Advances toward diagnostic tools for managing zoonotic visceral leishmaniasis

Visceral leishmaniasis (VL) is a life-threatening outcome of Leishmania infantum or Leishmania donovani infection. Dogs are the primary domestic reservoir of L. infantum parasites, and ownership of infected dogs increases the risk of human VL. Controlling infection within dog populations is regarded as critical to VL management in endemic countries, both preventing progression of canine disease and limiting parasite transmission to humans and dogs. Here we discuss various strategies that are used to diagnose canine visceral leishmaniasis (CVL) and the possibilities of adapting these for use within population screening and control programs. In addition, given the variable transmissibility of L. infantum to the sand fly vector, we outline some possibilities for the preferential identification of ‘super-spreader’ dogs among the overall infected population

Visceral Leishmaniasis’li Köpeklerde Dermal Lezyonların Biyofiziksel Muayenesi

Bu tez çalışmasında Leishmaniasisli hayvanlarda derinin korneometrik analizleri yapılıp hastalığın deriyi ne oranda etkilediği tespit edilerek uygulanacak sağaltım modülüne ışık tutması amaçlandı. Adnan Menderes Üniversitesi Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Kliniğine Canine Visceral Leishmaniasis pozitif (n=28) ve sağlıklı (n=10) olan farklı ırk, yaş ve cinsiyette toplamda 38 köpek çalışma kapsamına alındı. Leishmaniasis tanısında klinik bulgular ile birlikte hızlı test kitleri (ELISA), IFAT ve PZR yöntemleri kullanıldı. Evrelendirme Leishvet grubunun rehber bülteni ile uyumlu olarak idrar protein/ kreatinin ve kan kreatinin düzeyleri dikkate alınarak yapıldı. Çalışmaya alınan CVL’li köpekler 4 farklı gruptan birinde (her grupta n=7) değerlendirildi. Buna göre; I. grup: evre 1 (hafif), II. grup: evre 2 (orta şiddetli), III. grup: evre 3 (şiddetli), IV. grup: evre 4 (çok şiddetli) olarak değerlendirildi. Sağlıklı kontrol grubu (V. grup) ise ADÜ Veteriner Fakültesi İç Hastalıkları Anabilim Dalı Küçük Hayvan Kliniğine rutin kontrol amaçlı getirilen, klinik ve laboratuvar bulgularında herhangi bir anormallik saptanmayan farklı ırk, yaş ve cinsiyette köpeklerden (n=10) oluştu. Sağlıklı ve hasta hayvanlarda derinin hidrasyonu, elastikiyeti, sıcaklığı ve melanin miktarı ölçülerek biyofiziksel muayene gerçekleştirildi. Elde edilen verilerin istatistiksel analizleri sonucunda sağlıklı köpeklere göre tüm evrelerdeki Leishmaniasis’li köpeklerde pH değerinde azalma belirlenirken (p0.05). Sonuç olarak Visceral Leishmaniasis saptanan köpeklerde dermal lezyonların biyofiziksel muayenesinin önem teşkil ettiği ve uygulanacak sağaltım modülüne ışık tutabileceği öne sürülebilir.KABUL VE ONAY SAYFASI i TEŞEKKÜR ii İÇİNDEKİLER iii SİMGELER VE KISALTMALAR DİZİNİ vi ŞEKİLLER DİZİNİ ix RESİMLER DİZİNİ x TABLOLAR DİZİNİ xi ÖZET xii ABSTRACT xiii 1.GİRİŞ 1 2.GENEL BİLGİLER 3 2.1. Yaşam Döngüsü 3 2.2. Dağılım ve Epidemiyoloji 5 2.3. Klinik Bulgular ve Laboratuvar Anormallikleri 6 2.3.1. Dermatolojik Bulgular 10 2.4. Tanı 12 2.4.1. Moleküler Tanı 16 2.4.1.1. Parazit hedefleri 16 2.4.1.2. Numune kaynağı 19 2.4.1.3. POC (Point of care) moleküler testleri 19 2.4.2. Serolojik Tanı 21 2.4.2.1. Serodiyagnoz için kullanılan antijenler 22 2.5. Hastalığın Evrelendirmesi 24 2.5.1. Evre-1 (Hafif Şiddetli Hastalık) 24 2.5.2. Evre-2 (Orta Şiddetli Hastalık) 24 2.5.3. Evre-3 (Şiddetli Hastalık) 25 2.5.4. Evre-4 (Çok Şiddetli Hastalık) 26 2.6. Sağaltım ve Prognoz 26 2.7. Deri Muayenesine Ait Fiziksel Biyobelirteçler 27 2.7.1. Deri Nemi 27 2.7.2. Deri pH 28 3. GEREÇ VE YÖNTEM 29 3.1. Gereç 29 3.1.1.Hayvan Materyali 29 3.2. Yöntem 29 3.2.1. Gruplandırma Protokolü 29 3.2.2. Çalışmada Kullanılan Laboratuvar Yöntemleri 31 3.2.2.1. İndirekt floresan antikor testi (IFAT) 32 3.2.2.1.1. Leishmania IgG IFAT içeriği: 32 3.2.2.1.2. Test prosedürü ve uygulanması 33 3.2.2.1.3. Sonuçların yorumu 34 3.2.2.2. Serum biyokimyasal analizler 34 3.2.2.3. İdrar analizleri 35 3.2.2.4. Polimeraz zincir reaksiyonu 35 3.2.3. Derinin Biyofiziksel Muayenesi 36 3.2.3.1 Hidrasyon 37 3.2.3.2. Elastikiyet 38 3.2.3.3. Melanin 39 3.2.3.4. Sıcaklık 40 3.2.3.5. pH 41 3.2.4. İstatistiksel Analizler 42 4.BULGULAR 43 4.1. Sağlıklı ve Evrelendirme Yapılan Olgularda Demografik Bulgular 43 4.2. Dermatolojik Bulgular 44 4.2.1. V.Grup (Sağlıklı- Kontrol) 44 4.2.2. I.Grup (Evre 1 CVL) 45 4.2.3. II. Grup (Evre 2 CVL) 46 4.2.4. III. Grup (Evre 3 CVL) 47 4.2.5. IV. Grup (Evre 4 CVL) 48 4.3. Laboratuvar Bulguları 49 4.3.1. IFAT Bulguları 49 4.3.2 ELISA ve PZR Bulguları 50 4.3.3 Biyokimyasal Bulgular 50 4.3.4. İdrar Analiz Bulguları 51 4.4. Biyofiziksel Muayene Bulguları 51 5.TARTIŞMA 56 6. SONUÇ VE ÖNERİLER 64 KAYNAKLAR 65 EKLER 90 ÖZGEÇMİŞ 9

