Blood transcriptomics reveal the evolution and resolution of the immune response in tuberculosis.

Blood transcriptomics have revealed major characteristics of the immune response in active TB, but the signature early after infection is unknown. In a unique clinically and temporally well-defined cohort of household contacts of active TB patients that progressed to TB, we define minimal changes in gene expression in incipient TB increasing in subclinical and clinical TB. While increasing with time, changes in gene expression were highest at 30 d before diagnosis, with heterogeneity in the response in household TB contacts and in a published cohort of TB progressors as they progressed to TB, at a bulk cohort level and in individual progressors. Blood signatures from patients before and during anti-TB treatment robustly monitored the treatment response distinguishing early and late responders. Blood transcriptomics thus reveal the evolution and resolution of the immune response in TB, which may help in clinical management of the disease.

A modular transcriptional signature identifies phenotypic heterogeneity of human tuberculosis infection.

Whole blood transcriptional signatures distinguishing active tuberculosis patients from asymptomatic latently infected individuals exist. Consensus has not been achieved regarding the optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. Here we show a blood transcriptional signature of active tuberculosis using RNA-Seq, confirming microarray results, that discriminates active tuberculosis from latently infected and healthy individuals, validating this signature in an independent cohort. Using an advanced modular approach, we utilise the information from the entire transcriptome, which includes overabundance of type I interferon-inducible genes and underabundance of IFNG and TBX21, to develop a signature that discriminates active tuberculosis patients from latently infected individuals or those with acute viral and bacterial infections. We suggest that methods targeting gene selection across multiple discriminant modules can improve the development of diagnostic biomarkers with improved performance. Finally, utilising the modular approach, we demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.

A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants.

BACKGROUND: Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. METHODS: A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l'Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). RESULTS: By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. CONCLUSION: The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.

Artesunate-erythropoietin combination for murine cerebral malaria treatment.

Cerebral malaria is the most severe and rapidly fatal complication of Plasmodium falciparum infection. Despite appropriate anti-malarial treatment using quinine or artemisinin derivatives, 10-20% of mortality still occurs during the acute phase. To improve cerebral malaria outcome, adjunctive therapies are clearly needed. Most experiments in this area have been dedicated to immuno-modulation with various successes. Since erythropoietin has been shown to be highly effective in human ischemic stroke and in murine cerebral malaria, we addressed the issue of cerebral malaria outcome improvement by erythropoietin-artesunate drug combination. Compared to the previous study using erythropoietin high doses at the early beginning of the disease, erythropoietin treatment was decreased by six-fold and delayed to the pre-mortem phase. We studied effects on survival and on clinical recovery of the drug combination given from day 6 to day 8 post-infection to CBA/J mice infected by Plasmodium berghei ANKA. We showed that the artesunate-erythropoietin drug combination led to clinical recovery 24 h earlier for surviving mice, and to increase in the global survival rate compared to artesunate monotherapy (p<0.01). Since erythropoietin has no effect on parasite clearance, it could be stated that this drug combination is efficient and that erythropoietin could be a lead for the implementation of a new adjunctive therapy during the acute phase of cerebral malaria.

Features of apoptosis in Plasmodium falciparum erythrocytic stage through a putative role of PfMCA1 metacaspase-like protein.

The ability to undergo apoptosis, previously thought to be exclusive to multicellular organisms, has been demonstrated in unicellular parasites. On the basis of an observation that Plasmodium "crisis forms" were seen in vitro after cultivation in media containing an antimalarial drug, we attempted to determine whether Plasmodium falciparum has the ability to undergo apoptosis. By use of either the apoptosis-inducer etoposide or the antimalarial chloroquine, apoptosis in Plasmodium asexual stages was evident by the observation of DNA fragmentation and disruption of transmembrane mitochondrial potential. Next, we sought to determine whether Plasmodium produces specific cysteine proteases that can induce apoptosis. We hypothesized that the 2 metacaspase-like proteins present in the Plasmodium genome contained features typical of downstream execution steps and upstream signaling pathways such caspase activation and domain recruitment. We report that one of the metacaspase genes, PF13_0289, in addition to a universally conserved catalytic cysteine and histidine dyad required for catalysis activity, contains a putative caspase recruitment domain in the N-terminal amino acid sequence. This putative P. falciparum metacaspase protein has been designated PfMCA1. Our findings offer important insights into parasite survival strategies that could open new ways for therapeutic alternatives to drug resistance.

Multicenter proficiency study for detection of Toxoplasma gondii in amniotic fluid by nucleic acid amplification methods.

UNLABELLED: A proficiency panel was designed to assess the performance of nucleic acid amplification technologies for the detection of Toxoplasma gondii in amniotic fluid. METHODS: The proficiency panel consisted of five lyophilised coded samples in a range of concentration between 5 to 1000 parasites/ml and a negative control. The distribution also included a questionnaire on the applied methods. RESULTS: Thirty-three laboratories in 17 countries participated and returned a total of 38 data sets. The percentage of data sets achieving correct results on all panel samples was 42.1%, whereas two or more incorrect or equivocal results were reported for 36.8%. The lowest concentration (5 parasites/ml) was not identified correctly in 15 (39.5%) data sets. False positive results were reported by two laboratories both of which had not included a step in their procedure to rule out contamination. In 32 (84.2%) data sets an "in-house" method was used, and in 6 (15.8%) sets a commercial assay was applied. CONCLUSIONS: Overall, the results of this study demonstrate the need for improvements in both sensitivity and specificity of molecular detection methods of T. gondii and for the development of international reference materials to help laboratories with the development and validation of their assays.

