Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

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.

Human Transmission of Blastocystis by Fecal Microbiota Transplantation Without Development of Gastrointestinal Symptoms in Recipients.

BACKGROUND: Patients with multiple recurrent Clostridioides difficile infections (rCDI) are treated with fecal microbiota transplantation (FMT), using feces provided by healthy donors. Blastocystis colonization of donors is considered an exclusion criterion, whereas its pathogenicity is still under debate. METHODS: The introduction of molecular screening for Blastocystis sp. at our stool bank identified 2 donors with prior negative microscopies but positive polymerase chain reactions (PCRs). Potential transmission of Blastocystis sp. to patients was assessed on 16 fecal patient samples, pre- and post-FMT, by PCR and subtype (ST) analyses. In addition, clinical outcomes for the treatment of rCDI (n = 31), as well as the development of gastrointestinal symptoms, were assessed. RESULTS: There was 1 donor who carried Blastocystis ST1, and the other contained ST3. All patients tested negative for Blastocystis prior to FMT. With a median diagnosis at 20.5 days after FMT, 8 of 16 (50%) patients developed intestinal colonization with Blastocystis, with identical ST sequences as their respective donors. Blastocystis-containing fecal suspensions were used to treat 31 rCDI patients, with an FMT success rate of 84%. This success rate was not statistically different from patients transferred with Blastocystis sp.-negative donor feces (93%, 76/82). Patients transferred with Blastocystis sp.-positive donor feces did not report any significant differences in bowel complaints in the first week, after 3 weeks, or in the months following FMT. CONCLUSIONS: We demonstrated the first transmission of Blastocystis ST1 and ST3 from donors to patients by FMT. This did not result in gastrointestinal symptomatology or have any significant effect on rCDI treatment outcomes.

The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance. A nationwide survey and rationale for the DB-MSG 002 study protocol.

Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres.

In vivo efficacy of artesunate-amodiaquine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria: an open-randomized, non-inferiority clinical trial in South Kivu, Democratic Republic of Congo.

BACKGROUND: Between 2009 and 2012, malaria cases diagnosed in a Médecins sans Frontières programme have increased fivefold in Baraka, South Kivu, Democratic Republic of the Congo (DRC). The cause of this increase is not known. An in vivo drug efficacy trial was conducted to determine whether increased treatment failure rates may have contributed to the apparent increase in malaria diagnoses. METHODS: In an open-randomized non-inferiority trial, the efficacy of artesunate-amodiaquine (ASAQ) was compared to artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria in 288 children aged 6-59 months. Included children had directly supervised treatment and were then followed for 42 days with weekly clinical and parasitological evaluations. The blood samples of children found to have recurring parasitaemia within 42 days were checked by PCR to confirm whether or not this was due to reinfection or recrudescence (i.e. treatment failure). RESULTS: Out of 873 children screened, 585 (67 %) were excluded and 288 children were randomized to either ASAQ or AL. At day 42 of follow up, the treatment efficacy of ASAQ was 78 % before and 95 % after PCR correction for re-infections. In the AL-arm, treatment efficacy was 84 % before and 99.0 % after PCR correction. Treatment efficacy after PCR correction was within the margin of non-inferiority as set for this study. Fewer children in the AL arm reported adverse reactions. CONCLUSIONS: ASAQ is still effective as a treatment for uncomplicated malaria in Baraka, South Kivu, DRC. In this region, AL may have higher efficacy but additional trials are required to draw this conclusion with confidence. The high re-infection rate in South-Kivu indicates intense malaria transmission. Trial registration NCT02741024.

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.

Evaluation of four single-locus markers for Leishmania species discrimination by sequencing.

Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis.

Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands.

BACKGROUND: Blastocystis sp. are among the most commonly observed intestinal parasites in routine clinical parasitology. Blastocystis in humans consists of at least 9 genetic subtypes. Different subtypes of Blastocystis may be associated with differences in pathogenicity and symptomatology. METHODS: Advanced microscopy on two samples and sequence-confirmed PCR on a third sample from the same individual were used for Blastocystis diagnosis and subtype analyses on routine clinical samples in a university hospital. RESULTS: With a combined gold standard of sequence-confirmed PCR and positive advanced microscopy, 107 out of 442 (24.2%) patients were diagnosed with Blastocystis. infection, which is a high frequency of detection in comparison to previous reports from industrialized countries. The sensitivity of microscopy and sequence-confirmed PCR was 99.1% (106/107) and 96.3% (103/107), respectively.Among 103 typable samples, subtype 3 was most abundant (n = 43, 42%), followed by subtypes 1 and 2 (both n = 23, 22%), subtype 4 (n = 12, 12%), and single samples with subtypes 6 (1%) and subtype 7 (1%). The prevalence of Blastocystis infection was 38% in patients from the Department of Tropical Medicine and 18% in patients from other departments. CONCLUSIONS: A high prevalence of Blastocystis infection was found with both advanced microscopy and sequence-confirmed PCR in our patient population. Most cases were caused by subtypes ST1, ST2, ST3 and ST4. A significantly higher prevalence was found among patients with a history of recent travel to tropical countries.

