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Immunoglobulin E (IgE) is thought to have evolved to protect mammalian hosts against parasitic infections or toxins and plays a central role in the pathogenesis, diagnosis, and therapy of IgE-mediated allergy. Despite the prominence of IgE responses in most parasitic infections, and in stark contrast to its use in the diagnosis of allergy, this isotype is almost completely unexploited for parasite diagnosis. Here, we discuss the perceived or real limitations of IgE-based diagnosis in parasitology and suggest that the recent creation of a new generation of very sensitive cellular IgE-based reporters may represent a powerful new diagnostic platform, but needs to be based on a very careful choice of diagnostic allergens.

Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp. can cause devastating brain infections in humans which almost always result in death. The symptoms of the three infections overlap, but brain inflammation and the course of the disease differ, depending on the amoeba that is responsible. Understanding the differences between these amoebae can result in the development of strategies to prevent and treat these infections. Recently, numerous scientific advancements have been made in the understanding of pathogenicity mechanisms in general, and the basic biology, epidemiology, and the human immune response towards these amoebae in particular. In this review, we combine this knowledge and aim to identify which factors can explain the differences between the lethal brain infections caused by N. fowleri, B. mandrillaris, and Acanthamoeba spp.

Toxoplasmosis caused by the protozoan parasite Toxoplasma (T.) gondii, occurs worldwide. Infections range from asymptomatic to life-threatening disease. T. gondii infection is acquired either via bradyzoites in meat or via oocysts in the environment, but the relative importance of these routes and the different sources remains unclear. This study examined possible risk factors for toxoplasmosis in the Netherlands. A case-control study was conducted including persons with a recent infection and individuals with a negative test result for IgM and IgG to T. gondii between July 2016 and April 2021. Forty-eight cases and 50 controls completed the questionnaire. Food history and environmental exposures were compared using logistic regression. Consumption of different meat types was found to be related to recent infection. In the multivariable model, adjusted for age, gender and pregnancy, consumption of meat of large game animals (adjusted odds ratio (aOR) 8.2, 95% confidence interval 1.6-41.9) and sometimes (aOR 4.1, 1.1-15.3) or never (aOR 15.9, 2.2-115.5) washing hands before preparing food remained. These results emphasize the value of the advice to be careful with consumption of raw and undercooked meat. Good hand hygiene also could be promoted in the prevention of T. gondii infection.

In this clinical pearl an ectopic human Dioctophyma renale infection in the abdominal cavity is reported for the first time. The patient presented with a gastric perforation and the release of an adult Dioctophyma renale through an abdominal drain and three co-infections (Plasmodium malariae, Strongyloides stercoralis and Mansonella perstans).

Examination of viability of trypomastigotes before and after single-dose pentamidine treatment demonstrated that single-dose pentamidine substantially affected motility of trypomastigotes, a proxy of drug efficacy. This suggests that single-dose pentamidine may be of added value to bridge time until suramin will be available for treatment of human East Africa trypanosomiasis.

Background In falciparum malaria the total parasite biomass can be estimated by blood levels of histidine-rich protein 2 (PfHRP2), a Plasmodium falciparum-specific protein, which has been widely studied in malaria-endemic regions. This study investigates the usefulness of PfHRP2 as marker for disease severity in imported falciparum malaria. Methods A retrospective cohort analysis was done in 145 patients with imported falciparum malaria. Associations between PfHRP2, malaria disease severity and classic parameters of disease severity were examined by statistical analyses. Patients with different travel purposes were examined in two groups: visiting friends and relatives (VFRs) and other travel purposes (mainly tourists). Results High PfHRP2 levels were clearly associated with disease severity. VFRs status showed to be an independent determinant protecting against severe malaria. At similar PfHRP2 levels VFRs patients had significantly lower levels of peripheral blood parasitemia compared to other patients. Conclusion Our study confirms the association between PfHRP2 and disease severity in patients with imported falciparum malaria, but for proper interpretation of PfHRP2 levels as disease severity marker in travellers, the possible presence of pre-existing acquired anti-malarial immunity should be taken into account as the correlation between PfHRP2 levels and disease severity differed significantly between VFRs patients and patients with other travel purposes.

Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri. The amoeba migrates along the olfactory nerve to the brain, resulting in seizures, coma, and, eventually, death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several already-approved inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine., Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri. The amoeba migrates along the olfactory nerve to the brain, resulting in seizures, coma, and, eventually, death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several already-approved inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine, and valproic acid inhibited growth of N. gruberi. We then tested these compounds on N. fowleri and found etomoxir, perhexiline, and thioridazine to be effective growth inhibitors. Hence, not only are lipids the preferred food source for N. gruberi, but also oxidation of fatty acids seems to be essential for growth of N. fowleri. Inhibition of fatty acid oxidation could result in new treatment options, as thioridazine inhibits N. fowleri growth in concentrations that can be reached at the site of infection. It could also potentiate currently used therapy, as checkerboard assays revealed synergy between miltefosine and etomoxir. Animal testing should be performed to confirm the added value of these inhibitors. Although the development of new drugs and randomized controlled trials for this rare disease are nearly impossible, inhibition of fatty acid oxidation seems a promising strategy as we showed effectivity of several drugs that are or have been in use and that thus could be repurposed to treat PAM in the future.

Background Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. Methods and principal findings A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. Conclusions/Significance We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs., Author summary Tests that detect parasite DNA in human stool are increasingly being used for the diagnosis of infections with intestinal worms, including schistosomiasis. To ensure the quality in diagnostic testing of these parasitic worms, it is important that laboratories evaluate the diagnostic performance of their DNA-based tests. This can best be achieved by participating in an external quality assessment scheme (EQAS). An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. Currently, such an EQAS for parasitic worms is lacking. We therefore piloted an international EQAS for the diagnosis of parasitic worms involving 15 laboratories in Africa, Asia, Australia, Europe, and North America. Although most laboratories performed well, we could clearly identify those laboratories that may need to improve their test protocol. We found that laboratories were using many different test protocols, and further research should aim to verify whether this has an impact on the performance of the diagnostic outcomes.

Toll-like receptor 2 (TLR2) is an important pattern recognition receptor on the surface of host immune cells that binds a variety of ligands that are released by microorganisms as well as by damaged or dying host cells. According to the current concept, TLR2/1 and TLR2/6 heterodimers are activated by tri- or di-acylated ligands, respectively. However, also mono-acyl phospholipid containing lipid fractions derived from parasites, were reported to be able to activate TLR2. In order to provide conclusive evidence for the TLR2 activating capacity of mono-acyl phospholipids derived from pathogens, we developed a biosynthetic method to enzymatically convert commercially available phospholipids into several mono-acyl-phospholipid variants that were examined for their TLR2 activating capacity. These investigations demonstrated that 1-(11Z-eicosenoyl)-glycero-3-phosphoserine 20:1 (20:1 lyso-PS) is a true agonist of the TLR2/6 heterodimer and that its polar headgroup as well as the length of the acyl chain are crucial for TLR2 activation. In silico modelling further confirmed 20:1 mono-acyl PS as a ligand for TLR2/6 heterodimer, as this predicted that multiple hydrogen bonds are formed between the polar headgroup of 20:1 mono-acyl PS and amino acid residues of both TLR2 and TLR6. Future studies can now be performed to further assess the functions of 20:1 lyso-PS as an immunological mediator, because this enzymatic method enables its preparation in larger quantities than is possible by isolation from the parasite that naturally produces this compound, Schistosoma mansoni, the source of the original discovery (Van der Kleij et al., 2002).

Nontraumatic myelopathy causes severe morbidity and is not uncommon in Africa. Clinically, patients often present with paraplegia, and extrinsic cord compression and transverse myelitis are most common causes. Data on exact pathogenesis are scanty because of limitations in diagnostic methods. In Queen Elizabeth Central Hospital, Blantyre, Malawi, we recorded consecutive patients presenting with nontraumatic paraplegia for maximally 6 months between January and July 2010 and from March to December 2011. The diagnostic workup included imaging and examining blood, stool, urine, sputum, and cerebrospinal fluid (CSF) samples for infection. After discharge, additional diagnostic tests, including screening for virus infections, borreliosis, syphilis, and schistosomiasis, were carried out in the Netherlands. The clinical diagnosis was, thus, revised in retrospect with a more accurate final differential diagnosis. Of 58 patients included, the mean age was 41 years (range, 12-83 years) and the median time between onset and presentation was 18 days (range, 0-121 days), and of 55 patients tested, 23 (42%) were HIV positive. Spinal tuberculosis (n = 24, 41%), tumors (n = 16, 28%), and transverse myelitis (n = 6, 10%) were most common; in six cases (10%), no diagnosis could be made. The additional tests yielded evidence for CSF infection with Schistosoma, Treponema pallidum, Epstein-Barr virus (EBV), HHV-6, HIV, as well as a novel cyclovirus. The diagnosis of the cause of paraplegia is complex and requires access to an magnetic resonance imaging (MRI) scan and other diagnostic (molecular) tools to demonstrate infection. The major challenge is to confirm the role of detected pathogens in the pathophysiology and to design an effective and affordable diagnostic approach.

