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Schistosomiasis is a parasitosis caused by trematodes of the genus Schistosoma. Humans are infected when coming into contact with freshwater containing the parasites’ infective stages, which are amplified through freshwater-dwelling snails acting as intermediate hosts. Schistosomiasis has posed significant problems for troops exposed to freshwater in endemic regions ever since the Napoleonic wars. Schistosomiasis has substantial differences in clinical presentation, depending on the type of parasite, intensity of infection and reinfection, clinical form, and disease stage. It can remain undiagnosed for long periods of time, with well-known long-term morbidity and mortality risks. The diagnosis of schistosomiasis depends on its stage and relays on several tests, all with limitations in sensitivity and specificity. The diagnostic gold standard is the detection of eggs in urine, feces, or tissue biopsies, but this can raise problems in patients such as military personnel, in which the worm burden is usually low. Praziquantel is the drug of choice for schistosomiasis. Currently, there is no available commercial vaccine against any Schistosoma parasite. Avoiding freshwater exposure is the best prevention. Herein, we review the clinical presentation, diagnosis, treatment, and prevention of schistosomiasis in the military. This information may decrease the impact of schistosomiasis on this particular professional group.

Recently a number of broad-range stool parasite PCR assays have been developed. However, there is ongoing disagreement regarding their diagnostic performance, as various studies have produced contradictory results. In this study, we compared the diagnostic accuracy of the Seegene Allplex GI-Parasite and Allplex GI-Helminth assays (SA) with the conventional methods used at the travel clinic of the Institute of Tropical Medicine (ITM) including microscopy, antigen testing, and molecular detection in order to provide insights into the strengths and limitations of this diagnostic tool which may be crucial to select the most appropriate diagnostic tools for the suspected pathogen. A total of 97 native stool samples from 95 patients with suspected gastrointestinal illness were analyzed, including 26 from a frozen collection and 71 prospectively collected samples. The diagnostic performance of SA was notably superior to the conventional workflow in detecting Dientamoeba fragilis (sensitivity 100% vs. 47.4%) and Blastocystis hominis (sensitivity 95% vs. 77.5%). SA had a comparable performance with the conventional workflow in detecting pathogenic protozoa (sensitivity 90% vs. 95%). In contrast, SA had a much lower diagnostic performance in detecting helminths (59.1%) compared to the conventional workflow (100%). We conclude that the Seegene Allplex GI-Parasite assay may be useful for protozoa screening in low-endemic industrialized countries. However, the Allplex GI-Helminth assay is not recommended due to its suboptimal performance compared to microscopy.

Objectives: The origin and spread of dengue virus (DENV) circulating in Africa remain poorly characterized, with African sequences representing

Background: The presentation of mpox clade IIb during the 2022 outbreak overlaps with a range of other diseases. Understanding the factors associated with mpox is important for clinical decision making. Methods: We described the characteristics of mpox patients who sought care at Belgian sexual health clinic. Furthermore we compared their characteristics to those of patients with a clinical suspicion of mpox but who tested negative on polymerase chain reaction. Results: Between May 23 and September 20, 2022, 155 patients were diagnosed with mpox, and 51 patients with suspected symptoms tested negative. All mpox patients self-identified as men and 148/155 (95.5%) as gay or bisexual MSM. Systemic symptoms were present in 116/155 (74.8%) patients. All but 10 patients (145/155, 93.5%) presented with skin lesions. Other manifestations were lymphadenopathy (72/155, 46.5%), proctitis (50/155, 32.3%), urethritis (12/155, 7.7%), tonsillitis (2/155, 1.3%). Complications involved bacterial skin infection (13/155, 8.4%) and penile oedema with or without paraphimosis (4/155, 2.6%). In multivariable logistic regression models, the presence of lymphadenopathy (OR 3.79 95% CI 1.44–11.49), skin lesions (OR 4.35 95% CI 1.15–17.57) and proctitis (OR 9.41 95% CI 2.72–47.07) were associated with the diagnosis of mpox. There were no associations with age, HIV status, childhood smallpox vaccination, number of sexual partners and international travel. Conclusions: The presence of proctitis, lymphadenopathies and skin lesions should increase clinical suspicion of mpox in patients with compatible symptoms.