Molecular detection of Plasmodium knowlesi in a Dutch traveler by real-time PCR.

Plasmodium knowlesi infection with low parasitemia presents a diagnostic challenge, as rapid diagnostic tests are often negative and identification to the species level by microscopy is difficult. P. knowlesi malaria in a traveler is described, and real-time PCR is demonstrated to support fast and reliable diagnosis and identification to the species level.

Host prediction for disease-associated gastrointestinal cressdnaviruses.

Metagenomic techniques have facilitated the discovery of thousands of viruses, yet because samples are often highly biodiverse, fundamental data on the specific cellular hosts are usually missing. Numerous gastrointestinal viruses linked to human or animal diseases are affected by this, preventing research into their medical or veterinary importance. Here, we developed a computational workflow for the prediction of viral hosts from complex metagenomic datasets. We applied it to seven lineages of gastrointestinal cressdnaviruses using 1,124 metagenomic datasets, predicting hosts of four lineages. The Redondoviridae, strongly associated to human gum disease (periodontitis), were predicted to infect Entamoeba gingivalis, an oral pathogen itself involved in periodontitis. The Kirkoviridae, originally linked to fatal equine disease, were predicted to infect a variety of parabasalid protists, including Dientamoeba fragilis in humans. Two viral lineages observed in human diarrhoeal disease (CRESSV1 and CRESSV19, i.e. pecoviruses and hudisaviruses) were predicted to infect Blastocystis spp. and Endolimax nana respectively, protists responsible for millions of annual human infections. Our prediction approach is adaptable to any virus lineage and requires neither training datasets nor host genome assemblies. Two host predictions (for the Kirkoviridae and CRESSV1 lineages) could be independently confirmed as virus-host relationships using endogenous viral elements identified inside host genomes, while a further prediction (for the Redondoviridae) was strongly supported as a virus-host relationship using a case-control screening experiment of human oral plaques.

Treatment outcome of imported cutaneous leishmaniasis among travelers and migrants infected with Leishmania major and Leishmania tropica: a retrospective study in European centers 2013 to 2019.

OBJECTIVES: Cutaneous leishmaniasis (CL) in Asia, Northern, and Sub-Saharan Africa is mainly caused by Leishmania major and Leishmania tropica. We describe and evaluate the treatment outcome of CL among travelers and migrants in Europe. METHODS: We conducted a retrospective study of parasitological confirmed CL cases caused by L. major and L. tropica during 2013-2019 in Europe. Data were collected from medical records and databases within the LeishMan network. RESULTS: Of 206 included cases of CL, 75 were identified as L. major and 131 as L. tropica. Of patients with L. tropica infection, 80% were migrants, whereas 53% of patients with L. major infection had been visiting friends and relatives. Among patients with L. tropica, 48% were younger than 15 years. Pentavalent antimony cured 73% (L. major) and 78% (L. tropica) of patients. The cure rate for intralesional administration was 86% and 67% for systemic, on L. tropica. Liposomal amphotericin B had a cure rate of 44-63%. CONCLUSION: L. major infections were mostly found in individuals visiting friends and relatives, whereas L. tropica were mainly identified in migrants. No patients with L. major relapsed. Pentavalent antimony, liposomal amphotericin B, and cryotherapy had cure rates in accordance with previous studies.

Intestinal Microbiota in Children With Symptomatic Dientamoeba fragilis Infection: A Case-control Study.

BACKGROUND: Dientamoeba fragilis in children has been associated with gastrointestinal symptoms, like abdominal pain and diarrhea. The mechanism underlying these symptoms in children with D. fragilis remains unclear. We hypothesized that concomitant microbial alterations, which have been described in other parasitic infections, may be associated with gastrointestinal symptoms in D. fragilis. METHODS: In this case-control study performed in 2 centers, 19 children referred to a pediatrician because of gastrointestinal symptoms and with a positive fecal PCR for D. fragilis were included as cases. We included 19 healthy children as controls and matched for age and gender, selected from an existing cohort of 63 children. A PCR for D. fragilis was performed on fecal samples of the 19 controls to assess D. fragilis carriership in this asymptomatic group. Microbiota was analyzed with the IS-pro technique, and the intestinal microbiota composition and diversity were compared between the 2 groups. RESULTS: Microbiota of children with D. fragilis and gastrointestinal symptoms did not significantly differ in terms of composition and diversity compared with controls, both on phylum and species level. In the asymptomatic controls, a positive fecal PCR for D. fragilis was found in 16 of 19 (84.2%). CONCLUSION: Intestinal microbiota does not seem to play a key role in the presence of clinical symptoms in children with D. fragilis. The pathogenicity of D. fragilis and pathophysiologic pathways underlying the development of gastrointestinal symptoms remains yet to be clarified.

