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The biology of intraerythrocytic Babesia parasites presents unique challenges for the diagnosis of human babesiosis. Antibody-based assays are highly sensitive but fail to detect early stage Babesia infections prior to seroconversion (window period) and cannot distinguish between an active infection and a previously resolved infection. On the other hand, nucleic acid-based tests (NAT) may lack the sensitivity to detect window cases when parasite burden is below detection limits and asymptomatic low-grade infections. Recent technological advances have improved the sensitivity, specificity and high throughput of NAT and the antibody-based detection of Babesia. Some of these advances include genomics approaches for the identification of novel high-copy-number targets for NAT and immunodominant antigens for superior antigen and antibody-based assays for Babesia. Future advances would also rely on next generation sequencing and CRISPR technology to improve Babesia detection. This review article will discuss the historical perspective and current status of technologies for the detection of Babesia microti, the most common Babesia species causing human babesiosis in the United States, and their implications for early diagnosis of acute babesiosis, blood safety and surveillance studies to monitor areas of expansion and emergence and spread of Babesia species and their genetic variants in the United States and globally.

Background Complete malaria eradication and optimal use of transmission-reducing interventions require knowledge of submicroscopic infectious reservoirs among asymptomatic individuals. Even submicroscopic levels of Plasmodium falciparum gametocytes can infect mosquitoes and promote onward transmission. Most efforts to identify gametocyte carriers use polymerase chain reaction amplification of the gametocyte-specific transcript Pfs25. Methods To expand the repertoire of biomarkers available for superior gametocyte detection, we compared the gene expression profiles of gametocytes and asynchronous blood-stage P. falciparum parasites by microarray technology. This allowed the identification of 56 molecules abundantly expressed in the gametocyte stage of the parasite. The analytical sensitivity for gametocyte detection was evaluated for 25 genes with the highest expression levels. Results One candidate, Pfg17, exhibited superior analytical sensitivity against a panel of gametocyte-spiked whole blood, detecting 10 gametocytes/mL; in comparison, Pfs25 detected only 25.3 gametocytes/mL. Pfg17 also exhibited superior clinical sensitivity, identifying 19.1% more samples from blood-film microscopy-negative Ghanaian children and 40% more samples from asymptomatic adults as gametocyte positive. Conclusions Cumulatively, our results suggest Pfg17 is an excellent biomarker for detecting asymptomatic infectious reservoirs otherwise missed by the most sensitive molecular method available. Our study has also improved the repertoire of transmission-stage antigens available for evaluation as candidate vaccines.

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01(E) vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4(+) and CD8(+) T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4(+) T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4(+) T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαβ combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRβ expressing CD11b + Ly6G + neutrophils in the spleen during the early phase of infection. Measurement of TCRβ transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRβ and peripheral blood parasite burden. In addition, TCRβ + neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRβ − neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

Exposure to ionizing radiation has a profound effect on the viability and further replication of living cells. Radiation is used for the treatment of cancer [1], generation of attenuated vaccines [2], and in food safety [3]. Radiation attenuation has also been one of the earliest methods used to generate whole parasite–based malaria vaccines [4–6]. Given the importance of ionizing radiation in the treatment of different forms of cancer, a large body of scientific data has accumulated on the effects of ionizing radiation on the cell cycle [7]. Such studies have shed light on the molecular and biochemical aspects of radiation-mediated cell death and survival mechanisms [1, 8]. These studies have also provided insight into the cell cycle checkpoints and the complex signaling pathways and associated molecules that ensure that each phase of the cell cycle is completed without errors [7, 9, 10]. Ionizing radiation is detrimental to the growth and survival of Plasmodium parasites. In murine models, asexual blood-stage parasites exposed to high doses of radiation failed to cause blood-stage parasite infection [11], and immunization with radiation-attenuated asexual-stage parasites protected against parasitemia [11–13] and experimental cerebral malaria [11]. In preerythrocytic-stage malaria, exposure of Plasmodium sporozoites to a threshold level of γ-radiation allows their invasion into liver cells and early development. However, these parasites undergo arrested development and fail to produce infectious liver-stage merozoites and, consequently, blood-stage parasite infection [14]. Irradiation-attenuated malaria sporozoites protect mice [4, 15] and humans [5, 16] against malaria in experimental challenge studies. Accordingly, despite the limited success of subunit-based malaria vaccines in clinical testing [17], there has been considerable interest in using radiation exposure to create an attenuated sporozoite-stage vaccine [14, 16]. In this study, we measured the effects of different doses of γ-irradiation on P. falciparum in short-term and long-term cultures. These studies were performed to evaluate both the immediate effect on survival and the ability of rare parasite populations to recover and repopulate. We also studied the effect of γ-irradiation on parasite structure, by fluorescence microscopy and transmission electron microscopy. In addition, we performed extensive genome-wide transcriptional profiling in P. falciparum exposed to increasing doses of γ-radiation, to identify parasite proteins that could be correlated with survival and cell death, and, on the basis of the data set, created a functionally diverse molecular signature of growth attenuation. Last, bioinformatic analyses were performed to decipher the biological systems altered by γ-irradiation that are associated with growth regulation and survival.