Abstract Background Persistent fever, defined as fever lasting for 7 days or more at first medical evaluation, has been hardly investigated as a separate clinical entity in the tropics. This study aimed at exploring the frequencies and diagnostic predictors of the ubiquitous priority (i.e., severe and treatable) infections causing persistent fever in the tropics. Methods In six different health settings across four countries in Africa and Asia (Sudan, Democratic Republic of Congo [DRC], Nepal, and Cambodia), consecutive patients aged 5 years or older with persistent fever were prospectively recruited from January 2013 to October 2014. Participants underwent a reference diagnostic workup targeting a pre-established list of 12 epidemiologically relevant priority infections (i.e., malaria, tuberculosis, HIV, enteric fever, leptospirosis, rickettsiosis, brucellosis, melioidosis, relapsing fever, visceral leishmaniasis, human African trypanosomiasis, amebic liver abscess). The likelihood ratios (LRs) of clinical and basic laboratory features were determined by pooling all cases of each identified ubiquitous infection (i.e., found in all countries). In addition, we assessed the diagnostic accuracy of five antibody-based rapid diagnostic tests (RDTs): Typhidot Rapid IgM, Test-itTM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-itTM Leptospira IgM Lateral Flow Assay and SD Bioline Leptospira IgG/IgM for leptospirosis. Results A total of 1922 patients (median age: 35 years; female: 51%) were enrolled (Sudan, n = 667; DRC, n = 300; Nepal, n = 577; Cambodia, n = 378). Ubiquitous priority infections were diagnosed in 452 (23.5%) participants and included malaria 8.0% (n = 154), tuberculosis 6.7% (n = 129), leptospirosis 4.0% (n = 77), rickettsiosis 2.3% (n = 44), enteric fever 1.8% (n = 34), and new HIV diagnosis 0.7% (n = 14). The other priority infections were limited to one or two countries. The only features with a positive LR ≥ 3 were diarrhea for enteric fever and elevated alanine aminotransferase level for enteric fever and rickettsiosis. Sensitivities ranged from 29 to 67% for the three RDTs targeting S. Typhi and were 9% and 16% for the two RDTs targeting leptospirosis. Specificities ranged from 86 to 99% for S. Typhi detecting RDTs and were 96% and 97% for leptospirosis RDTs. Conclusions Leptospirosis, rickettsiosis, and enteric fever accounted each for a substantial proportion of the persistent fever caseload across all tropical areas, in addition to malaria, tuberculosis, and HIV. Very few discriminative features were however identified, and RDTs for leptospirosis and Salmonella Typhi infection performed poorly. Improved field diagnostics are urgently needed for these challenging infections. Trial registration NCT01766830 at ClinicalTrials.gov.

We described a case of Plasmodium malariae malaria in a traveler returning from the Democratic Republic of Congo to Belgium. This occurred despite successful artemether-lumefantrine treatment for Plasmodium falciparum three weeks earlier and in the absence of re-exposure in an endemic area. We discuss possible explanations for this unusual observation.

Abstract We report the levels of neutralising antibodies against Wuhan, Delta and Omicron variants in unimmunized infected (group 1), immunised and boosted (group 2) and infected immunised and boosted (group 3) adult individuals. Our observations support the rapid administration of a booster vaccine dose to prevent infection and disease caused by Omicron.

We report 3 confirmed autochthonous tick-borne encephalitis cases in Belgium diagnosed during summer 2020. Clinicians should include this viral infection in the differential diagnosis for patients with etiologically unexplained neurologic manifestations, even for persons without recent travel history.

A couple presented with fever and strikingly similar symptoms starting exactly on the same day after returning from an adventurous journey in Peru. Symptom onset was 12 days after exposure to bats from a hollow tree. The further evolution underscores the disparate disease course of Histoplasmosis in different individuals, despite similar radiological findings. Our case highlights the importance of careful history taking in returning travelers since exposure to bat (or fowl) excrement can be easily overlooked.

Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax. Several genetic surveillance applications (‘use cases’) have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP within-country barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/µL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33-SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy.

Abstract Background Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. Methods Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. Results The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0–95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. Conclusions Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).

In a world with an increasing population at risk of exposure to arthropod-borne flaviviruses, access to timely and accurate diagnostic tests would impact profoundly on the management of cases. Twenty peptides previously identified using a flavivirus proteome-wide microarray were evaluated to determine their discriminatory potential to detect dengue virus (DENV) infection. This included nine peptides recognized by IgM antibodies (PM peptides) and 11 peptides recognized by IgG antibodies (PG peptides). A bead-based multiplex peptide immunoassay (MPIA) using the Luminex technology was set-up to determine Ab binding levels to each of these peptides in a panel of 323 carefully selected human serum samples. Sera are derived from individuals either infected with different viruses, namely, the four DENV serotypes, Zika virus (ZIKV), yellow fever virus (YFV), chikungunya virus (CHIKV), West Nile virus (WNV) and Human immunodeficiency virus (HIV), or receiving vaccination against YFV, tick-borne encephalitis (TBEV), and Japanese encephalitis virus (JEV). Additionally, a set of healthy controls were included. We targeted a minimum specificity of 80% for all the analysis. The PG-9 peptide had the best sensitivity (73%) when testing DENV sera from acute patients (A-DENV; 10 days since symptom onset) the FPG-1 peptide was the best seromarker with a sensitivity of 86%. When combining all A-DENV and C-DENV samples, peptides PM-22 and FPG-1 had the best-diagnostic performance with a sensitivity of 60 and 61.1%, and areas under the curve (AUC) of 0.7865 and 0.8131, respectively. A Random forest (RF) algorithm was used to select the best combination of peptides to classify DENV infection at a targeted specificity >80%. The best RF model for PM peptides that included A-DENV and C-DENV samples, reached a sensitivity of 72.3%, while for PG peptides, the best RF models for A-DENV only, C-DENV only and A-DENV + C-DENV reached a sensitivity of 88.9%, 89.1%, and 88.3%, respectively. In conclusion, the combination of multiple peptides constitutes a founding set of seromarkers for the discrimination of DENV infected individuals from other flavivirus infections.