Background Liver injury is a known feature of severe malaria, but is only incidentally investigated in uncomplicated disease. In such cases, drug-induced hepatotoxicity is often thought to be the primary cause of the observed liver injury, and this can be a major concern in antimalaria drug development. We investigated liver function test (LFT) abnormalities in patients with imported uncomplicated malaria, and in Controlled Human Malaria Infection (CHMI) studies. Methods Clinical and laboratory data from 484 imported malaria cases and 254 CHMI participants were obtained from the Rotterdam Malaria Cohort database, and the Radboud University Medical Center database (between 2001 and 2017), respectively. Routine clinical LFTs, clinical profiles, parasite densities, hematological, and inflammation parameters were assessed in 217 patients with imported falciparum malaria upon admission, and from longitudinal data of 187 CHMI participants. Findings Upon admission, the proportion of patients with imported uncomplicated malaria and elevated liver enzymes was 128/186 (69%). In CHMI, 97/187 (52%) participants showed LFT abnormalities, including mild (64%, >1.0 ≤ 2.5× upper limit of normal (ULN)), moderate (20%, >2.5 ≤ 5.0xULN) or severe (16%, >5.0xULN). LFT abnormalities were primarily ALT/AST elevations and to a lesser extent γGT and ALP. LFT abnormalities peaked shortly after initiation of treatment, regardless of drug regimen, and returned to normal within three to six weeks. Positive associations were found with parasite burden and inflammatory parameters, including cumulative inflammatory cytokine responses and oxidative stress markers (r = 0·65, p = 0·008, and r = −0·63, p = 0·001, respectively). Interpretation This study shows that reversible liver injury is a common feature of uncomplicated falciparum malaria, most likely caused by an enduring pro-inflammatory response post treatment. The recognition of this phenomenon is of clinical relevance for individual patient care as well as clinical development of (new) antimalarial drugs. Fund PATH Malaria Vaccine Initiative (MVI)

Primary Amoebic Meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoebaNaegleria fowleri. The disease mostly affects healthy children and young adults after contaminated water enters the nose, generally during recreational water activities. The amoeba migrate along the olfactory nerve to the brain, resulting in seizures, coma and eventually death. Previous research has shown thatNaegleria gruberi, a close relative ofN. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine and valproic acid inhibited growth ofN. gruberi. Furthermore, additive effects were seen when drugs were combined. Both thioridazine and valproic acid inhibit in vitro growth ofN. gruberiin concentrations that can be obtained at the site of infection, which is doubtful with the currently used drugs amphotericin B and miltefosine. Both thioridazine and valproic acid have already been used for other diseases. As the development of new drugs and randomized controlled trials for this rare disease is nearly impossible, repurposing drugs is the most promising way to obtain additional drugs to combat PAM. Thioridazine and valproic acid are available drugs without major side-effects and can, therefore, be used as new complementary options in PAM therapy.

We present a unique case of disseminated Leishmaniasis in an HIV patient. Two different Leishmania species were identified by genomic sequencing in both bone marrow and skin. The Leishmania infection could be suppressed but not cured, despite a high dose of amphotericin B of nearly 65 g over more than 6 years.

Background After a controlled human malaria infection (CHMI), presentation of clinical signs and symptoms and host responses is heterogeneous. Transforming growth factor-beta (TGF-β) is the first serum cytokine that changes in malaria-naive volunteers after CHMI. We studied a possible relation between TGF-β changes, pro-inflammatory cytokines, activation of haemostasis and endothelial cells and clinical symptoms. Methods A panel of cytokines including TGF-β, and markers of activation of haemostasis and endothelial cells were measured in blood samples of 15 volunteers at baseline before CHMI and during CHMI at day of treatment. The change of the parameters on the day of treatment was examined for a significant alteration during infection. Results Nine of 15 volunteers showed a significant decrease in TGF-β compared to baseline, with concomitant increased concentrations of D-dimer (p = 0.012), Von Willebrand factor (p = 0.017), IL-6 (p = 0.012) and IFN-γ (0.028) and a significantly decreased platelet count (p = 0.011). In contrast, 6 of 15 volunteers showed sustained or increased TGF-β concentrations without change in the aforementioned parameters. The sustained responders presented with less moderate and severe clinical symptoms than the negative responders (p = 0.036) and had a higher baseline lymphocyte count (p = 0.026). TGF-β concentrations did not correlate with the parasitaemia on day of treatment. Conclusion Early decreases of serum TGF-β might function a marker for a pro-inflammatory host response and downstream clinical symptoms and pathology during CHMI.

