30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Prof Dr

Diagnostic value of PCR-based detection of Sarcoptes scabiei in scabies outbreak situations

Abstract text Introduction: Scabies, a dermatological condition caused by reactions to the mite Sarcoptes scabiei, is an important public health issue in institutions such as nursing/ residential homes, childcare facilities, prisons and hospitals. Scabies outbreaks occurring among patients, visitors and staff are common, subject to diagnostic delay and hard to control. Verifying the clinical suspicion remains highly challenging. Time-consuming microscopic examination and dermoscopy are recommended to visualize the parasite to verify human scabies. However, microscopy has a suboptimal sensitivity, dermoscopy has low specificity and is affected by the dermatologist"s experience. Objectives: To evaluate a new laboratory-based molecular assay as a diagnostic method for scabies, we compared PCR to microscopy and dermoscopy in a prospective study. Patients &amp; Methods: 163 symptomatic patients, aged 9 weeks to 99 years, and 369 asymptomatic contacts were examined. All patients underwent clinical and dermoscopic examination. To obtain skin scales, adhesive tape was applied to suspicious lesions before and after skin scraping. Tapes were mounted onto slides to enable microscopy prior to DNA extraction and Sarcoptes scabiei real-time PCR targeting the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Specificity of the PCR assay was verified by using DNA extracted from other common mite species. Results: Mite or mite products were detected by microscopy in only 8% of the patients. By dermoscopy, mites were detected in 88% (95% CI: 82·3 – 93·0) and Sarcoptes scabiei DNA was detected by PCR in 87% of patients (95% CI: 80·0 – 91·4). No cross-reactivity to other relevant mites was detected. Scabies PCR offers an improvement in sensitivity to verify scabies compared to microscopy and performed equivalently to dermoscopy (p-value 0·628). Skin scraping before applying adhesive tape significantly increased success (87% vs 56%; p &lt;0·001). Conclusion: Our PCR-based method from skin scrapings is nontraumatic, non-expert-dependent and reproducible. S. scabiei PCR is specific, sensitive and as successful as dermoscopy, but requires no specific equipment or expertise on-site. This PCR-based diagnostic method has the potential to accelerate diagnosis and treatment, ultimately limiting or preventing scabies outbreaks in care homes or other close communal environments. PCR-based verification can also be used as an objective diagnostic criterion for scabies in therapeutic trials.

Clinical Parasitology

Emerging Parasitic Diseases

Talk

Prof.

Improved Sero-Diagnosis of Visceral Leishmaniasis in East Africa and development of a new therapy by testing the Leishmania proteasome as target molecule

Abstract text Reliable and field suitable sero-diagnosis of visceral Leishmaniasis (VL) in East Africa is till today a big challenge due to low sensitivity and crossreactivity with other pathogens. To improve VL sero-diagnosis, new and field suitable POC tests were developed on the basis of a recombinant kinesin antigen from L. infantum (rKLi8.3). The new rkLi8.3 based sero-diagnostic devices revealed improved sensitivity and no cross-reactivity when tested with sera from patients originating from Sudan, India and South America, suffering from VL, malaria, tuberculosis and trypanosomiasis. We could show that rKLi8.3 based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other endemic areas, compared to currently commercial available sero-diagnostic tests. Besides diagnosis, the current treatment of visceral leishmaniasis is also far from optimal, as many drugs induce resistent Leishmania strains and have severe side effects. It has been shown that the family of kinetoplastida (T. brucei, T. cruzi and Leishmania spp.) are highly susceptible to the proteasome inhibitor, GNF-6702. Characteristic sequence variations within the proteasome beta 4 and beta 5 subunits of kinetoplastida have been made responsible for the specific binding of GNF-6702 to the proteasome of kinetoplastia but not humans. To test whether these proteasome subunits are conserved throughout various Leishmania isolates and differ from the human proteasome, we analyzed the beta 4 and beta 5 proteasome subunits of 15 different Leishmania strains and found conserved AA substitutions in the beta 4 proteasome subunit of all strains analayzed, indicating that GNF-6702 is a potential drug for VL. Further, analyzing the parasite survival by treating pro-and amastigote L. donovani with inhibitors for constitutive - (GNF-6702) or immuno-proteasomes (ONX-0914) revealed that ONX-0914 had no effect on promastigote parasites but strong effects on amastigotes. This finding strongly suggests that the pressure of the intracellular lifestyle alters the proteasome composition from a constitutive to an immuno-like form. We are currently characterizing the proteasome subunit composition of pro-and amastigote Leishmania.