Dengue is a major public health problem in tropical and sub-tropical regions worldwide. Since the Zika epidemic and the increased co-circulation of other arboviruses, the serology-based diagnosis of dengue has become more problematic due to the high antigenic resemblance, especially among the flavivirus family. Therefore, a more comprehensive understanding of the diversity, specificity and temporal evolution of the antibody response following dengue infection is needed. In order to close this knowledge gap, we used a high-density peptide microarray of 9,072 linear peptides covering the entire proteome diversity of dengue, Zika, yellow fever and chikungunya viruses. The IgM and IgG antibody responses were measured against the designed microarray in symptomatic dengue infected individuals from an arbovirus endemic area in Peru and in overseas travelers returning to Belgium, as representatives of multiple-exposed and primary infections, respectively. Serum samples were collected longitudinally across four time points over the period of six months in Peru and over two time points in travelers. We show that epitopes eliciting the strongest flavivirus cross-reactive antibodies, in both primary and secondary infections were concentrated in the capsid, E, NS1, NS3 and NS5 proteins. The IgG antibody responses against NS1 and NS3 followed a rise-and-fall pattern, with peak titers between two to four weeks after onset of illness. The response to the E and NS5 proteins increased rapidly in the acute phase and was maintained at stable levels until at least 6 months after illness. A more scattered IgM antibody reactivity across the viral proteome was observed in the acute phase of the disease and that persisted through the 6-month window. The magnitude, breadth (i.e. number of unique epitopes targeted) and depth (i.e. number of epitope variants recognized) of the IgG response was higher in secondary infections compared to primary infections. For IgM antibodies, the magnitude of the response was higher in primary infected individuals whereas the breadth and depth of the response was lower in this group compared with the endemic subjects. Finally, through this arboviral proteome-wide epitope mapping, we were able to identify IgM and IgG dengue-specific epitopes which can be useful serological markers for dengue diagnosis and serostatus determination.

Abstract Background The Belgian Reference Laboratory for Plasmodium offers a free-of-charge reference testing of malaria-positive or doubtful samples to clinical laboratories. Methods The final malaria diagnosis from the Reference Laboratory (microscopy, rapid diagnostic tests (RDTs) and Plasmodium species-specific PCR) were compared with the final diagnosis from peripheral Belgian laboratories. The Reference Laboratory reports were analysed for all samples submitted between 2013 and 2017. Criteria assessed included the diagnosis of malaria, Plasmodium species identification including mixed infections, and in case of Plasmodium falciparum, the parasite density and the presence of sexual and asexual stages. Results A total of 947 non-duplicate samples were included. Reference testing confirmed 96.3% (893/927) and 90.0% (18/20) samples submitted as positive and negative, respectively, the two missed diagnoses were samples with Plasmodium ovale and Plasmodium malariae. Submitting laboratories had correctly identified P. falciparum in 95.1% (508/534) samples with P. falciparum single infection. They had correctly diagnosed the species in 62.9% (95/151) single non-falciparum samples and had reported ‘non-falciparum’ in another 26 (17.2%) samples; most errors occurred among P. malariae (n = 8/21, 38.1%) and P. ovale (n = 14/51, 27.5%). Only one of the 21 mixed Plasmodium species infections had been diagnosed as such by the submitting laboratories; in three of them, P. falciparum had been overlooked. Taken single and mixed infections together, P. falciparum was diagnosed in 98.6% (546/554) samples. Among 471 single P. falciparum samples available for comparison, laboratories had correctly reported parasite densities above 2% in 87.5% (70/80) samples; they had incorrectly reported parasite densities > 2% in an extra 52 (8.9%) samples. Laboratories had correctly reported P. falciparum schizonts and gametocytes in 25.6% (11/43) and 56.7% (17/30) samples, respectively. Conclusion Diagnostic laboratories in a malaria non-endemic setting provided excellent diagnosis of malaria and P. falciparum, reasonably good diagnosis of non-falciparum infections and acceptable calculation of P. falciparum parasite density.