Clinical diversity and treatment results in Tegumentary Leishmaniasis: A European clinical report in 459 patients.

BACKGROUND: Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management. METHODOLOGY: Demographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients. PRINCIPAL FINDINGS: Compared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%). CONCLUSION/SIGNIFICANCE: CL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates.

Entamoeba and Giardia parasites implicated as hosts of CRESS viruses.

Metagenomic techniques have enabled genome sequencing of unknown viruses without isolation in cell culture, but information on the virus host is often lacking, preventing viral characterisation. High-throughput methods capable of identifying virus hosts based on genomic data alone would aid evaluation of their medical or biological relevance. Here, we address this by linking metagenomic discovery of three virus families in human stool samples with determination of probable hosts. Recombination between viruses provides evidence of a shared host, in which genetic exchange occurs. We utilise networks of viral recombination to delimit virus-host clusters, which are then anchored to specific hosts using (1) statistical association to a host organism in clinical samples, (2) endogenous viral elements in host genomes, and (3) evidence of host small RNA responses to these elements. This analysis suggests two CRESS virus families (Naryaviridae and Nenyaviridae) infect Entamoeba parasites, while a third (Vilyaviridae) infects Giardia duodenalis. The trio supplements five CRESS virus families already known to infect eukaryotes, extending the CRESS virus host range to protozoa. Phylogenetic analysis implies CRESS viruses infecting multicellular life have evolved independently on at least three occasions.

Carriage of Blastocystis spp. in travellers - A prospective longitudinal study.

INTRODUCTION: A lack of prospective and longitudinal data on pre- and post-travel carriage of Blastocystis spp. complicates interpretation of a positive test post-travel. Therefore we studied dynamics of Blastocystis carriage in a cohort of Dutch travellers. METHODS: From the prospective, multicentre COMBAT study among 2001 Dutch travellers, a subset of 491 travellers was selected based on travel destination to 7 subregions (70 or 71 travellers each). Faecal samples taken directly before and after travel were screened for Blastocystis with qPCR, followed, when positive, by sequence analysis to determine subtypes. RESULTS: After exclusion of 12 samples with missing samples or inhibited qPCR-reactions, stool samples of 479 travellers were analysed. Before travel, 174 of them (36.3%) carried Blastocystis and in most of these, the same subtype was persistently carried. However, in 48/174 of those travellers (27.6%; CI95 20.8-36.6%) no Blastocystis or a different subtype was detected in the post-travel sample, indicating loss of Blastocystis during travel. Only 26 (5.4%; CI95 3.7%-8.0%) of all travellers acquired Blastocystis, including two individuals that were already positive for Blastocystis before travel but acquired a different subtype during travel. DISCUSSION: This study shows that Blastocystis carriage in travellers is highly dynamic. The observed acquisition and loss of Blastocystis could either be travel-related or reflect the natural course of Blastocystis carriage. We demonstrate that the majority of Blastocystis detected in post-travel samples were already carried before travel.

Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker.

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

Frequent occurrence of human-associated microsporidia in fecal droppings of urban pigeons in amsterdam, the Netherlands.

Human-associated microsporidia were frequently observed in fecal samples of 331 feral pigeons in Amsterdam, The Netherlands, obtained during high- and low-breeding periods. Thirty-six of 331 samples (11%) contained the human pathogens Enterocytozoon bieneusi (n = 18), Encephalitozoon hellem (n = 11), Encephalitozoon cuniculi (n = 6), and Encephalitozoon intestinalis (n = 1); 5 samples contained other microsporidia. Pigeon feces can be an important source of human microsporidian infection.

Case Report: Ocular Microsporidiosis: Case in a Patient Returning from India and Review of the Literature.

Microsporidia are protists close to the kingdom of fungi that may cause eye infections. Most cases are reported in Asia and affect both immunocompromised and immunocompetent patients. Here, we report a rare case of microsporidial keratoconjunctivitis in an immunocompetent French patient 3 weeks after returning from India. In our patient, Weber trichrome staining of conjunctival scrapings revealed rounded elements approximately 1-3 µm in size. Conventional polymerase chain reaction analysis by ribosomal RNA subunit sequencing showed 100% identity with Vittaforma corneae. Treatment by corneal debridement combined with fluoroquinolone eye drops allowed complete resolution of the lesions. Although rare, ocular microsporidiosis should be investigated in a patient who is native to Asia or has returned from an endemic area and presents with keratoconjunctivitis of undetermined etiology.