4.6; 95% CI 2.0-10.7). Similarly, analysis of DU participants revealed that moderate anaemia (aOR 2.5; 95% CI 1.1-5.4) and fever within the past week (aOR 5.8; 95% CI 2.4-13.9) were strongly associated with peripheral and/or placental parasitaemia. Malaria-related admissions were more frequent among pregnant women in Bastar, the district with greater malaria prevalence (51% vs. 11%, p < 0.0001). CONCLUSIONS: Given the overall low prevalence of malaria, a strategy of enhanced anti-vector measures coupled with intermittent screening and targeted treatment during pregnancy should be considered for preventing malaria-associated morbidity in central India.

Immune responses of mice with different genetic backgrounds to improved multiepitope, multitarget malaria vaccine candidate antigen FALVAC-1A.

FALVAC-1A is a second-generation multitarget, multiepitope synthetic candidate vaccine against Plasmodium falciparum, incorporating elements designed to yield a stable and immunogenic molecule. Characteristics of the immunogenicity of FALVAC-1A were evaluated in congenic (H-2(b), H-2(k), and H-2(d)) and outbred strains of mice. The influences of four adjuvants (aluminum phosphate, QS-21, Montanide ISA-720, and copolymer CRL-1005) on different aspects of the immune response were also assessed. FALVAC-1A generated strong antibody responses in all mouse strains. The highest mean enzyme-linked immunosorbent assay (ELISA) antibody concentrations against FALVAC-1A were observed in the outbred ICR mice, followed by B10.BR, B10.D2, and C57BL/6 mice, though this order varied for the different adjuvants, with no statistical differences between mouse strains. In all mouse strains, the highest anti-FALVAC-1A antibody titers in ELISAs were induced by FALVAC-1A in copolymer and ISA-720 formulations, followed by QS-21 and AlPO4. These antibodies were of all four subclasses, though immunoglobulin G1 (IgG1) predominated, with the exception of FALVAC-1A with the QS-21 adjuvant, which induced predominantly IgG2c responses. Both sporozoites and blood stages of P. falciparum were recognized by anti-FALVAC-1A sera in the immunofluorescence assay. In addition to antibody, cellular immune responses were detected; these responses were studied by examining spleen cells producing gamma interferon and interleukin-4 in enzyme-linked immunospot assays. In summary, FALVAC-1A was found to be highly immunogenic and elicited functionally relevant antibodies that can recognize sporozoites and blood-stage parasites in diverse genetic backgrounds.

Comparative flow cytometric analysis of term placental intervillous and peripheral blood from immediate postpartum women in Western kenya.

Understanding maternal immune responses in the placenta is critical for management of pregnancy failures and haematogenous infections during pregnancy. However, it is unknown whether maternal placental intervillous blood (IVB) mononuclear cell populations are distinct from those found in maternal peripheral blood (PB). In this study, cell populations in the IVB and PB from immediate postpartum women were compared by flow cytometry. While levels of B and CD4+ and CD8+ T lymphocytes were similar, IVB contained significantly higher levels of monocytes (10.9+/-5.9 versus 5.5+/-2.5 per cent, respectively) and natural killer cells (14.3+/-9.6 versus 5.9+/-3.2 per cent, respectively) than the PB. Expression of the early activation marker CD69 was increased on T cells in the IVB, whereas levels of HLA-DR, a late activation marker, were similar between IVB and PB. These results suggest that maternal cells that circulate through the intervillous compartment may be subject to local influences that affect their distribution, phenotype and function. Further comparative study of these blood compartments will be necessary to elucidate the mechanisms by which the local placental milieu influences the IVB.

Tumor necrosis factor-alpha promoter variant 2 (TNF2) is associated with pre-term delivery, infant mortality, and malaria morbidity in western Kenya: Asembo Bay Cohort Project IX.

A polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene, with a guanine to adenine nucleotide change at position -308, TNF2 is associated with increased TNF-alpha production. TNF2 homozygotes have a higher risk of severe disease and/or death due to cerebral malaria and other infectious diseases. We investigated the impact of this allele on malaria morbidity and mortality in young children who participated in an immuno-epidemiologic cohort study of malaria in an area of intense perennial Plasmodium falciparum transmission in western Kenya. A total of 1,048 children were genotyped. Poisson regression and Cox proportional hazards models were used to determine the relationship between TNF-308 variants and morbidity and mortality. The gene frequencies of the TNF1 and TNF2 alleles were 0.90 and 0.10, respectively. TNF2 homozygosity was associated with pre-term birth when compared with TNF1 homozygotes [relative risk (RR) 7.3, 95% CI, 2.85-18.9, P = 0.002) and heterozygotes (RR 6.7, 95% CI 2.0-23.0, P = 0.008). Among children born prematurely, the TNF2 allele was significantly associated with a higher risk of death in infancy compared with TNF1 (RR 7.47, 95% CI 2.36-23.6). The risk of death was higher among TNF2 homozygotes than among heterozygotes. The TNF2 allele was significantly associated with high density P. falciparum parasitemia (RR 1.11, 95% CI 1.0-1.24). Among low birth weight children, the TNF2 allele was associated with severe anemia (RR 2.16, 95% CI 1.17-4.01) and showed a trend toward a risk for severe malaria anemia (RR 1.99, 95% CI 0.89-4.46). These data suggest that TNF2 is a risk factor for pre-term birth and early childhood mortality and malaria morbidity in children in this region. Further understanding of the pathogenic mechanisms underlying this association is required.

Longitudinal study of natural immune responses to the Plasmodium falciparum apical membrane antigen (AMA-1) in a holoendemic region of malaria in western Kenya: Asembo Bay Cohort Project VIII.

We investigated the development and maintenance of proliferative and antibody responses to apical membrane antigen-1 (AMA-1) epitopes in a holoendemic area of western Kenya. Young children (< 10 years), older children (10-17 years), and adults (> or = 18 years) were followed longitudinally for antibody and T-cell responses at 3 time points with an interval of 3-4 months. The proliferative responses against the AMA-1 T epitopes (PL171, PL172, PL173, PL186, PL191, and PL192) were not stable during follow-up; however, response to mycobacterial antigen PPD was highly stable. The responder frequencies were similar in all 3 time points except for epitope PL192. The younger and older children responded more frequently to T-cell epitopes, but the differences were not significant. A positive proliferative response to PL191 was associated with a significantly lower risk of parasitemia at subsequent follow-up (relative risk, 0.5; P = 0.03). The presence of antibody response to B epitopes PL169, PL170, PL173, PL187, and PL192 in one time point was associated with a subsequent response (P = 0.0001-0.008) suggesting a stable response. Younger (P = 0.046) and older children (P = 0.017) more frequently responded to epitope PL169 than did adults, and adults responded more frequently to PL187 than did younger children (P = 0.009). Responses to AMA-1 T-cell epitopes were short lived, and antibody responses were relatively stable.

Immunity to placental malaria. II. Placental antigen-specific cytokine responses are impaired in human immunodeficiency virus-infected women.

An association was demonstrated recently between elevated in vitro production of interferon (IFN)-gamma by intervillous blood mononuclear cells (IVBMCs) and protection against placental malaria (PM). Because human immunodeficiency virus (HIV)-infected pregnant women have increased susceptibility to PM, loss of the IFN-gamma response in these women may impair their ability to control PM. Measurement of cytokines in culture supernatants by ELISA revealed that IFN-gamma responses by HIV-positive IVBMCs were impaired, especially after malarial antigen stimulation. Interleukin (IL)-4 and IL-10 responses also were reduced in HIV-positive persons, the latter more so in HIV-positive, PM-positive persons. In contrast, tumor necrosis factor-alpha production generally was enhanced in PM-positive and HIV-positive persons. Overall, cytokine production was reduced in HIV-positive persons with CD4 T cell counts <500/microL, particularly in response to malarial antigen. Thus, HIV-mediated cytokine dysregulation and impairment of the protective IFN-gamma response may contribute to the increased susceptibility of HIV-positive pregnant women to malaria.