Background: The incidence rate of Zika virus (ZIKV) infection in travellers from non-endemic areas to the Americas during the ZIKV outbreak in 2016 is unknown. Methods: Belgian adults who planned to travel to South America, Central America, and the Caribbean were recruited prospectively to study the incidence and characteristics of ZIKV. Demographic data and sera were collected at baseline. Participants were trained to collect capillary blood on filter paper (BFP). When ill during travel, the participants completed a questionnaire and they sampled BFP for post-hoc analysis. All symptomatic participants were screened for ZIKV using ZIKV-specific RT-PCR on serum or urine, or BFP, and antibody detection assays (ELISA). Follow-up sera of asymptomatic travellers, obtained at least 20 days post travel, were tested by ZIKV ELISA only. All positive ELISA results were subject to confirmation by virus neutralization testing (VNT). Results: Forty-nine participants completed follow-up: 38 women and 11 men, with a median age of 32 years (range 19–64 years). Travel destinations were countries in South America (n = 20), Central America (n = 24), and the Caribbean (n = 5). The total travel duration was 67.8 person-months. Illness was reported by 24 participants (49.0%). ZIKV infection was confirmed in nine cases, by RT-PCR (n = 5) and by VNT (n = 4). Only one of nine ZIKV cases (11.1%) was asymptomatic. The ZIKV incidence rate was 17.0% (95% confidence interval 7.8–32.2%) per month of travel. Conclusions: The ZIKV incidence rate in adult travellers from non-endemic countries to the epidemic territories during the 2016 outbreak was high. Asymptomatic ZIKV infection was rare in this population. Keywords: Zika virus, Americas, Incidence rate, Prospective cohort study, Enzyme-linked immunosorbent assay, Virus neutralization testing, Real-time reverse transcription polymerase chain reaction, Filter paper

We present a case of infective endocarditis (IE) on a prosthetic pulmonary valve in a 36-year-old patient with tetralogy of Fallot (TOF). The patient underwent valve replacement surgery and active antibiotic treatment against Gram-negative cocci (Piperacillin Tazobactam then Ceftriaxone) for a total duration of 42 days with a favourable outcome. The causative agent was Neisseria mucosa which was identified on the infected valve by sequencing of 16S ribosomal RNA. To our knowledge, this is the first described case of a N. mucosa infective endocarditis on a pulmonary valve. Initially, serologies performed in clinical settings by immunofluorescence for Coxiella burnetii antibodies showed a major increase in phase I IgG titers at 1024 (normal values

Background: We report a rare case of imported and asymptomatic Strongyloides fuelleborni infection in a Belgian student, returning from the Democratic Republic of the Congo. Strongyloidiasis, one of the most neglected tropical diseases, is estimated to affect more than 100 million people worldwide. The exact number of cases attributable to Strongyloides fuelleborni is not known, as correct identification of this parasite can be difficult. S. fuelleborni is a parasitic helminth, known to infect both humans and non-human primates. S. fuelleborni eggs in the stools of infected patients may be mistaken for hookworm eggs. Correct identification of these eggs is essential, as the recommended treatment for hookworm infection is different. Case report: A Belgian student, who had returned from the Democratic Republic of the Congo, was treated at the outpatient clinic of the Institute of Tropical Medicine in Antwerp, Belgium, because of an episode of fever, rash and diarrhea. Microscopic examination of a stool sample demonstrated Trichuris trichiura eggs and Strongyloides eggs, but no helminth larvae. A Strongyloides fuelleborni infection was suspected and subsequently confirmed using molecular techniques. The patient was treated with ivermectin and mebendazole. No helminth eggs could be detected in a follow-up stool sample. Conclusions: Imported human Strongyloides fuelleborni infection is very rarely reported. When hookworm-like eggs are found during parasitological stool examination, Strongyloides spp. should be kept in mind as a possibility. Careful measurement of eggs, together with embryonation level and stool sample consistency, are important criteria to ensure correct initial identification, while molecular testing can be used for confirmation. This case should raise awareness about Strongyloides fuelleborni as a possible cause of infection in travelers returning from endemic regions, something which should be kept in mind in order to avoid erroneous treatment.

BackgroundNucleic 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 findingsA 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/significanceWe 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.

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Plasmodium ovale malaria is often neglected due to its less severe course compared to Plasmodium falciparum. In 2011–2012, Belgian Armed Forces identified a cluster of P. ovale cases among military personnel after deployment in the Democratic Republic of Congo (DRC). In this retrospective, monocentric, observational study, clinical and biological features of soldiers diagnosed with P. ovale after deployment in DRC were reviewed. Species diagnosis was based on polymerase chain reaction (PCR) and/or thick blood smear. Medical records of 149 soldiers screened at the Queen Astrid Military Hospital after deployment were reviewed. Eight cases (seven P. ovale infections and one P. ovale—falciparum coinfection) were identified. All had positive thick smears, and seven were confirmed by PCR. Chemoprophylaxis was mefloquine in all subjects. Median time of disease onset was 101 days after return from the endemic region. Median delay between return and diagnosis was 103 days. All P. ovale bouts were uncomplicated. None had relapses after primaquine treatment. This military cohort highlights a hotspot of P. ovale in Eastern DRC. Non-specific symptoms, the less severe presentation, the lack of sensitive parasitological tools in the field and long delays between infection and symptoms probably lead to underestimation of P. ovale cases.

