An evaluation of a lateral flow rapid diagnostic test for Strongyloides stercoralis infection.

Strongyloides stercoralis in humans often presents as a chronic asymptomatic infection. Diagnosis can be challenging due to the limited sensitivity of faecal-based parasitological techniques. A prototype lateral flow rapid diagnostic test (RDT) for the detection of specific antibodies against Strongyloides stercoralis (SsRapid) was evaluated using 143 samples from the serum bank of the Swiss Tropical and Public Health Institute. Group 1 (n = 30) comprised serum samples from larvae-positive individuals; the RDT's diagnostic sensitivity was 97 % (29/30). Group II comprised serum samples from patients with other parasitic infections (n = 86) and Swiss blood donors (n = 27); the RDT's diagnostic specificity for this group was 90 % (102/113). The RDT showed good diagnostic performance and is a promising point-of-care test for detecting human Strongyloides stercoralis infection.

Human Taenia martis Neurocysticercosis, Switzerland.

Neurocysticercosis is almost exclusively caused by Taenia solium tapeworms. We describe a case of neurocysticercosis in Switzerland caused by infection with Taenia martis, the marten tapeworm, and review all 5 published cases of human infection with the marten tapeworm. In epidemiologically nonplausible cases of neurocysticercosis, zoonotic spillover infections should be suspected.

Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia.

Leptospirosis is among the most important zoonotic diseases in (sub-)tropical countries. The research objective was to evaluate the accuracy of the Serion IgM ELISA EST125M against the Microscopic Agglutination Test (MAT = imperfect reference test); to assess its ability to diagnose acute leptospirosis infections and to detect previous exposure to leptospires in an endemic setting. In addition, to estimate the overall Leptospira spp. seroprevalence in the Wiwa indigenous population in North-East Colombia. We analysed serum samples from confirmed leptospirosis patients from the Netherlands (N = 14), blood donor sera from Switzerland (N = 20), and sera from a cross-sectional study in Colombia (N = 321). All leptospirosis ELISA-positive, and a random of negative samples from Colombia were tested by the MAT for confirmation. The ELISA performed with a sensitivity of 100% (95% CI 77% - 100%) and a specificity of 100% (95% CI 83% - 100%) based on MAT confirmed Leptospira spp. positive and negative samples. In the cross-sectional study in Colombia, the ELISA performed with a sensitivity of 100% (95% CI 2-100%) and a specificity of 21% (95% CI 15-28%). Assuming a 5% Leptospira spp. seroprevalence in this population, the positive predictive value was 6% and the negative predictive value 100%. The Leptospira spp. seroprevalence in the Wiwas tested by the ELISA was 39%; however, by MAT only 0.3%. The ELISA is suitable to diagnose leptospirosis in acutely ill patients in Europe several days after onset of disease. For cross-sectional studies it is not recommended due to its low specificity. Despite the evidence of a high leptospirosis prevalence in other study areas and populations in Colombia, the Wiwa do not seem to be highly exposed to Leptospira spp.. Nevertheless, leptospirosis should be considered and tested in patients presenting with febrile illness.

Aberrant stromal tissue factor localisation and mycolactone-driven vascular dysfunction, exacerbated by IL-1ß, are linked to fibrin formation in Buruli ulcer lesions.

Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an 'indirect' route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone's mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. There was little evidence of primary haemostasis, perhaps due to mycolactone-dependent depletion of endothelial von Willebrand factor. Instead, fibrin staining appeared to be linked to the extrinsic pathway activator, tissue factor (TF). There was significantly greater than expected fibrin staining around vessels that had TF staining within the stroma, and this correlated with the distance it extended from the vessel basement membrane. TF-induced fibrin deposition in these locations would require plasma proteins outside of vessels, therefore we investigated whether mycolactone could increase vascular permeability in vitro. This was indeed the case, and leakage was further exacerbated by IL-1ß. Mycolactone caused the loss of endothelial adherens and tight junctions by the depletion of VE-cadherin, TIE-1, TIE-2 and JAM-C; all Sec61-dependent proteins. Taken together, our findings suggest that both vascular and lymphatic vessels in BU lesions become "leaky" during infection, due to the unique action of mycolactone, allowing TF-containing structures and plasma proteins into skin tissue, ultimately leading to local coagulopathy and tissue ischemia.

Surveillance of leishmaniasis cases from 15 European centres, 2014 to 2019: a retrospective analysis.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

Immunohistochemistry: A Method to Analyze M. ulcerans Infected Skin Tissue.

Immunohistochemistry (IHC) is a combination of immunological, biochemical, and pathological methods to visualize the presence and distribution of specific epitopes in tissue sections. Selected antigens are stained by differently labeled antibodies binding to their target antigens in situ. Here we describe sample preparation and sample staining in order to diagnose and analyze tissue samples infected with M. ulcerans from human as well as animal source.

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity.