Host-pathogen interactions in emerging and re-emerging infectious diseases: a genomic perspective of tuberculosis, malaria, human immunodeficiency virus infection, hepatitis B, and cholera.

On exposure to a pathogen, a host may resist infection, become subclinically infected, or progress through several stages from mild to severe infection. Chronic sequelae may or may not occur. Host factors, particularly host genes, influence many of these stages. We have used a model of the continuum of pathogenesis of infectious diseases to consider the effect of host genes on five pathogens of significant public health burden: Mycobacterium tuberculosis, Plasmodium species, human immunodeficiency virus, hepatitis B virus, and Vibrio cholerae. The relationships between these infections and polymorphisms in human leukocyte antigen, cytokines, other immune response, or pathogen receptor genes are reviewed. We discuss gene-gene interactions and their effects in complex settings, such as coinfections with several pathogens. Priorities for prevention and control of these pathogens include vaccines and antimicrobial drugs. Research on how host genes can influence vaccine responses and the efficacy of drugs or other interventions, as well as further research into the relationship of host genes to infectious disease outcomes, may lead to new strategies for prevention and control.

Development, expression, and murine testing of a multistage Plasmodium falciparum malaria vaccine candidate.

A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.

Immunologic memory in the placenta: a lymphocyte recirculation hypothesis.

The placenta is an immunologically unique organ where a balance between maternal immunity and fetoplacental well-being must be maintained for successful pregnancy to occur. The intervillous blood is important in this context, yet little is known about local immunologic processes, particularly how placenta-specific memory immune responses are maintained. Using malaria as an illustrative case, we describe an hypothetical model in which recirculation of memory T lymphocytes from the intervillous blood to local lymphoid tissue facilitates maintenance of local memory immunity. This explains how memory cells might be retained when the placenta is expelled at parturition and thus remain available for rapid recall from the local lymphoid tissue to the intervillous space when exposure to the same antigenic stimulus occurs in subsequent pregnancies. Study of cell-mediated immunity to infections like malaria in the intervillous blood and the use of animal models will be necessary to provide proof for this hypothesis.

Field studies of cytotoxic T lymphocytes in malaria infections: implications for malaria vaccine development.

The search for a cytotoxic T lymphocyte (CTL)-inducing malaria vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines. Development of CTL-inducing vaccine candidates has taken center stage based on the observation that CTL-mediated protection might be the dominant mechanism by which sterile immunity is achieved in irradiated sporozoite immunization experiments in humans and laboratory animals. However, studies in naturally infected individuals living in endemic areas, as reviewed here by Michael Aidoo and Venkatachalam Udhayakumar, have revealed that CTL induction might be influenced by factors such as parasite variants, host genes, other infections and transmission patterns. The influence of these factors on CTL induction has been demonstrated individually and in various combinations in controlled animal experiments. However, in naturally infected humans, they are presented in a complex host-parasite-environment interaction, in a manner that is not easily achieved in laboratory-based experiments. Understanding these interactions is crucial for the development and testing of CTL-inducing vaccines for humans.

Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission I. Description of study site, general methodology, and study population.

A large-scale longitudinal cohort project was initiated in western Kenya in June 1992. The primary purpose of the project was to study Plasmodium falciparum malaria in a highly endemic area using a comprehensive and multidisciplinary approach, which included epidemiology, entomology, and immunology. Between June 1992 and July 1994, pregnant women living in 15 rural villages were identified during a monthly census and 1,164 were enrolled. The women were followed-up throughout their pregnancy and they, along with their newborn infants and direct siblings of the infants' less than 15 years of age, were monitored over time. As of May 1995, 1,017 infants had been born to these women. This paper presents the design and general methodology used in this study and describes the initial experience with intense monitoring of a large population over a prolonged period.