COVID-19 Antibody Detecting Rapid Diagnostic Tests (COVID-19 Ab RDTs) are the preferred tool for SARS-CoV-2 seroprevalence studies, particularly in low- and middle-income countries. The present study challenged COVID-19 Ab RDTs with pre-pandemic samples of patients exposed to tropical pathogens. A retrospective study was performed on archived serum (n = 94) and EDTA whole blood (n = 126) samples obtained during 2010–2018 from 196 travelers with malaria (n = 170), schistosomiasis (n = 25) and dengue (n = 25). COVID-19 Ab RDTs were selected based on regulatory approval status, independent evaluation results and detecting antigens. Among 13 COVID-19 Ab RDT products, overall cross-reactivity was 18.5%; cross-reactivity for malaria, schistosomiasis and dengue was 20.3%, 18.1% and 7.5%, respectively. Cross-reactivity for current and recent malaria, malaria antibodies, Plasmodium species and parasite densities was similar. Cross-reactivity among the different RDT products ranged from 2.7% to 48.9% (median value 14.5%). IgM represented 67.9% of cross-reactive test lines. Cross-reactivity was not associated with detecting antigens, patient categories or disease (sub)groups, except for schistosomiasis (two products with ≥60% cross-reactivity). The high cross-reactivity for malaria, schistosomiasis and—to a lesser extent—dengue calls for risk mitigation when using COVID-19 Ab RDTs in co-endemic regions.

Abstract Background Rickettsiosis is a potential life threatening infectious disease in travelers. Clinical recognition is not always straightforward, as typical manifestations such as rash and/or eschar may be absent. Definite diagnosis is based on seroconversion and therefore often delayed until the convalescent phase of disease. Case presentation In this case report, we describe four patients with severe travel-related rickettsiosis (two patients with murine- and two patients with scrub typhus), in whom acute- phase diagnosis was possible by real-time polymerase chain reaction on serum or blood. Conclusions Despite its limitations, we think that polymerase chain reaction can contribute significantly to the early diagnosis and treatment of rickettsial disease in travelers.

BACKGROUND:Pathogens causing acute fever, with the exception of malaria, remain largely unidentified in sub-Saharan Africa, given the local unavailability of diagnostic tests and the broad differential diagnosis. METHODOLOGY:We conducted a cross-sectional study including outpatient acute undifferentiated fever in both children and adults, between November 2015 and June 2016 in Kinshasa, Democratic Republic of Congo. Serological and molecular diagnostic tests for selected arboviral infections were performed on blood, including PCR, NS1-RDT, ELISA and IFA for acute, and ELISA and IFA for past infections. RESULTS:Investigation among 342 patients, aged 2 to 68 years (mean age of 21 years), with acute undifferentiated fever (having no clear focus of infection) revealed 19 (8.1%) acute dengue-caused by DENV-1 and/or DENV-2 -and 2 (0.9%) acute chikungunya infections. Furthermore, 30.2% and 26.4% of participants had been infected in the past with dengue and chikungunya, respectively. We found no evidence of acute Zika nor yellow fever virus infections. 45.3% of patients tested positive on malaria Rapid Diagnostic Test, 87.7% received antimalarial treatment and 64.3% received antibacterial treatment. DISCUSSION:Chikungunya outbreaks have been reported in the study area in the past, so the high seroprevalence is not surprising. However, scarce evidence exists on dengue transmission in Kinshasa and based on our data, circulation is more important than previously reported. Furthermore, our study shows that the prescription of antibiotics, both antibacterial and antimalarial drugs, is rampant. Studies like this one, elucidating the causes of acute fever, may lead to a more considerate and rigorous use of antibiotics. This will not only stem the ever-increasing problem of antimicrobial resistance, but will-ultimately and hopefully-improve the clinical care of outpatients in low-resource settings. TRIAL REGISTRATION:ClinicalTrials.gov NCT02656862.

Visceral pentastomiasis is usually found incidentally during surgery. We describe a case of visceral pentastomiasis discovered during inguinoscrotal hernia surgery for a man from Benin, Africa. Because surgical removal of nymphs is needed for symptomatic patients only, this patient’s asymptomatic pentastomiasis was not treated and he recovered from surgery uneventfully.