Introduction. Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. It is endemic to the American continent due to the distribution of its insect vectors. The disease is occasionally imported to other continents by travel of infected individuals. It is rarely diagnosed in the Netherlands and exact numbers of infected individuals are unknown. Clinical manifestations can start with an acute phase of 4–8 weeks with non-specific, mild symptoms and febrile illness. In the chronic phase, it can lead to fatal cardiac and gastro-intestinal complications. Case presentation. We describe a case of a 40-year-old man with end-stage cardiomyopathy due to Chagas disease. He lived in Surinam for more than 20 years and had an unremarkable medical history until he was hospitalized due to pneumonia and congestive heart failure. Despite antibiotic treatment and optimizing cardiac medication, his disease progressed to end-stage heart failure for which cardiac transplantation was the only remaining treatment. A left ventricular assist device (LVAD) was implanted as a bridge to transplantation. Tissue analysis after LVAD surgery revealed ongoing myocarditis caused by Chagas disease. Based on a literature review, a scheme for follow up and treatment after transplantation was postulated. Conclusion. Chagas disease should be taken into account in patients from endemic countries who have corresponding clinical signs. Heart transplantation in patients with Chagas cardiomyopathy is accompanied by specific challenges due to the required immunosuppressive therapy and the thereby increased risk of reactivation of a latent T. cruzi infection.

Background: Real-time PCR methods are increasingly used in routine patient care settings not only to determine the presence or absence of pathogens in patient materials, but also to obtain semiquantitative results to estimate the pathogen load. However, it is so far unknown how well these methods are harmonized among different laboratories. Methods: Sets of stool samples were distributed three to four times per year to ca. 25–40 participating laboratories within the European Union as part of an external quality assessment scheme (EQAS) for the detection of gastrointestinal protozoa. This paper presents the results obtained over a 3-year period for Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, Cryptosporidium species and Dientamoeba fragilis. Results: Although both false-positive and false-negative results were reported, the overall sensitivity and specificity were high. The substantial differences in the quantitative output of the real-time PCR assays could be traced back to differences in DNA isolation procedures between different laboratories. Conclusions: Participation in an EQAS proved to be important as it provides information on how the real-time PCR methods used by the participant compares to the generally reported results and indicates how procedures could be improved. Semiquantitative results of real-time PCR methods are not exchangeable between laboratories as long as the diagnostic procedures are not harmonized. Intralaboratory comparison of semiquantitative real-time PCR results seems only possible by the use of calibration curves derived from well-validated standards in clinical material and not by spiking solutions with purified DNA.

The hydrophobic molecules of the metabolome - also named the lipidome - constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host-pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs.

To the Editor: Cutaneous leishmaniasis (CL) is a protozoan disease transmitted by sand flies that usually runs a relatively mild course. Classic CL starts as a red papule at the place of the insect bite; it gradually enlarges into a painless nodule or plaque-like lesion, which eventually becomes encrusted. When the crust falls off, a typical ulcer with raised and indurated border becomes apparent. CL can cause considerable illness and may leave disfiguring and disabling scars after healing. The interplay between Leishmania species and host immune response is complex, and, as a result, disease manifestations may vary substantially among species as well as among infected persons (1,2). An estimated 0.7–1.2 million new CL cases occur annually in tropical and subtropical regions of the world. CL is currently endemic in >98 countries worldwide; Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica, and Peru together account for up to 75% of global estimated CL incidence (3).

Contains fulltext : 177607.pdf (Publisher’s version ) (Closed access) Malaria sporozoites must first undergo intrahepatic development before a pathogenic blood-stage infection is established. The success of infection depends on host and parasite factors. In healthy human volunteers undergoing controlled human malaria infection (CHMI), we directly compared three clinical Plasmodium falciparum isolates for their ability to infect primary human hepatocytes in vitro and to drive the production of blood-stage parasites in vivo. Our data show a correlation between the efficiency of strain-specific sporozoite invasion of human hepatocytes and the dynamics of patent parasitemia in study subjects, highlighting intrinsic differences in infectivity among P. falciparum isolates from distinct geographical locales. The observed heterogeneity in infectivity among strains underscores the value of assessing the protective efficacy of candidate malaria vaccines against heterologous strains in the CHMI model.

