Case report: Chronic Candida albicans meningitis: a rare entity diagnosed by metagenomic next-generation sequencing.

The aetiology of chronic aseptic meningitis is difficult to establish. Candida meningitis in particular is often diagnosed late, as cerebrospinal fluid (CSF) work-up and imaging findings are nonspecific. A 35-year-old patient with chronic aseptic meningitis, for which repeated microbiological testing of CSF was unrevealing, was finally diagnosed with Candida albicans (C. albicans) meningitis with cauda equina involvement using metagenomic next-generation sequencing (mNGS). This report highlights the diagnostic challenges and the difficulties of treating shunt-associated fungal meningitis.

The trajectory of anti-recEm18 antibody levels determines follow-up after curative resection of hepatic alveolar echinococcosis.

INTRODUCTION: Recurrence after curative resection of hepatic alveolar echinococcosis remains a clinical challenge. The current study tested if assessment of anti-recEm18 allows for postsurgical patient surveillance. METHODS: A retrospective study with patients undergoing liver resection for alveolar echinococcosis (n = 88) at the University Hospital Bern from 2002 to 2020 and at the University Hospital and Medical Center Ulm from 2011 to 2017 was performed. Analysis was directed to determine a potential association of pre- and postoperative values of anti-recEm18 with clinical outcomes. RESULTS: Anti-recEm18 had a linear correlation to the maximum lesion diameter (R2 = 0.558). Three trajectories of anti-recEm18 were identified based on a threshold of 10 AU/ml: "Em18-low" (n = 31), "responders" (n = 53) and "residual disease" (n = 4). The decline of anti-recEm18 in "responders" reached a plateau after 10.9 months at which levels decreased by 90%. The only patient with recurrence in the entire population was also the only patient with a secondary increase of anti-recEm18. CONCLUSION: In patients with preoperative elevated values, anti-recEm18 confirms curative surgery at 12 months follow-up and allows for long-term surveillance.

A novel multiplex real-time polymerase chain reaction for the molecular diagnosis of metacestode infections in human patients.

OBJECTIVES: The diagnosis of larval cestodiases in humans primarily depends on using imaging techniques in combination with serological tests. However, in case of atypical imaging results, negative serology results due to immunosuppression, or infection with rare taeniid species, traditional diagnostic tools may not provide a definitive species-level diagnosis. We aimed to validate a rapid, reliable, and cost-effective single-step real-time PCR method that can identify and differentiate larval cestodiases from biopsy material. METHODS: We validated a real-time PCR technique able to distinguish Echinococcus multilocularis, E. granulosus sensu lato (s.l.), and Taenia spp. from biopsy or cytology material in a single-step analysis. Further Sanger sequencing of E. granulosus s.l. and Taenia spp. amplicons enables differentiation of various Echinococcus and Taenia species. The assay was validated on (a) a reference sample collection of 69 clinical and veterinary cases confirmed by imaging, serology, and morphological analysis, (b) 38 routine human patient samples confirmed for aforementioned pathogens by a conventional end-point PCR, and (c) 127 samples from patients with suspected echinococcosis that were submitted to our laboratory for diagnostic analysis. RESULTS: Compared to a conventional reference end-point PCR approach, the quadruplex real-time PCR exhibited a lower limit of detection in a serial dilution with 5-log dilutions for all three targets (2 log for E. multilocularis, 1 log for E. granulosus s.s., and 1 log for T. saginata). We were able to detect DNA from E. multilocularis, E. granulosus s.l. (E. granulosus s.s., E. canadensis, E. ortleppi, and E. felidis), a wide range of Taenia spp., as well as from non-echinococcal metacestodes such as Hydatigera taeniaformis, Hymenolepis spp., Versteria sp., and Spirometra erinaceieuropaei. DISCUSSION: We suggest that the presented real-time PCR method is a suitable tool to be routinely used in a clinical microbiology laboratory to rapidly detect and identify larval cestodiases in human tissue.

Case Report: Diagnostic Challenges in the Detection of a Mixed Plasmodium vivax/ovale Infection in a Non-Endemic Setting.

In clinical practice, mixed-species malaria infections are often not detected by light microscopy (LM) or rapid diagnostic test, as a low number of parasites of one species may occur. Here, we report the case of an 8-year-old girl migrating with her family from Afghanistan with a two-species mixed infection with Plasmodium vivax and Plasmodium ovale. This case demonstrates the significance of molecular testing in the detection of mixed-species malaria infections and highlights the importance of a detailed data analysis during the medical validation procedure to prevent underestimation of mixed-species infections. To our knowledge, this is the first case report of a two-species mixed infection comprising both P. vivax and P. ovale confirmed by LM and different real-time polymerase chain reaction (PCR) approaches.

Proteomic analysis of exported chaperone/co-chaperone complexes of P. falciparum reveals an array of complex protein-protein interactions.

Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure. To address this, we have utilized chemical cross-linking followed by mass spectrometry and immunoblotting to isolate and characterize proteins complexes containing an exported Hsp40 (PFE55), and the only known exported Hsp70 (PfHsp70x). Our data reveal that both of these proteins are contained in high molecular weight protein complexes. These complexes are found both in the infected erythrocyte, and within the parasite-derived compartment referred to as the parasitophorous vacuole. Surprisingly, our data also reveal an association of PfHsp70x with components of PTEX, a putative protein translocon within the membrane of the parasitophorous vacuole. Our results suggest that the P. falciparum- infected human erythrocyte contains numerous high molecular weight protein complexes, which may potentially be involved in host cell modification.

Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton.

Adherence of Plasmodium falciparum-infected erythrocytes to host endothelium is conferred through the parasite-derived virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major contributor to malaria severity. PfEMP1 located at knob structures on the erythrocyte surface is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion of infected erythrocytes to the endothelial receptor CD36. Adhesion to other endothelial receptors was less affected or even unaltered by PFE1605w depletion, suggesting that PHIST proteins might be optimized for subsets of PfEMP1 variants. PFE1605w does not play a role in PfEMP1 transport, but it directly interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P. falciparum.

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface.

Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 µM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.

Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export.

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.