Background: Strongyloides stercoralis, a nematode endemic in all (sub)tropical regions, can cause life-threatening disease, especially in immunosuppressed patients. Many countries with high HIV-prevalence rates are also highly S. stercoralis endemic, and co-infection may occur. Methods: Retrospective study based on serological testing for S. stercoralis in all HIV-infected migrants followed at the Institute of Tropical Medicine, Antwerp, Belgium. If untested, serologic testing was performed on stored samples, dating from the first HIV viral load determination. The epidemiological, clinical and laboratory features of patients with and without strongyloidiasis were retrieved from the electronic medical files. Results: Of the 2846 HIV patients in active follow-up, 723 (25.4%) had a migration background. Thirty-six patients (5.1%) were diagnosed with Strongyloides co-infection, including 29 during their medical evaluation and seven retrospectively. Patients had a median age of 35.3 years (IQR 30.3–44.4), 28 patients (78%) originated from Sub-Saharan Africa and median time in Belgium was 3.5 years (IQR 0.8–5.7). Symptoms compatible with strongyloidiasis were present in 17 (47%) patients, of whom two were diagnosed retrospectively. Eosinophilia (eosinophil cell count > 450/µL) was observed in 19 (53%) participants. Median CD4 level was 386 /µL (IQR 299–518) at diagnosis of co-infection. Of note, 8 (22%) patients with strongyloidiasis had no reported symptoms nor eosinophilia. None of the patients developed hyperinfection syndrome. There were no differences in age, gender, geographic origin, clinical presentation, CD4 level or viral load between patients with and without strongyloidiasis. Only eosinophilia was strongly correlated with the presence of Strongyloides in multivariate analysis (OR 10.74 (95% CI 5.19–22.25), p < 0.001); the positive likelihood ratio (LR+) of eosinophilia for strongyloidiasis was 5.38 (95% CI 3.66–7.91). Conclusion: Strongyloidiasis was diagnosed in 5.1% of HIV-infected migrants. Eosinophilia had good confirming power for the presence of the disease. However, a sizeable proportion (22%) of co-infected individuals were asymptomatic and had normal eosinophil count, supporting universal screening of all HIV-positive patients native to tropical countries.

The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN® Hantavirus Puumala IgM/IgG ELISA assay (Darmstadt, Germany). Methods: The sensitivity was evaluated with a panel of 68 samples from patients with an acute infection (n = 44) or a past infection (n = 24). Specificity was evaluated with a panel of 62 samples from patients with potentially false borderline results (n = 7) (no seroconversion), seronegative samples (n = 25) and potentially cross reacting samples (n = 30). Discordances were resolved by immunoblot. Substantial agreement was calculated using Cohen kappa coefficient. Results: The RIDASCREEN® kit showed a higher specificity (IgM: 94.3%; IgG: 94.4%) than the Progen kit (IgM: 77.0% IgG: 93.0%). The sensitivity for IgM ELISA was 100% for both assays. IgG sensitivity was, respectively, 98.3% and 100% for Progen and RIDASCREEN®. A Cohen kappa coefficient of 0.76 and 0.90 was found between Puumala IgM and IgG, respectively. Conclusions: This study showed a higher specificity for the RIDASCREEN® kit than the Progen kit, while the sensitivity was as good as for the Progen kit.

BACKGROUND:Amebiasis is a protozoal infection caused by Entamoeba histolytica, while the morphologically indistinguishable E. dispar is considered as non-pathogenic. Polymerase chain reaction (PCR) assays are necessary to differentiate both species. The most common clinical presentations of E. histolytica disease are amebic colitis and amebic liver abscess, but asymptomatic infection is also possible. We assessed the frequency and pattern of clinical symptoms and microscopic features in travelers/migrants associated with E. histolytica intestinal infection and compared them to those found in individuals with E. dispar infection. METHODS:We conducted a retrospective study at the travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium on travelers/migrants found from 2006 to 2016 positive for Entamoeba histolytica/dispar through antigen detection and/or through microscopy confirmed by PCR. All files of individuals with a positive PCR for E. histolytica (= cases) and a random selection of an equal number of Entamoeba dispar carriers (= controls) were reviewed. We calculated the sensitivity, specificity and likelihood ratios (LRs) of clinical symptoms (blood in stool, mucus in stool, watery diarrhea, abdominal cramps, fever or any of these 5 symptoms) and of microscopic features (presence of trophozoites in direct and in sodium acetate-acetic acid-formalin (SAF)-fixed stool smears) to discriminate between E. histolytica and E. dispar infection. RESULTS:Of all stool samples positive for Entamoeba histolytica/dispar for which PCR was performed (n = 810), 30 (3.7%) were true E. histolytica infections, of which 39% were asymptomatic. Sensitivity, specificity and positive LRs were 30%, 100% and 300 (p 0.007) for presence of blood in stool; 22%, 100% and 222 (p 0.03) for mucus in stool; 44%, 90% and 4.7 (p 0.009) for cramps and 14%, 97% and 4.8 (p = 0.02) for trophozoites in direct smears. For watery diarrhea, fever and for trophozoites in SAF fixated smears results were non-significant. CONCLUSIONS:E. histolytica infection was demonstrated in a small proportion of travelers/migrants with evidence of Entamoeba histolytica/dispar infection. In this group, history of blood and mucus in stool and cramps had good to strong confirming power (LR+) for actual E. histolytica infection. Trophozoites were also predictive for true E. histolytica infection but in direct smears only.