Background Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on diagnostic test results and infectious Ebola virus (EBOV) titers. Methods Serum and whole-blood samples were treated with 0.1% or 1% sodium dodecyl sulfate (SDS) or 0.1% Triton X-100 and assayed for clinical chemistry and malaria antigen detection. Infectious EBOV titers were determined in SDS-treated plasma and whole blood from EBOV-infected nonhuman primates (NHPs). Infectious titers of EBOV or herpes simplex virus type 1 (HSV-1) in detergents-treated cell culture medium containing various serum concentrations were determined. Results Laboratory test results were not affected by 0.1% detergent treatment of blood samples, in contrast with 1% SDS treatment. However, 0.1% detergent treatment did not inactivate EBOV in blood samples from infected NHPs. Experiments with cell culture medium showed that virus inactivation by detergents is annulled at physiological serum concentrations. Conclusions Treatment of blood samples with 0.1% SDS or Triton X-100 does not inactivate EBOV. Inactivation protocols for HFV should be validated with serum and whole blood.

Background Levels of both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) correlate with malaria disease severity and are proposed as biomarkers and possible therapeutic targets. To establish their role in malaria, a systematic review was performed of the literature on Ang-1 and Ang-2 with regard to their potential as biomarkers in malaria and discuss their possible place in adjuvant treatment regimens. Methods Ten electronic databases were systematically searched to identify studies investigating Ang-1 and Ang-2 in human and murine malaria in both clinical and experimental settings. Information about the predictive value of Ang-1 and Ang-2 for disease severity and their regulatory changes in interventional studies were extracted. Results Some 579 studies were screened; 26 were included for analysis. In all five studies that determined Ang-1 levels and in all 11 studies that determined Ang-2 in different disease severity states in falciparum malaria, a decline in Ang-1 and an increase of Ang-2 levels was associated with increasing disease severity. All nine studies that determined angiopoietin levels in Plasmodium falciparum patients to study their ability as biomarkers could distinguish between multiple disease severity states; the more the disease severity states differed, the better they could be distinguished. Five studies differentiating malaria survivors from non-survivors with Ang-2 as marker found an AUROC in a range of 0.71–0.83, which performed as well or better than lactate. Prophylactic administration of FTY720, rosiglitazone or inhalation of nitric oxide (NO) during malaria disease in mice resulted in an increase in Ang-1, a decrease in Ang-2 and an increased survival. For rosiglitazone, a decrease in Ang-2/Ang-1 ratio was observed after post-infection treatment in mice and humans with malaria, but for inhalation of NO, an effect on Ang-1 and survival was only observed in mice. Conclusion Both Ang-1 and Ang-2 levels correlate with and can distinguish between malaria disease severity states within the group of malaria-infected patients. However, distinct comparisons of disease severity states were made in distinct studies and not all distinctions made had clinical relevance. Changes in levels of Ang-1 and Ang-2 might also reflect treatment effectiveness and are promising therapeutic targets as part of multi-targeted therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1624-8) contains supplementary material, which is available to authorized users.