Immunity to placental malaria. I. Elevated production of interferon-gamma by placental blood mononuclear cells is associated with protection in an area with high transmission of malaria.

In areas in which malaria is holoendemic, primigravidae and secundigravidae, compared with multigravidae, are highly susceptible to placental malaria (PM). The nature of gravidity-dependent immune protection against PM was investigated by measuring in vitro production of cytokines by placental intervillous blood mononuclear cells (IVBMC). The results demonstrated that interferon (IFN)-gamma may be a critical factor in protection against PM: production of this cytokine by PM-negative multigravid IVBMC was elevated compared with PM-negative primigravid and secundigravid and PM-positive multigravid cells. Low IFN-gamma responsiveness to malarial antigen stimulation, most evident in the latter group, was balanced by increased interleukin (IL)-4 production, suggesting that counter-regulation of these two cytokines may be a crucial determinant in susceptibility to PM. A counter-regulatory relationship between IL-10 and tumor necrosis factor-alpha was also observed in response to malarial antigen stimulation. These data suggest that elevated production of IFN-gamma, as part of a carefully regulated cytokine network, is important in the control of PM.

Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites.

Using a flow cytometry-based parasite growth inhibition assay (GIA) and an antibody-dependent cellular inhibition (ADCI) assay, we have assessed the differential effect and interaction of monocytes, immune sera, and purified immunoglobulins from Kenyan adults on the growth of Plasmodium falciparum parasites in vitro. We found that monocytes from 14 different normal, healthy, non-malaria-exposed donors had varying effects on parasite growth, i.e., inhibition or enhancement of parasitemia, suggesting heterogeneity in anti-parasitic activities of monocytes from individual donors. Twenty-two serum samples collected from clinically immune adults from western Kenya inhibited growth of P. falciparum after 48 hr in culture. In contrast, all IgG preparations, except one, purified from the same serum samples enhanced parasite growth. In ADCI experiments, of the 22 purified IgG samples used, 11 showed ADCI activities with specific growth inhibition (SGI) of more than 10%, with the highest at 27.6%, and the remaining 11 IgG samples had an SGI of less than 10%. Our results also showed that the ratio of IgG1 to IgG3 antibodies, as determined by an indirect immunofluorescence assay, was higher in the high ADCI response group than in the low response group, suggesting that a higher concentration of IgG1 antibodies with a higher IgG1/IgG3 ratio might be associated with ADCI activities. The present study has resulted in the development of simple, reproducible flow cytometry-based GIA and ADCI assays, and also provides baseline information for further investigation of the role of ADCI activity in naturally acquired immune protection against malaria.

A low interleukin-10 tumor necrosis factor-alpha ratio is associated with malaria anemia in children residing in a holoendemic malaria region in western Kenya.

The balance between Th1 cytokines (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma) and Th2 cytokines (interleukin [IL]-10, -4) may be critical in the development of severe falciparum malaria. Therefore, plasma concentrations of these cytokines were determined in children with various manifestations of malaria. Plasma levels of IFN-gamma and IL-4 were undetectable in most children. However, TNF-alpha and IL-10 were significantly elevated in children with high-density parasitemia and malaria anemia compared with children in control groups. In children with mild malaria, IL-10, but not TNF-alpha, was significantly elevated. While the highest concentrations of TNF-alpha were found in children with malaria anemia, IL-10 levels were highest in children with high-density uncomplicated malaria. The mean ratio of IL-10 to TNF-alpha was significantly higher in children with mild and high-density parasitemia (4.64, P<.005) than in children with malaria anemia (1.77). Thus, higher levels of IL-10 over TNF-alpha may prevent development of malaria anemia by controlling the excessive inflammatory activities of TNF-alpha.

Immunogenicity of Plasmodium falciparum and Plasmodium vivax circumsporozoite protein repeat multiple antigen constructs (MAC).