BACKGROUND:Chikungunya virus (CHIKV) emerged in Aruba for the first time in 2014. We studied the clinical presentation of acute CHIKV infection and the contribution of serologic and molecular assays to its diagnosis. In a cohort of confirmed CHIKV cases, we analysed the frequency, duration and predictors of post-chikungunya chronic polyarthralgia (pCHIK-CPA), defined as joint pains lasting longer than 6 weeks or longer than 1 year. METHODOLOGY:Patient sera obtained within 10 days of symptom onset were tested for CHIKV, using an indirect immunofluorescence test for the detection of CHIKV-specific Immunoglobulin M (IgM) and post-hoc, by reverse-transcription polymerase chain reaction (RT-PCR). CHIKV was isolated from selected samples and genotyped. For confirmed CHIKV cases, clinical data from chart review were complemented by a Telephone survey, conducted 18-24 months after diagnosis. When joint pain was reported, the duration, presence of inflammatory signs, type and number of joints affected, were recorded. Joint involvement was scored according to the 2010 'American College of Rheumatology/ European League Against Rheumatism' criteria for seronegative rheumatoid arthritis (ACR-score). Risk factors for pCHIK-CPA were identified by logistic regression. PRINCIPAL FINDINGS:Acute CHIKV infection was diagnosed in 269 of 498 sera, by detection of IgM (n = 105), by RT-PCR (n = 59), or by both methods (n = 105). Asian genotype was confirmed in 7 samples. Clinical data were complete for 171 of 248 (69.0%) patients, aged 15 years or older (median 49.4 [35.0-59.6]). The female-to-male ratio was 2.2. The main acute symptoms were arthralgia (94%), fever (85%), myalgia (85%), headache (73%) and rash (63%). In patients with arthralgia (n = 160), pCHIK-CPA longer than 6 weeks was reported by 44% and longer than 1 year by 26% of cases. Inflammatory signs, stiffness, edema and redness were frequent (71%, 39% and 21%, respectively). Joints involved were knees (66%), ankles (50%), fingers (52%), feet (46%), shoulders (36%), elbows (34%), wrists (35%), hips (31%), toes (28.1%) and spine (28.1%). Independent predictors of pCHIK-CPA longer than 1 year were female gender (OR 5.9, 95%-CI [2.1-19.6]); high ACR-score (7.4, [2.7-23.3]), and detection of CHIKV-RNA in serum beyond 7 days of symptom onset (6.4, [1.4-34.1]. CONCLUSIONS:We identified 269 CHIKV patients after the first outbreak of Asian genotype CHIKV in Aruba in 2014-2015. RT-PCR yielded 59 (28%) additional CHIKV diagnoses compared to IgM antibody detection alone. Arthralgia, fever and skin rash were the dominant acute phase symptoms. pCHIK-CPA longer than 1 year affected 26% of cases and was predicted by female gender, high ACR-score and CHIKV-RNA detection beyond 7 days of symptom onset.

INTRODUCTION:In non-endemic settings, expertise in malaria microscopy is limited and rapid diagnostic tests (RDTs) are an adjunct to malaria diagnosis. AIM:We performed an External Quality Assessment (EQA) on reading and interpretation of malaria RDTs in a non-endemic setting. METHODS:Participants were medical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs; they received (i) 10 high-resolution photographs presenting test line combinations of RDTs with interpretations listed in a multiple choice format and (ii) a questionnaire about their practices of malaria diagnosis. RESULTS:Among 135 subscribing laboratories, 134 (99.3%) used 139 RDT products (11 different products from 8 brands). After exclusion of the results of one laboratory, analysis was done for 133 laboratories using 137 RDT products. Scores of 10/10, 9/10 and 8/10 were achieved for 58.4%, 13.1% and 8.0% of 137 RDT products respectively. For three-band P. falciparum-pan-Plasmodium RDTs (113 (82.5%) products, 6 brands), most frequent errors were (1) disregarding faint test lines (18.6%), (2) reporting invalid instead of P. falciparum (16.8%) and (3) reporting "Plasmodium spp., no further differentiation possible" without mentioning the presence or absence of P. falciparum (11.5%). For four-band RDTs (21 (15.3%) products, 1 brand), errors were (4) disregarding faint P. vivax test lines (47.6%) and (5) reporting "Plasmodium spp., no further differentiation possible" without mentioning the presence of P. falciparum and P. vivax (28.6%). Instructions for use (IFU) of only 4 out of 10 RDT products mentioned to interpret faint-intensity test lines as positive (conducive to errors 1 and 4) and IFU of 2 products displayed incorrect information (conducive to errors 2 and 5). Outside of office hours, 36.1% of participants relied on RDTs as the initial diagnostic test; 13.9% did not perform microscopic confirmation. CONCLUSION:Reading and interpretation of malaria RDTs was satisfactory, but errors were embedded in the instructions for use of the products. Relying on RDTs alone for malaria diagnosis (about one third of participants) is not a recommended practice.