To the Editor: Balamuthia mandrillaris is a free-living ameba that has a worldwide distribution in soil and was first reported in 1990 (1). Approximately 200 B. mandrillaris meningoencephalitis cases have been described, mostly from warm climate areas in South America. Its prevalence in the United States is estimated to be 1 case/year (2). However, B. mandrillaris meningoencephalitis has not been reported in Africa, and only 4 cases have been reported in Europe (3–6). Transmission occurs through the respiratory tract or the skin or by organ transplant, and the incubation period varies from weeks to months after primary infection (7). After an indolent, subacute phase with aspecific symptoms, the amebae invade the central nervous system, and illness rapidly progresses, leading almost invariably to death (7). Because B. mandrillaris is difficult to detect in soil, its specific geographic distribution around the world is unknown and is estimated on the basis of where illnesses have been reported (7). This report addresses fatal B. mandrillaris meningoencephalitis in a woman from the Netherlands who had visited The Gambia. In December 2013, a previously healthy 61-year-old white woman in the Netherlands sought care for fever, headaches, and muscle pains she had experienced for 1 week. That year, she had traveled 4 times to The Gambia, the last visit being 1 month before her hospitalization (Technical Appendix Table 1). After she returned from her visit in September 2013, fatigue, diarrhea, fever, and pustular skin lesions on her back and lower extremities developed. A wound swab culture showed Staphylococcus aureus, for which she was treated successfully with oral clarithromycin and topical fucidin ointment. On admission in December, her physical and neurologic examination results were unremarkable. Malaria was excluded; because of persisting headaches, a cerebral computed tomography scan without contrast was performed but showed no abnormalities. In the following days, high fevers, altered mental status, and nuchal rigidity without focal neurologic deficits developed. Cerebrospinal fluid (CSF) examination showed mononuclear pleocytosis, highly elevated protein levels, and low glucose levels (Technical Appendix Table 2). Serial cerebral computed tomography and magnetic resonance imaging scans showed development of an asymmetric hydrocephalus and diffuse leptomeningeal and subependymal contrast enhancement, especially around the brainstem, without signs of intracerebral mass lesions (Technical Appendix Figure). Presumed diagnosis was tuberculous meningitis, and she was treated with tuberculostatic drugs (isoniazid, rifampin, pyrazinamide, and ethambutol) combined with intravenous acyclovir, ceftriaxone, and co-trimoxazole for other infectious causes of meningoencephalitis. Despite external lumbar and ventricular (both lateral ventricles and fourth ventricle) CSF drainage, her neurologic condition deteriorated. Multiple cranial nerve palsies developed, and she became comatose and died 11 days after admission. Informed consent for postmortem examination was obtained, and macroscopic pathologic examination showed uncal and cerebellar herniation caused by increased intracranial pressure. Microscopic brain tissue examination showed signs of acute granulomatous inflammation, multiple hemorrhagic infarctions, and angiitis in the presence of numerous amebic trophozoites and cysts (Figure), which showed granulomatous hemorrhagic necrotic amebic meningoencephalitis. Real-time PCR and subsequent sequencing on brain biopsy and CSF specimens showed B. mandrillaris to be the causative ameba (8,9). Figure Postmortem pathologic findings for woman in the Netherlands who died of Balamuthia mandrillaris meningoencephalitis after returning from travel to The Gambia. A) Macroscopic coronal central section scan showing hemorrhagic necrotizing lesions of the subependymal, ... The infection could have been acquired in The Gambia or the Netherlands because the patient had intensive soil contact in The Gambia, where she frequently cultivated land, and in the Netherlands, where she worked in glass horticulture. She may have been infected through the skin after contact with contaminated soil, but her skin lesions were atypical for B. mandrillaris, and postmortem examinations failed to identify B. mandrillaris except in the central nervous system. The lack of reported B. mandrillaris cases from Africa might indicate a low number of postmortem examinations and little access to advanced diagnostics, rather than a low environmental prevalence of B. mandrillaris. The few reported cases in Europe might be related to lack of awareness and to clinical signs and symptoms that mimic tuberculous meningitis: a lymphocytic pleocytosis with an elevated protein level and a low glucose level in CSF, together with a hydrocephalus and subependymal and leptomeningeal contrast enhancement on magnetic resonance imaging (10). Also, B. mandrillaris meningoencephalitis imaging findings are often nonspecific, including cerebral edema, hydrocephalus, multiple space-occupying and ring-enhancing lesions, leptomeningeal enhancement, or formation of mycotic aneurysms (2). Furthermore, amebic trophozoites are seldom detected in CSF by microscopy (2,3). Consequently, B. mandrillaris meningoencephalitis could be underdiagnosed, especially where this infection has no or only sporadic reports. B. mandrillaris should be considered in refractory or unexplained cases of meningoencephalitis, even outside the Americas and in immunocompetent patients. Detecting B. mandrillaris by PCR in CSF seems most likely to enable early diagnosis and timely treatment. However, appropriate therapy is not well defined; success has been sparsely reported with the simultaneous use of azoles, flucytosine, pentamidine, sulfazidine, macrolide antimicrobial drugs, phenothiazines, and miltefosine (2,7,10). Technical Appendix. Timeline of relevant events, laboratory investigations during hospitalization, and imaging results for Dutch traveler who died of Balamuthia mandrillaris meningoencephalitis after returning from The Gambia. Click here to view.(248K, pdf)