Drug Development/Target Identification

Current progress in reverse vaccinology towards in silico vaccine discovery for parasitic diseases

Abstract text Introduction Reverse vaccinology (RV) was first described in 2000 as an in silico process that starts from the genomic sequence of the pathogen and ends with a list of potential vaccine candidates. Subsequently, other in silico related processes with overlapping workflows have also emerged with terms such as subtractive proteomics, computational vaccinology, and immunoinformatics. From the perspective of a new RV practitioner, determining the appropriate workflow steps and bioinformatics tools is a time consuming and overwhelming task. Objectives We document the current knowledge on RV and its usage in the research community by a comprehensive survey of scientific papers published with "reverse vaccinology" (RV) in the title from 2015 to 2021. Materials &amp; methods All papers published with "reverse vaccinology" in their title were manually reviewed (140 papers in total, source: Web of Science). There were, however, 205 additional papers from the same period with RV in the abstract but not in the title. We made no attempt to capture current RV status in an unknown number of papers using an in silico vaccine discovery approach but with no reference to RV in the title or abstract. Results 287 different bioinformatics tools were used in one or more of the RV workflows and some are more popular than others. 95.6% of the workflows rely on online tools. The RV pipelines developed so far mainly predict proteins naturally exposed to the immune system. The workflow for selecting candidates in 87.8% of the latest publications involves a consecutive filtering process and not machine learning (ML). To automate the RV process, software pipelines were developed and made freely available since 2006. There are currently 11 known RV-related pipelines and almost all of them perform candidate ranking by ML for this purpose. They are not frequently used. Currently, only 12.2% of the latest publications report tests on animal models providing a measure of success on whether an RV-derived candidate induces a protective response. Conclusion The majority of surveyed publications use classical RV as only one stage in a broader series of workflow stages to computationally identify vaccine candidates. Installation of a standalone program and/or adapt an API is a major disincentive to RV practitioners. We recommend "in silico vaccine discovery" should be consistently used in titles, abstracts, and/or keywords of future publications to unify the discipline area of RV.

Diagnosis and Vaccinatio

Molecular Parasitology

Use of species-customized Rat Basophilic Leukemia (RBL) IgE reporters for diagnosis of Echinococcus granulosus infection and Taenia solium cysticercosis

Abstract text Echinococcus granulosus is the causative agent of cystic echinococcosis in humans infected as accidental hosts. Similarly, cysticercosis is caused by accidental ingestion of Taenia solium eggs by pigs and humans. Diagnosis is usually achieved using computerized imaging technologies, complemented by serological analyses. However, the latter have serious limitations in terms of specificity and, to a lesser extent, sensitivity. Even though Echinococcus infection induces a strong IgE response in infected hosts including human, this isotype is currently underexploited by current serological diagnostic techniques, which are mostly based on detection of parasite-specific IgG. Here, we show proof-of-principle that humanized IgE reporter systems can be used advantageously for diagnosis of cystic echinococcosis. We introduce our new RBL NPY-mRFP reporter system, which requires neither expensive substrates nor overnight incubation for detection of activation. We are also adapting the reporter systems for detection of IgE in dogs and pigs. Our data obtained using raw cyst fluid as antigen demonstrate the high discriminating power of IgE-based reporter systems for cystic echinococcosis diagnosis. We are currently employing several immunological and bioinformatic techniques, together with recombinant expression and protein purification, to identify known and novel Echinococcus granulosus and Taenia solium allergens in cyst fluid and oncospheres and assessing their suitability as diagnostic antigens using our reporter systems.

Session XIV
Diagnosis, Vacination and Clinical Parasitology

Appointment

Chairs

Programme

Session XIVDiagnosis, Vacination and Clinical Parasitology

Appointment

Chairs

Programme

Session XIV
Diagnosis, Vacination and Clinical Parasitology