The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3-/-/perk-/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A1; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Chronic wounds in Sierra Leone: Searching for Buruli ulcer, a NTD caused by Mycobacterium ulcerans, at Masanga Hospital.

BACKGROUND: Chronic wounds pose a significant healthcare burden in low- and middle-income countries. Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, causes wounds with high morbidity and financial burden. Although highly endemic in West and Central Africa, the presence of BU in Sierra Leone is not well described. This study aimed to confirm or exclude BU in suspected cases of chronic wounds presenting to Masanga Hospital, Sierra Leone. METHODOLOGY: Demographics, baseline clinical data, and quality of life scores were collected from patients with wounds suspected to be BU. Wound tissue samples were acquired and transported to the Swiss Tropical and Public Health Institute, Switzerland, for analysis to detect Mycobacterium ulcerans using qPCR, microscopic smear examination, and histopathology, as per World Health Organization (WHO) recommendations. FINDINGS: Twenty-one participants with wounds suspected to be BU were enrolled over 4-weeks (Feb-March 2019). Participants were predominantly young working males (62% male, 38% female, mean 35yrs, 90% employed in an occupation or as a student) with large, single, ulcerating wounds (mean diameter 9.4cm, 86% single wound) exclusively of the lower limbs (60% foot, 40% lower leg) present for a mean 15 months. The majority reported frequent exposure to water outdoors (76%). Self-reports of over-the-counter antibiotic use prior to presentation was high (81%), as was history of trauma (38%) and surgical interventions prior to enrolment (48%). Regarding laboratory investigation, all samples were negative for BU by microscopy, histopathology, and qPCR. Histopathology analysis revealed heavy bacterial load in many of the samples. The study had excellent participant recruitment, however follow-up proved difficult. CONCLUSIONS: BU was not confirmed as a cause of chronic ulceration in our cohort of suspected cases, as judged by laboratory analysis according to WHO standards. This does not exclude the presence of BU in the region, and the definitive cause of these treatment-resistance chronic wounds is uncertain.

Exposure to Leptospira spp. and Associated Risk Factors in the Human, Cattle and Dog Populations in Bhutan.

Leptospirosis is a neglected worldwide zoonotic bacterial disease with a high prevalence in subtropical and tropical countries. The prevalence of Leptospira spp. in humans, cattle and dogs is unknown in Bhutan. Therefore, we sought to find out whether humans, cattle or dogs had been infected in the past with leptospires by measuring antibodies in the serum. We therefore collected blood from 864 humans ≥13 years of age, 130 bovines and 84 dogs from different rural and urban areas in Bhutan and tested the serum for antibodies specific for leptospires with a screening of enzyme-linked immunosorbent assays (ELISA) and a confirmatory microscopic agglutination test (MAT). In humans, 17.6% were seropositive by ELISA and 1.6% by MAT. The seropositivity was stronger in bovines (36.9%) and dogs (47.6%). "Having had a fever recently" (OR 5.2, p = 0.004), "working for the military" (OR 26.6, p = 0.028) and "being unemployed" (OR 12.9, p = 0.041) (reference category = housemaker) were statistically significantly associated with seropositivity when controlled for the effects of other risk factors. However, due to the small number of positive test results, the findings on risk factors should be interpreted with caution. Based on the serogroups found in the three species, dogs could be a source of infection for humans, or dogs and humans are exposed to the same environmental risk factors Clinical leptospirosis in humans and domestic animals should be investigated by testing blood and urine for the presence of leptospires by molecular methods (qPCR).

Strongyloides stercoralis prevalence and diagnostics in Vientiane, Lao People's Democratic Republic.

BACKGROUND: Despite the high prevalence of strongyloidiasis in the Laotian population, Laotian hospitals still lack diagnostic capacity to appropriately diagnose Strongyloides stercoralis infections. This cross-sectional hospital-based study was conducted to assess the prevalence of Strongyloides stercoralis infection among hospitalized patients treated at Mahosot Hospital, the primary reference hospital of Lao People's Democratic Republic (Lao PDR), and to validate feasible methods for diagnosing S. stercoralis infection at hospital's laboratory. METHODS: Between September and December 2018, stool samples of 104 inpatients were investigated for S. stercoralis infection by wet smear, Baermann technique, Koga Agar plate culture (KAPC), and real-time detection polymerase chain reaction (RTD-PCR) at the Infectious Diseases Ward of the Mahosot Hospital in Vientiane. The sensitivity, the specificity, the negative predictive value (NPV) of each diagnostic test, as well as their combination(s) was calculated using a composite reference standard (CRS). The correlation of the different test methods was assessed by chi-square or Fisher's exact test. Cohen's kappa coefficient was used to assess the diagnostic agreement of the different test methods. RESULTS: The overall prevalence of S. stercoralis infections among the study population was 33.4%. The cumulative infection prevalence statistically significantly increased from the lowest age group of 40 years and below (22.4%), to the medium (40.0%) and to the oldest age group of 61 year and above (72.7%)(P = 0.003). The cumulative infection prevalence of CRS was considerably higher in male (40.4%) compared to female patients (28.1%), but not statistically different (P = 0.184). The diagnostic sensitivity of Baermann technique, KAPC, RTD-PCR, and the combination of Baermann technique and KAPC were 60.0, 60.0, 74.3, and 77.1%, respectively. Only 13 patients (37.1%) of the total 35 S. stercoralis patients diagnosed with any technique had a simultaneously positive diagnostic test with Baermann, KAPC and RTD-PCR. CONCLUSIONS: We identified Baermann technique and KAPC to be currently the most feasible and implementable standard methods for diagnosing S. stercoralis at a hospital setting such as Mahosot Hospital and provincial and district hospitals in Lao PDR and other low- and middle income countries in Southeast Asia. TRIAL REGISTRATION: This study was approved by the National Ethics Committee for Health Research in Lao PDR (reference no. 083/NECHR) and by the Ethics Committee Northwest and Central Switzerland (reference no. 2018-00594).