Until now, two studies investigated whether the long-term survival after heart transplantation is affected by the Toxoplasma serostatus of the recipient and the donor (1, 2). These studies, however, yielded conflicting results as to the recipient serostatus. Arora et al. reported that a Toxoplasma seropositive status in heart transplant recipients was associated with an increased risk of all-cause mortality as well as risk of death by cardiac allograft vasculopathy and Doesch et al. reported that a Toxoplasma seronegative status was associated with an increased mortality risk (1, 2). In both studies, no association of donor serostatus and mortality was found. Toxoplasma gondii is an obligate intracellular protozoan parasite with a worldwide distribution that can invade and replicate in almost all nucleated cells of warm-blooded mammals. After the primary acute infection, the parasite remains in the body in a quiescent state by the formation of tissue cysts in predominantly muscle and brain tissue. Therefore, toxoplasmosis is a lifelong infection and the prevalence of human T. gondii infections thus increases with age. Its prevalence is reported to be more than 50% in many parts of the world (3). In immunocompetent individuals, the infection is usually asymptomatic, although cases of severe disease due to unusual T. gondii genotypes from South and Central America have been reported (4). In immunocompromised patients, however, toxoplasmosis can be a severe disease (3). Solid-organ transplant recipients are immunocompromised due to the required high dose of immunosuppressive medication. Heart transplant recipients are known to be at risk for toxoplasmosis, especially when recipients are seronegative (and thus naive for T. gondii) and receive a heart transplant, containing tissue cysts, from a T. gondii–infected donor (5). In these cases, prophylactic treatment is indicated to prevent toxoplasmic myocarditis and fatal disseminated toxoplasmosis (5, 6). Also, antibiotic drugs, administered prophylactically to Toxoplasma mismatched recipients, could have a direct or indirect immunomodulatory effect and could negatively influence patient outcome by their side effects (7). Cardiac allograft vasculopathy, a process in which chronic inflammation and immune activation play an important role, is a major cause of death after heart transplantation (8, 9). A T. gondii infection might influence the host immunologic status and thereby affect the risk for cardiac allograft vasculopathy. Therefore, the effects of a T. gondii infection on the survival after heart transplantation have been subject of study. Because of the earlier conflicting results of studies, which involved rather small numbers of patients and a limited follow-up duration, we performed a study on our whole population of heart transplant recipients to evaluate the effects of a T. gondii infection in the recipient and/or the donor on survival after heart transplantation.

Background Acute kidney injury (AKI) is a frequently encountered complication of imported Plasmodium falciparum infection. Markers of structural kidney damage have been found to detect AKI earlier than serum creatinine-based prediction models but have not yet been evaluated in imported malaria. This pilot study aims to explore the predictive performance of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) for AKI in travellers with imported P. falciparum infection. Methods Thirty-nine patients with imported falciparum malaria from the Rotterdam Malaria Cohort with available serum and urine samples at presentation were included. Ten of these patients met the criteria for severe malaria. The predictive performance of NGAL and KIM-1 as markers for AKI was compared with that of serum creatinine. Results Six of the 39 patients (15 %) developed AKI. Serum and urine NGAL and urine KIM-1 were all found to have large areas under the receiver operating characteristics curves (AUROC) for predicting AKI. Urine NGAL was found to have an excellent performance with positive predictive value (PPV) of 1.00 (95 % CI 0.54–1.00), a negative predictive value (NPV) of 1.00 (95 % CI 0.89–1.00) and an AUROC of 1.00 (95 % CI 1.00–1.00). Conclusion A good diagnostic performance of NGAL and KIM-1 for AKI was found. Particularly, urine NGAL was found to have an excellent predictive performance. Larger studies are needed to demonstrate whether these biomarkers are superior to serum creatinine as predictors for AKI in P. falciparum malaria. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1516-y) contains supplementary material, which is available to authorized users.

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynolds layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.

To identify unknown human viruses, we analyzed serum and cerebrospinal fluid samples from patients with unexplained paraplegia from Malawi by using viral metagenomics. A novel cyclovirus species was identified and subsequently found in 15% and 10% of serum and cerebrospinal fluid samples, respectively. These data expand our knowledge of cyclovirus diversity and tropism.

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of 13C-enriched and 14C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.

Mitochondria are usually considered to be the powerhouses of the cell and to be responsible for the aerobic production of ATP. However, many eukaryotic organisms are known to possess anaerobically functioning mitochondria, which differ significantly from classical aerobically functioning mitochondria. Recently, functional and phylogenetic studies on some enzymes involved clearly indicated an unexpected evolutionary relationship between these anaerobically functioning mitochondria and the classical aerobic type. Mitochondria evolved by an endosymbiotic event between an anaerobically functioning archaebacterial host and an aerobic α–proteobacterium. However, true anaerobically functioning mitochondria, such as found in parasitic helminths and some lower marine organisms, most likely did not originate directly from the pluripotent ancestral mitochondrion, but arose later in evolution from the aerobic type of mitochondria after these were already adapted to an aerobic way of life by losing their anaerobic capacities. This review will focus on some biochemical and evolutionary aspects of these fermentative mitochondria, with special attention to fumarate reductase, the synthesis of the rhodoquinone involved, and the enzymes involved in acetate production (acetate : succinate CoA–transferase and succinyl CoA–synthetase).