Development of new diagnostic tools to identify canine reservoir super-spreaders of Leishmania infantum

Zoonotic visceral leishmaniasis (ZVL) is a vector-borne infection induced by protozoan parasite Leishmania infantum and transmitted from animal reservoir to human. Domestic dogs are the main proven reservoir, and the detection of their transmission potential is a research priority. Longitudinal xenodiagnosis studies of natural infection in dogs showed that a large fraction of transmission events to the sand fly vector is due to a small fraction of the infected canine reservoir population, known as superspreaders. The management of visceral leishmaniasis requires a different approach to current blanket control operations that otherwise require extremely high intervention coverage to successfully include the super-spreaders. The aims of the study were to discriminate super-spreaders in a mixed reservoir population by developing novel diagnostic tools, and to complete mathematical models based on collected data including transmission potential and tool-implementation in the field. Existing and novel anti-Leishmania antigens were tested in enzyme-linked immunosorbent assays (ELISA) on archived sera collected from a naturally infected cohort population of Brazilian dogs. Their transmission potential was measured by xenodiagnoses during a two years longitudinal study. Results from serological assays showed that carefully selected threshold-based antigens allowed a more specific test towards reducing transmission; and some of the novel proteins (rK28, K26, rK34) out-performed the currently available test antigens for infection. These antigens were tested for the serodetection of infectiousness and were able to discriminate super-spreaders of Leishmania within the mixed canine population. Based on these results, a prototype of rapid diagnostic test (RDT) was developed based on a brand-new antigen, KL914, and specifically designed for detecting superspreaders. The aim was to setup a field-friendly screening method. The impact of the novel diagnostic tool to detect and remove super-spreaders from the population before the onset of infectiousness was modelled and quantified under different population dynamic scenarios. The mathematical model offered a notice on the diagnostic tool to be applied in the field, and pointed out the limitations and the possible improvement. This project was funded by the European Union’s Horizon2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement

Aplicação do antígeno recombinante LBK39 e seus respectivos peptídeos PepLi379 e PepLi409 sintéticos no diagnóstico e protocolos vacinais das leishmanioses