In this study we characterized the immunogenic properties of three different multispecies multiple antigen constructs (MACs) carrying the circumsporozoite protein (CSP) repeats of human malaria parasites, Plasmodium falciparum and P. vivax. We synthesized tetrameric MACs containing the antigenic repeats from the CSP of P. vivax-like parasite in two arms and CSP repeat sequences of either P. vivax type-1 (vivax-like/vivax type-1 MAC), P. vivax type-2 (vivax-like/vivax type-2 MAC), or P. falciparum (vivax-like/falciparum MAC) in the other two arms. Mice of four different genetic backgrounds (H-2a, H-2b, H-2d, and H-2k) were immunized with these MACs in Freund's adjuvant. All three MAC preparations were found to elicit antibodies to P. vivax-like CSP repeats in B10.BR, B10.A, and C57BL/6 mice. On the other hand, in B10.D2 mice only vivax-like/vivax type-1 MAC, but not the other two MACs induced antibodies to the P. vivax-like CSP repeats. In mice immunized with vivax-like/vivax type-1 MAC, antibodies to P. vivax type-1 CS repeat peptides were induced in B10.BR, B10.A, and C57BL/6 mice, but not in B10.D2 mice. Antibody responses to P. vivax type-2 repeats were not induced in any of the four strains of mice that were immunized with vivax-like/vivax type-2 MAC. While B10.BR, B10.A, and C57BL/6 mice produced antibodies to NANP repeats of P. falciparum CSP following immunization with vivax-like/falciparum MAC, B10.D2 mice failed to elicit antibodies to this repeat. All the sera that showed positive reactivity to peptides in enzyme-linked immunosorbent assay were found to react with sporozoites by IFA. In conclusion, these results showed that naturally immunogenic epitopes from different species of malaria parasites can be incorporated in a single vaccine construct to induce immune responses against multiple epitopes.

A longitudinal investigation of IgG and IgM antibody responses to the merozoite surface protein-1 19-kiloDalton domain of Plasmodium falciparum in pregnant women and infants: associations with febrile illness, parasitemia, and anemia.

This study was aimed at delineating characteristics of naturally acquired immunity against the merozoite surface antigen-1 (MSP-1) of Plasmodium falciparum, a candidate malaria vaccine antigen. A case/control study was performed on 75 case/control pairs of infants with febrile illness at the time of the first detected infection indicating a clinical case. The presence and level of antibodies at one month prior to the first infection and at the time of the first infection in the afebrile group was significantly higher than in the febrile group. Decreased parasite density and decreased infection-related loss of hemoglobin was seen in infants with anti-MSP-1(19kD) IgG antibodies. In addition, mothers who were positive for the presence of these antibodies conferred protection against placental infection and infection in their infants. In this study, development of anti-MSP-1(19kD) antibody responses in 24 infants were studied longitudinally using monthly serum samples collected from birth until approximately one year of age. In addition, umbilical cord blood sera and respective mothers' sera were analyzed. Longitudinal studies of antibody responses revealed several short-lived IgG and IgM peaks throughout an infant's first year that correlated with detection of parasitemia. The protection against parasitemia and febrile illness was observed in infants when anti-MSP-1(19kD) antibodies were present; when infants were negative for IgG, they had a 10-times greater risk of becoming parasitemic. These data from a longitudinal and prospective study of malaria suggest a protective role for anti-MSP-1(19kD) antibodies in infants and pregnant women.

Cytotoxic T cell reactivity and HLA-B35 binding of the variant Plasmodium falciparum circumsporozoite protein CD8+ CTL epitope in naturally exposed Kenyan adults.

In this study, we have investigated the extent of natural polymorphism in the CD8+ cytotoxic T lymphocyte (CTL) determinant (amino acids 368-390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)-B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA-B35. When we tested the effect of natural variation in this octamer sequence on HLA-B35 binding, it became evident that SP13 with D --> N substitution retained its binding specificity to HLA-B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA-B35. The most interesting finding was the observation that a D --> G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E --> Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA-class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA-B35.