Background Both in endemic countries and in imported malaria, changes in total and differential leukocyte count during Plasmodium falciparum infection have been described. To study the exact dynamics of differential leukocyte counts and their ratios, they were monitored in a group of healthy non-immune volunteers in two separate Controlled Human Malaria Infection (CHMI) studies. Methods In two CHMI trials, CHMI-a and CHMI-b, 15 and 24 healthy malaria-naïve volunteers, respectively, were exposed to bites of infected mosquitoes, using the P. falciparum research strain NF54 and the novel clones NF135.C10 and NF166.C8. After mosquito bite exposure, twice-daily blood draws were taken to detect parasitaemia and to monitor the total and differential leukocyte counts. All subjects received a course of atovaquone–proguanil when meeting the treatment criteria. Results A total of 39 volunteers participated in the two trials. Thirty-five participants, all 15 participants in CHMI-a and 20 of the 24 volunteers in CHMI-b, developed parasitaemia. During liver stage development of the parasite, the median total leukocyte count increased from 5.5 to 6.1 × 109 leukocytes/L (p = 0.005), the median lymphocyte count from 1.9 to 2.2 (p = 0.001) and the monocyte count from 0.50 to 0.54 (p = 0.038). During the subsequent blood stage infection, significant changes in total and differential leukocyte counts lead to a leukocytopenia (nadir median 3.3 × 109 leukocytes/L, p = 0.0001), lymphocytopenia (nadir median 0.7 × 109 lymphocytes/L, p = 0.0001) and a borderline neutropenia (nadir median 1.5 × 109 neutrophils/L, p = 0.0001). The neutrophil to lymphocyte count ratio (NLCR) reached a maximum of 4.0. Significant correlations were found between parasite load and absolute lymphocyte count (p

The hydrophobic molecules of the metabolome – also named the lipidome – constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host–pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs., The lipidome constitutes a major part of the entire cellular metabolome with functions in cell structure, membrane dynamics and organization, energy supply and storage, and cellular signaling. Pathogens have adapted in different ways to take advantage of the complexity of the host cell lipidome in order to escape the immune system and to allow their survival and replication. Here we review the lipid targeting by various types of pathogens and provide interaction maps, allowing visualization of differences between viruses, bacteria, fungi and parasites.

Background: The incidence of transfusion-transmitted malaria is very low in non-endemic countries due to strict donor selection. The optimal strategy to mitigate the risk of transfusion-transmitted malaria in non-endemic countries without unnecessary exclusion of blood donations is, however, still debated and asymptomatic carriers of Plasmodium species may still be qualified to donate blood for transfusion purposes. Case description: In April 2011, a 59-year-old Dutch woman with spiking fevers for four days was diagnosed with a Plasmodium malariae infection. The patient had never been abroad, but nine weeks before, she had received red blood cell transfusion for anaemia. The presumptive diagnosis of transfusion-transmitted quartan malaria was made and subsequently confirmed by retrospective PCR analysis of donor blood samples. The donor was a 36-year-old Dutch male who started donating blood in May 2006. His travel history outside Europe included a trip to Kenya, Tanzania and Zanzibar in 2005, to Thailand in 2006 and to Costa Rica in 2007. He only used malaria prophylaxis during his travel to Africa. The donor did not show any abnormalities upon physical examination in 2011, while laboratory examination demonstrated a thrombocytopenia of 126 × 10 9 /L as the sole abnormal finding since 2007. Thick blood smear analysis and the Plasmodium PCR confirmed an ongoing subclinical P. malariae infection. Chloroquine therapy was started, after which the infection cleared and thrombocyte count normalized. Fourteen other recipients who received red blood cells from the involved donor were traced. None of them developed malaria symptoms. Discussion: This case demonstrates that P. malariae infections in non-immune travellers may occur without symptoms and persist subclinically for years. In addition, this case shows that these infections pose a threat to transfusion safety when subclinically infected persons donate blood after their return in a non-endemic malaria region. Since thrombocytopenia was the only abnormality associated with the subclinical malaria infection in the donor, this case illustrates that an unexplained low platelet count after a visit to malaria-endemic countries may be an indicator for asymptomatic malaria even when caused by non-falciparum Plasmodium species.