Toward a Single-Dose Cure for Buruli Ulcer.

A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans.

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.

Gambiense Human African Trypanosomiasis Sequelae after Treatment: A Follow-Up Study 12 Years after Treatment.

The clinical presentation of Human African Trypanosomiasis (HAT) due to Trypanosoma brucei gambiense is well known, but knowledge on long-term sequelae is limited. In the frame of studies conducted between 2004 and 2005 in the Democratic Republic of the Congo (DRC), the prevalence of HAT related signs and symptoms were evaluated before the start of treatment and at the end of treatment. To explore possible long-term sequelae, the same clinical parameters were assessed in 2017 in 51 first stage and 18 second stage HAT patients. Signs and symptoms 12-13 years after treatment were compared to before and immediately after treatment and to controls matched for sex and age (±5 years). In first stage HAT patients, the prevalence of all signs and symptoms decreased compared to before treatment but were still higher after 12-13 years than immediately at the end of treatment and in the control group. In second stage HAT patients, all HAT-specific findings had continuously decreased to the point where they were in the range of the healthy control group. In a selection of oligosymptomatic first stage HAT patients, no trypanosomes were detected in the blood by microscopic examination or PCR. An oligosymptomatic presentation of HAT due to the persistence of parasites in compartments, where first stage HAT medications do not penetrate, could not be ruled out.

Case Report: Human Subcutaneous Sparganosis in a Thai Migrant.

Human sparganosis is a cestode infection which is neglected as a differential diagnosis outside endemic countries. Diagnosis and therapy may be challenging depending on the clinical presentation and anatomic localization. The disease manifests predominantly as subcutaneous nodule(s) or intracranial mass lesion(s). Infection is primarily acquired by ingesting raw or undercooked amphibian or reptile flesh or by drinking water containing copepods. We report an unusual case of subcutaneous Spirometra erinaceieuropaei sparganosis presenting with two nonmigratory nodules in close proximity to each other on the right thigh of a Thai woman living in Switzerland.

Case Report: Paragonimiasis Presenting with Pericardial Tamponade.

We report an unusual case of paragonimiasis in a Nepali patient presenting with massive pericardial effusion and pericardial tamponade. The patient reported neither the consumption of crabs or crayfish nor the consumption of wild animal meat, which are the usual sources of infection. It is suspected that the source of infection was instead the ingestion of raw live slugs as part of a traditional medicine treatment.

A blind passenger: a rare case of documented seroconversion in an Angiostrongylus cantonensis induced eosinophilic meningitis in a traveler visiting friends and relatives.

BACKGROUND: Eosinophilic meningitis (EOM) is a rare condition that is caused by various communicable and non-communicable factors. The rat-lungworm Angiostrongylus cantonensis, which is associated with consumption of raw or undercooked paratenic or intermediate hosts, is the most common cause of parasitic eosinophilic meningitis worldwide. While the majority of A. cantonensis cases are reported from endemic regions, cases in travelers pose a challenge to clinicians in non-endemic countries. Here we report a rare case of eosinophilic meningitis caused by A. cantonensis in a Swiss traveler who was diagnosed after returning from Thailand. CASE PRESENTATION: A 33-year old woman with a travel history to rural north-eastern Thailand presented to an emergency department in Switzerland with severe headache and vomiting. Eosinophilic meningitis was confirmed as the cause of the symptoms; however, serologic investigations failed to confirm an A. cantonensis infection on the first evaluation. Nevertheless, empirical treatment with an anthelminthic and steroid regimen led to a rapid alleviation of symptoms. Repeated serology confirmed seroconversion 2 weeks after treatment initiation. DISCUSSION: Parasitic etiology must be considered in returning travelers who present with symptoms compatible with a central nervous system infection. A thorough medical history, including types of food consumed, is paramount and can often suggest differential diagnosis. Neuroangiostrongyliasis is rare and might be missed if serology does not cover possible seroconversion.

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.