OBJECTIVES:The aim of this study was to characterize by classical biotyping and Multi-Locus variable number tandem repeats (VNTR) Analysis (MLVA) all Brucella spp. derived from human cases in Belgium from 1996 to 2015. Final goals were to determine the species and biovar, to trace-back on genetic grounds the origin of each strain when patient history and risk factors were missing, and to survey for particular trends at the national level. METHODS:A total of 37 Brucella strains, isolated from 37 patients in Belgium, were analyzed by both classical biotyping and MLVA, and the genetic patterns compared to those of human strains isolated worldwide. RESULTS:Classical biotyping revealed that isolates were mainly Brucella melitensis. Most of them belonged to biovar 3, the most abundant biovar in the Mediterranean region. MLVA confirmed that Brucella melitensis is too diverse in VNTRs to be able to make clusters associated to each biovar, but it allowed retrieving precious epidemiological information. The analysis highlighted the imported nature of the strains from all over the world with a dominant part from the Mediterranean countries. Findings of the MLVA11 testing were in line with the travel history of patients coming from Italy, Turkey, Lebanon and Peru. The analysis was particularly useful because it suggested the geographical origin of the infection for 12/16 patients for whom no case history was available. CONCLUSION:Classical biotyping and MLVA analysis are not exclusive but remain complementary tools for Brucella melitensis strain surveillance. MLVA11 is sufficient for Brucella-free countries such as Belgium to trace the geographical origin of infection, but complete MLVA16 is needed to search for links with endemic areas.

Chikungunya fever has spread through several Indian Ocean islands and India, including popular travel destinations. To compare usefulness of diagnostic tests and to understand reasons for the magnitude and severity of an outbreak, we used 3 diagnostic methods to test 720 samples from 680 patients returning to Europe from the Indian Ocean region in 2006. Chikungunya infection was confirmed for 24.4% patients in the first half of the year and for 9.9% in the second half. Reverse transcription–PCR was positive for all samples taken up to day 4 after symptom onset. Immunofluorescence detected immunoglobulin (Ig) M on day 1 and IgG on day 2 for some patients, and in all patients from day 5 onward. Soon after onset of symptoms, patients had IgG and IgM and high viral loads (some >109 copies/mL plasma). These data will help healthcare providers select diagnostic tests for returning travelers.

Although infection with Toxocara canis or T. catis (commonly referred as toxocariasis) appears to be highly prevalent in (sub)tropical countries, information on its frequency and presentation in returning travelers and migrants is scarce. In this study, we reviewed all cases of asymptomatic and symptomatic toxocariasis diagnosed during post-travel consultations at the reference travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium. Toxocariasis was considered as highly probable if serum Toxocara-antibodies were detected in combination with symptoms of visceral larva migrans if present, elevated eosinophil count in blood or other relevant fluid and reasonable exclusion of alternative diagnosis, or definitive in case of documented seroconversion. From 2000 to 2013, 190 travelers showed Toxocara-antibodies, of a total of 3436 for whom the test was requested (5.5%). Toxocariasis was diagnosed in 28 cases (23 symptomatic and 5 asymptomatic) including 21 highly probable and 7 definitive. All but one patients were adults. Africa and Asia were the place of acquisition for 10 and 9 cases, respectively. Twelve patients (43%) were short-term travelers (< 1 month). Symptoms, when present, developed during travel or within 8 weeks maximum after return, and included abdominal complaints (11/23 symptomatic patients, 48%), respiratory symptoms and skin abnormalities (10 each, 43%) and fever (9, 39%), often in combination. Two patients were diagnosed with transverse myelitis. At presentation, the median blood eosinophil count was 1720/μL [range: 510-14160] in the 21 symptomatic cases without neurological complication and 2080/μL [range: 1100-2970] in the 5 asymptomatic individuals. All patients recovered either spontaneously or with an anti-helminthic treatment (mostly a 5-day course of albendazole), except both neurological cases who kept sequelae despite repeated treatments and prolonged corticotherapy. Toxocariasis has to be considered in travelers returning from a (sub)tropical stay with varying clinical manifestations or eosinophilia. Prognosis appears favorable with adequate treatment except in case of neurological involvement.

BACKGROUND: Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence. METHODOLOGY/PRINCIPAL FINDINGS: The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces). CONCLUSION/SIGNIFICANCE: The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.