Orientadora: Profa. Dra. Vanete Thomaz SoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 28/11/2020Inclui referênciasResumo: A Leishmaniose visceral possui importância mundial por ser uma zoonose. Esta doença possui como reservatório principalmente os cães e é causada Leishmania infantum. Os cães possuem papel importante na manutenção do parasito e infecção em humanos. Esses animais são capazes de se moverem por diversas regiões, o que contribui para a dispersão do parasito. Há cerca 1,3 milhão de novos casos/ano, ocasionando de 20 a 30 mil mortes. Os testes diagnósticos utilizados atualmente apresentam baixa sensibilidade e especificidade, e não são capazes de detectar presença da doença em fases assintomáticas, apresentando falsos negativos. Para contribuir com a melhoria e acesso ao diagnóstico nosso grupo desenvolveu previamente um novo antígeno recombinante relacionado à cinesina de Leishmania braziliensis, a Lbk39, no qual mostrou 59% de aminoácidos idênticos à L. infantum. A proteína foi testada como antígeno em diagnóstico de leishmaniose visceral canina. A Lbk39 foi sintetizada utilizando o vetor pLEXSY-sat2 e transfectada em células de Leishmania tarentolae por eletroporação. No presente trabalho, tanto a Lbk39 quanto seus peptídeos PepLi379 e PepLi409 foram utilizados como antígenos e determinadas a sensibilidade e especificidade dos mesmos. A Lbk39 apresentou 100% de sensibilidade e 96,1% de especificidade, e seus peptídeos, PepLi379 e PepLi409, 95% e 87,8% de sensibilidade, e 100% e 95% de especificidade, respectivamente. Mostrando que ambos os antígenos têm potencial para serem utilizados em testes para diagnóstico em cães. Com isso, tanto a Lbk39 quanto seus peptídeos apresentaram potenciais para serem utilizados em diagnóstico de leishmaniose visceral canina. Em relação a leishmaniose humana visceral, os peptídeos, PepLi379 e PepLi409, obtiveram 41,7 e 83,3% de sensibilidade e 95,8% e 100% de especificidade. Já para leishmaniose cutânea humana, apresentaram 42,9 e 93,5% de sensibilidade e 91,7 e 87,5% de especificidade. Mostrando que apenas o PepLi409 apresenta potencial para uso diagnóstico, tanto para leishmaniose visceral humana quanto para cutânea. A partir desses resultados, foi estudado o potencial vacinal destes peptídeos. Foram utilizados camundongos BALB/c, os quais receberam duas doses de imunização por via intranasal, com intervalo de 7 dias entre elas. Após 7 dias da última dose da vacina, os animais foram desafiados com 2x106 promastigota de L. infantum no coxim plantar da pata direita via subcutânea. O perfil clínico da doença foi verificado pela observação física e integridade dos animais. A carga parasitária do baço, do linfonodo poplíteo e do fígado foram quantificados pela técnica de diluição limitante nos dias 60 e 90 pós-desafio, com a eutanásia dos animais. O perfil das citocinas foi avaliado com o plasma dos camundongos. O grupo que recebeu as composições PepLi409 e Mix dos peptídeos, apresentaram diferença significativa na carga parasitária dos órgãos em relação ao seu controle e aos demais grupos vacinais e, a análise do perfil de citocinas no soro dos animais, mostrou um aumento da produção de IFN-?. Esses resultados sugerem que estas composições são candidatos ideias para uso vacinal contra leishmaniose humana.Abstract: Visceral Leishmaniasis has worldwide importance because it is a zoonosis. This disease has as a reservoir mainly dogs and is caused by Leishmania infantum. Dogs play an important role in maintaining the parasite and infection in humans. These animals are able to move through different regions, which contributes to the dispersion of the parasite. There are about 1.3 million new cases / year, causing 20 to 30 thousand deaths. The diagnostic tests currently used have low sensitivity and specificity, and are not able to detect the presence of the disease in asymptomatic phases, presenting false negatives. To contribute to the improvement and access to diagnosis, our group developed a new recombinant antigen related to Leishmania braziliensis kinesin, Lbk39, in which it showed 59% of amino acids identical to Leishmania infantum. The protein was tested as an antigen in the diagnosis of canine visceral leishmaniasis. Lbk39 was synthesized using the vector pLEXSY-sat2 and transfected into Leishmania tarentolae cells by electroporation. In the present work, both Lbk39 and its peptides PepLi379 and PepLi409 were used as antigens and their sensitivity and specificity were determined. Lbk39 showed 100% sensitivity and 96.1% specificity, and its peptides, PepLi379 and PepLi409, showed 95% and 87.8% sensitivity, and 100% and 95% specificity, respectively. Showing that both antigens have the potential to be used in diagnostic tests in dogs. Thus, both Lbk39 and its peptides showed potential to be used in the diagnosis of canine visceral leishmaniasis. In relation to human visceral leishmaniasis, the peptides, PepLi379 and PepLi409, obtained 41.7, 83.3% sensitivity, 95.8%, and 100% specificity. As for human cutaneous leishmaniasis, they had 42.9, 93.5% sensitivity, and 91.7 and 87.5% specificity. Showing that only PepLi409 has potential for diagnostic use, both for human visceral and cutaneous leishmaniasis. From these results, the vaccine potential of these peptides was studied. BALB / c mice were used, which received two doses of immunization intranasally, with an interval of 7 days between them. 7 days after the last dose of the vaccine, the animals were challenged with a 2x106 L. infantum promastigote in the footpad of the right leg, subcutaneously. The clinical profile of the disease was verified by the physical observation and integrity of the animals. The parasitic load of the spleen, popliteal lymph node and liver were quantified by the limiting dilution technique on days 60 and 90 post-challenge, with the euthanasia of the animals. The cytokine profile was evaluated with the plasma of the mice. The group that received the PepLi409 and Mix peptide compositions showed a significant difference in the parasitic load of the organs in relation to their control and the other vaccine groups, and the analysis of the cytokine profile in the animals' serum showed an increase in IFN-? production. These results suggest that these compositions are ideal candidates for vaccine use against human leishmaniasis