Recombinant human erythropoietin prevents the death of mice during cerebral malaria.

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface.

Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites.

Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii.

OBJECTIVE: Our purpose was to evaluate Toxoplasma gondii concentration in amniotic fluid (AF) samples as a prognostic marker of congenital toxoplasmosis. STUDY DESIGN: A retrospective study was carried out in 88 consecutive AF samples from 86 pregnant women, which were found positive by prospective polymerase chain reaction (PCR) testing. Parasite AF concentrations were estimated by real-time quantitative PCR and analyzed in relation to the clinical outcome of infected fetuses during pregnancy and at birth, taking into account the gestational age at maternal infection. RESULTS: A significant negative linear regression was observed between gestational age at maternal infection and T gondii DNA loads in AF. After adjusting for time at maternal seroconversion by multivariate analysis, higher parasite concentrations were significantly associated with a severe outcome of congenital infection (odds ratio [OR]=15.38/log (parasites/mL AF) [95% CI=2.45-97.7]). CONCLUSION: PCR quantification of T gondii in AF can be highly contributive for early prognosis of congenital toxoplasmosis. Maternal infections acquired before 20 weeks with a parasite load greater than 100/mL of AF have the highest risk of severe fetal outcome.

Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins.

Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites.

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum.

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.

Transformation of sporozoites into early exoerythrocytic malaria parasites does not require host cells.

Malaria parasite species that infect mammals, including humans, must first take up residence in hepatic host cells as exoerythrocytic forms (EEF) before initiating infection of red blood cells that leads to malaria disease. Despite the importance of hepatic stages for immunity against malaria, little is known about their biology and antigenic composition. Here, we show that sporozoites, the parasites' transmission stage that resides in the mosquito vector salivary glands, can transform into early EEF without intracellular residence in host hepatocytes. The morphological sequence of transformation and the expression of proteins in the EEF appear indistinguishable from parasites that develop within host cells. Transformation depends on temperature elevation to 37 degrees C and serum. Our findings demonstrate that residence in a host hepatocyte or specific host cell-derived factors are not necessary to bring about the profound morphological and biochemical changes of the parasite that occur after its transmission from vector to mammalian host.

The Plasmodium sporozoite journey: a rite of passage.

Sporozoites are the most versatile of the invasive stages of the Plasmodium life cycle. During their passage within the mosquito vector and the vertebrate host, sporozoites display diverse behaviors, including gliding locomotion and invasion of, migration through and egress from target cells. At the end of the journey, sporozoites invade hepatocytes and transform into exoerythrocytic stages, marking the transition from the pre-erythrocytic to the erythrocytic part of the life cycle. This article discusses recent work, mostly done with rodent malaria parasites, that has contributed to a better understanding of the sporozoites' complex biology and which has opened up new avenues for future sporozoite research.

Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.

Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction.

The incidence of imported malaria cases in travellers returning from endemic areas has considerably increased over the last few years. The microscopical examination of stained blood films is the gold standard method to confirm clinical suspicion of malaria but diagnosis is difficult in the case of mixed infections, low-grade parasitaemia, or forms altered by uncompleted treatment. We have developed a real-time polymerase chain reaction (PCR) for the simultaneous identification of the 4 human Plasmodium spp. and quantification of Plasmodium DNA in human blood. The rapid turnaround and reduction in the risk of PCR product carryover are major advantages compared with conventional PCR. In combination with conventional tests, this method could be a powerful tool for the diagnosis of malaria infections among travellers from endemic areas and during the follow-up of patients in reference centres involved in travel and tropical medicine. Quantitative real-time PCR could also be used for the follow-up of patients during drug resistance studies managed by national malaria programmes, the testing of new drugs, and vaccine trials.

Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage.

Injection of Plasmodium salivary gland sporozoites into the vertebrate host by Anopheles mosquitoes initiates malaria infection. Sporozoites develop within oocysts in the mosquito midgut and then enter and mature in the salivary glands. Although morphologically similar, oocyst sporozoites and salivary gland sporozoites differ strikingly in their infectivity to the mammalian host, ability to elicit protective immune responses, and cell motility. Here, we show that differential gene expression coincides with these dramatic phenotypic differences. Using suppression subtractive cDNA hybridization we identified highly up-regulated mRNAs transcribed from 30 distinct genes in salivary gland sporozoites. Of those genes, 29 are not significantly expressed in the parasite's blood stages. The most frequently recovered transcript encodes a protein kinase. Developmental up-regulation of specific mRNAs in the infectious transmission stage of Plasmodium indicates that their translation products may have unique roles in hepatocyte infection and/or development of liver stages.