scholarly journals A comparative study of nested-PCR and direct agglutination test (DAT) for the detection of Leishmania infantum infection in symptomatic and asymptomatic domestic dogs

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yonas Yimam Ayene ◽  
Mehdi Mohebali ◽  
Homa Hajjaran ◽  
Behnaz Akhoundi ◽  
Saeedeh Shojaee ◽  
...  

Abstract Objective Canine visceral leishmaniasis (CVL) is the main source of human visceral leishmaniosis (HVL) in Mediterranean region, including Iran and is spread from domestic dogs to Phlebotomine sand flies vectors to humans. To control the transmission of HVL, early and accurate detection of infected dogs is paramount importance despite it remains a confronting challenge. Herein, we evaluated the performance of direct agglutination test (DAT) against gold standard nested polymerase chain reaction (nested-PCR) for CVL diagnosis in symptomatic and asymptomatic domestic dogs from endemic areas of Iran. Results Venous blood samples were collected from dogs without clinical signs (n  =  30) and with clinical signs (n  =  35) suggestive of Leishmania infantum infection. Among 65 samples examined, Leishmania DNA was detected by nested-PCR in 89.23% (58/65). Furthermore, 86.15% (56/65) nested-PCR positive samples were also DAT positive. The results of the DAT sensitivity test were 96.43% and 96.67% in symptomatic and asymptomatic dogs, respectively, while the specificity was 100.00% and 60.00% in symptomatic and asymptomatic dogs, respectively. The results of this study also pointed out substantial concordance between DAT test and nested-PCR method in both symptomatic dogs (Κ  =  0.783; P  <  0.001) and asymptomatic dogs (Κ  =  0.618; P  <  0.001). Thus, DAT represents as a simple and economic tool for initial diagnosis of CVL particularly in endemic areas of the disease.

2015 ◽  
Vol 10 (3) ◽  
pp. 102-110
Author(s):  
H. Nahrevania ◽  
P. Ghaffarine ◽  
M. Farahmand ◽  
M. Mohebali ◽  
F. Zaboli ◽  
...  

Author(s):  
Bibi Razieh HOSSEINI FARASH ◽  
Mehdi MOHEBALI ◽  
Bahram KAZEMI ◽  
Abdolmajid FATA ◽  
Reza RAOOFIAN ◽  
...  

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). Conclusion: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended


2021 ◽  
Vol 49 ◽  
Author(s):  
Adam Leal-Lima ◽  
Tiago Cunha Ferreira ◽  
Társsila Mara Vieira Ferreira ◽  
Pedro Covas Coelho ◽  
Diana Célia Sousa Nunes-Pinheiro

 Background: Canine vector borne diseases (CVBD) are common in tropical countries where the climate favors arthropods abundance. Comorbidity with one or more CVBD are reported as clinical complication and worsen prognostic. Canine visceral leishmaniosis (CanL) is an endemic zoonotic disease in Brazil caused by Leishmania infantum, with several restrictions to canine treatment and suggestion of reservoirs euthanasia for disease control. Heart worm (HW) is a helminthic disease caused by Dirofilaria immitis infection in dogs. It is a chronic heart disease, which can lead to death by congestive heart failure. Canine ehrlichiosis (CE) is caused by Ehrlichia canis bacterial infection with a zoonotic potential and fatal to dogs in acute and chronic presentations. Exposed the above, this study aims to describe a successful treatment and management of a dog with CanL, CE, and HW comorbidity. Case: A 3-year-old male uncastrated black Labrador dog, weighing 35 kg, was admitted to the veterinary clinic due to immunochromatographic CanL positive test performed by municipal zoonosis control center active surveillance in August 2014. Clinical exam showed a mild shedding, intermittent eye white/yellow discharge and popliteal lymph nodes enlargement. After positive for CanL, veterinary requested more laboratorial exams. IFAT and ELISA were positive for CanL, blood smear showed presence of microfilaria, and bone marrow cytology showed Ehrlichia spp. morulae and microfilaria. Initial treatment prescribed was oral doxycycline, omeprazole, ranitidine, and domperidone for 30 days, and allopurinol and ivermectin until further recommendation. Additionally, repellent collar, repellent spray and vitamin supplement was indicated. After first month, marbofloxacin for 30 days and three doses of immune stimulant protocol were administrated. After three months of treatment, dog still positive for heartworm, ehrlichiosis, and CanL. Doxycycline protocol was repeated. Dog became consistently negative for all pathogens one year later with persistent thrombocytopenia but without clinical signs, ergo allopurinol and ivermectin were discontinued. After four years of follow up, the animal had an acute pancreatitis and died, with unremarkable total blood count and negative for all pathogens. Discussion: CVBD coinfections are commonly reported as worsen prognostic in endemic regions. The pathogens reported here share a host immunomodulation competence. L. infantum and Ehrlichia spp.downregulates Th1 response, whereas D. immitis increase as Th2 profile. The therapeutic protocol was iniciated by staging CanL. Since the patient had clinical signs, allopurinol was prescribed as a well-established drug for CanL. Marbofloxacin was added due to its high safety drug in clinical improvement of infected dogs with and without renal disease and in vitro effectiveness against L. infantum. Domperidone was used to promote Th1 cytokine profile as INF-γ, IL-2, IL-12, and TNF-α. We used an immunostimulant protocol to favor polarization to the Th1 profile comprised by 30 days of domperidone protocol followed by a vaccine and an immunomodulator. Doxycycline was used successfully for Ehrlichia spp. and HE clearance after two treatment courses and one year of ivermectin every 15 days. The animal presented intermittent coughing episodes on the first treatment course, but no medical intervention was needed besides exercise restriction. Our report shows the successful management of one dog with CanL, CE and HE comorbidity. This success was possible due to early detection and good therapeutic choice.Keywords: canine visceral leishmaniosis, coinfection, Dirofilaria immitis, Erhlichia canis, Leishmania infantum, treatment.