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S -transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum -infected young African children as diagnostic markers of CM.

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected withPlasmodium falciparummalaria will progress to CM. We used thePlasmodium bergheiANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin α1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin α1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.

Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of theSaccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP119) of the murine malaria parasitePlasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP119in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP119emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP119, 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund’s adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP119), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge. The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P= 0.007, chi square) and reduced parasite burden in infected mice. In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+and CD8+T cells in vaccinated mice had no significant effect on immunity. On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection. Inclusion of CpG ODN 1826 in the yMSP119plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity. Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates.

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (∼80%) is much greater than that of Homo sapiens (∼59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1 42 ) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 μg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

BackgroundGiven the considerable geographic overlap in the endemic regions for malaria and tuberculosis, it is probable that co-infections with Mycobacterium tuberculosis and Plasmodium species are prevalent. Thus, it is quite likely that both malaria and TB vaccines may be used in the same populations in endemic areas. While novel vaccines are currently being developed and tested individually against each of these pathogens, the efficacy of these vaccines has not been evaluated in co-infection models. To further assess the effectiveness of these new immunization strategies, we investigated whether co-infection with malaria would impact the anti-tuberculosis protection induced by four different types of TB vaccines in a mouse model of pulmonary tuberculosis.Principal findingsHere we show that the anti-tuberculosis protective immunity induced by four different tuberculosis vaccines was not impacted by a concurrent infection with Plasmodium yoelii NL, a nonlethal form of murine malaria. After an aerogenic challenge with virulent M. tuberculosis, the lung bacterial burdens of vaccinated animals were not statistically different in malaria infected and malaria naïve mice. Multi-parameter flow cytometric analysis showed that the frequency and the median fluorescence intensities (MFI) for specific multifunctional T (MFT) cells expressing IFN-γ, TNF-α, and/or IL-2 were suppressed by the presence of malaria parasites at 2 weeks following the malaria infection but was not affected after parasite clearance at 7 and 10 weeks post-challenge with P. yoelii NL.ConclusionsOur data indicate that the effectiveness of novel TB vaccines in protecting against tuberculosis was unaffected by a primary malaria co-infection in a mouse model of pulmonary tuberculosis. While the activities of specific MFT cell subsets were reduced at elevated levels of malaria parasitemia, the T cell suppression was short-lived. Our findings have important relevance in developing strategies for the deployment of new TB vaccines in malaria endemic areas.

Although clinically benign, malaria fever is thought to have significant relevance in terms of parasite growth and survival and its virulence which in turn may alter the clinical course of illness. In this article, the historical literature is reviewed, providing some evolutionary perspective on the genesis and biological relevance of malaria fever, and the available molecular data on the febrile-temperature-inducible parasite factors that may contribute towards the regulation of parasite density and alteration of virulence in the host is also discussed. The potential molecular mechanisms that could be responsible for the induction and regulation of cyclical malaria fevers caused by different species of Plasmodium are also discussed.

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as “hypothetical.” We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the “crisis forms” and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.