2014 ◽  
Vol 63 (1) ◽  
pp. 106-110
Author(s):  
Saul José Semião-Santos ◽  
Laura Barral Veloso ◽  
Paulo Paes de Andrade ◽  
Marcia Almeida de Melo ◽  
Luis Miguel Lourenço Martins ◽  
...  

We compared the performance of a locally produced β-mercaptoethanol-modified promastigote antigen (β-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for β-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for β-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen’s κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of β-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.


2019 ◽  
Vol 28 (1) ◽  
pp. 172-176 ◽  
Author(s):  
Filipa Teixeira Rodrigues ◽  
Catarina Pereira ◽  
Jitender Prakash Dubey ◽  
Miguel Nóvoa ◽  
Miguel Quaresma ◽  
...  

Abstract Toxoplasma gondii and Leishmania infantum are zoonotic protozoal parasites. Serum samples were obtained from 186 donkeys (Equus africanus asinus) from Portugal and assessed for antibodies to T. gondii by the modified agglutination test (MAT). For titration of antibodies to Leishmania spp. the direct agglutination test was used (DAT). Eleven donkeys were seropositive for T. gondii with titres of 20 (n = 7), 80 (n = 2), 640 (n = 1) and ≥ 2560 (n = 1). One donkey was seropositive for Leishmania spp. (titre of 800). Donkeys in Portugal are exposed to and can be infected with T. gondii and Leishmania spp.


2012 ◽  
Vol 81 (4) ◽  
pp. 327-331 ◽  
Author(s):  
Marián Prokeš ◽  
Dagmar Zendulková ◽  
Kateřina Rosenbergová ◽  
František Treml ◽  
Anna Ondrejková ◽  
...  

The aim of our study was to map the situation of swine mycoplasmoses on four farms in the region of Eastern Slovakia. The primary agent of Enzootic pneumonia of swine isMycoplasma hyopneumoniae. After reviewing the health status of conventional herds and evaluation of clinical symptoms, paired samples of nasal swabs and venous blood samples were collected from 38 pigs with clinical signs of respiratory disease. Nasal swab samples were tested by nested PCR, while blood samples were used to detect antibodies againstM. hyopneumoniaeby blockingELISA. The presence ofM. hyopneumoniaewas confirmed by nested PCR in four pigs (10.5%) and by blocking ELISA in 16 pigs (42.1%) of all four farms. This work presents for the first time comparison of different methods to diagnoseM. hyopneumoniaeinfection on pig farms in Eastern Slovakia.


2018 ◽  
Author(s):  
Netranapha Pandey ◽  
Suradej Siripattanapipong ◽  
Saovanee Leelayoova ◽  
Jipada Manomat ◽  
Mathirut Mungthin ◽  
...  

ABSTRACTLeishmaniasis is a neglected tropical disease causing opportunistic infection among patients with HIV/AIDS. The fatal form of this disease is visceral leishmaniasis (VL). DNA of Leishmania can be detected in saliva, for which the collection is noninvasive and requires little expertise. This study aimed to evaluate the sensitivity and specificity of a nested-PCR to amplify the Internal Transcribed Spacer 1 (ITS1) to detect Leishmania DNA in paired saliva and buffy coat samples of 305 Thai patients with HIV/AIDS in Trang Hospital, Trang Province, southern Thailand. For asymptomatic Leishmania infection among Thai patients with HIV/AIDS, the sensitivity and specificity of the nested-PCR-ITS1 in buffy coat were 73.9 and 100%, respectively. However, the sensitivity in saliva was 26.1% and specificity was 100%. Using the nested-PCR-ITS1, saliva and buffy coat samples showed positive agreement in only 52.0% of patients. Saliva tested results with the nested-PCR-ITS1 showed positive agreement with the Direct Agglutination Test (DAT) in 46.5% of patients. Only 12.1% of the samples showed positive agreement for Leishmania infection among all the three tests: saliva, buffy coat and DAT results. Using nucleotide sequencing, at least three species of Leishmania infection were identified in saliva, i.e., L. siamensis (n=28), L. martiniquensis (n=9), and L. donovani complex (n=1). As a result, buffy coat still appears to be a better specimen to diagnose asymptomatic VL infection among individuals with HIV. However, the use of both buffy coat and saliva together as clinical specimens would increase the sensitivity of Leishmania detection.


2020 ◽  
Author(s):  
Maria Alfonsa Cavalera ◽  
Roberta Iatta ◽  
Pietro Laricchiuta ◽  
Giuseppe Passantino ◽  
Francesca Abramo ◽  
...  

Abstract Background A large number of animal species are susceptible to Leishmania infantum (Kinetoplastida, Trypanosomatidae) in endemic areas, including domestic and wild felids such as tigers (Panthera tigris). Knowledge on the infection of this endangered species is still at its infancy, and therefore this study aims to identify clinical presentation and clinicopathological findings of tigers naturally infected by L. infantum. Results Tigers either L. infantum-positive (group A) or -negative (group B) were apparently healthy or presented visceral leishmaniasis unrelated conditions, except for one animal in which a large non-healing cutaneous lesion was observed. However, histological exam and immunohistochemistry carried out on the lesion excluded the presence of L. infantum amastigotes. Biochemical analysis showed that the average concentration of total proteins, globulins and haptoglobin were significantly higher (p<0.01, p=0.01 and p=0.02, respectively), while the albumin/globulin ratio significantly lower (p=0.05) in group A compared with group B. The biochemical alterations were partially confirmed by the serum protein electrophoresis results revealing a significant increase in the total protein value (p=0.01) and hypergammaglobulinemia (p=0.03) but an unmodified albumin/globulin ratio in group A.Conclusions In this study tigers infected by L. infantum have shown to be mainly asymptomatic. The absence of clinical signs may lead veterinarians to overlook leishmaniasis in animals kept in captivity. Therefore, diagnostic and screening tests as serology should be part of routinely surveillance programs to be performed on tigers in zoological gardens located in endemic areas. Though only few protein-related laboratory abnormalities were recorded in infected animals, they could provide diagnostic clues for a first suspicion of L. infantum infection in tigers. Indeed, considering the high risk of zoonotic transmission in heavily frequented environment as zoos, a prompt diagnosis of L. infantum infection is of pivotal importance.


2020 ◽  
Vol 41 (3) ◽  
pp. 897
Author(s):  
Jeane Martinha dos Anjos Cordeiro ◽  
Paula Elisa Brandão Guedes ◽  
Alexandre Dias Munhoz ◽  
Fabiana Lessa Silva

Ehrlichiosis is an emerging zoonosis worldwide and has had several adverse effects on public health. Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, has the tick Rhipicephalus sanguineus as the vector. The main clinical signs in affected dogs are fever, apathy, anorexia, weight loss, and neurological signs. The diagnosis is made through the association of clinical signs with parasitological, serological, and molecular tests. The aim of this study was to evaluate the occurrence of E. canis infection in dogs from the city of Itabuna-Bahia, as well as to identify the risk factors related to infection. For this, 405 dogs from the Center for Zoonoses Control (CCZ), non-governmental organizations (NGOs), and dogs domiciled and semi-domiciled in the city of Itabuna, southern Bahia, were evaluated. After initial physical evaluation of the dogs, blood samples were collected by venipuncture for subsequent DNA extraction and E. canis testing using the nested Polymerase Chain Reaction (nested-PCR) technique. In addition, an epidemiological questionnaire that included questions related to the animals was administered to the dog owners to identify the risk factors for exposure to the etiological agent and to the vector. Approximately 17% of the dogs in the municipality of Itabuna-Bahia tested positive for E. canis by nested-PCR, a result higher than that found in other studies conducted in the same municipality. Among the factors associated with E. canis infection, contact with other dogs (p = 0.0226) was an important factor for the dissemination of CME, since dogs are reported to be reservoirs of E. canis. Male dogs (p = 0.0016) presented lower risk for E. canis infection. Other studies, however, describe no association between animal gender and infection by E. canis. Preventive measures to reduce exposure to the vector of ehrlichiosis are necessary.


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