Leishmania exposure in dogs from two endemic countries from New and Old Worlds (Brazil and Portugal): evaluation of three serological tests using Bayesian Latent Class Models.

BACKGROUND: Zoonotic leishmaniosis caused by Leishmania infantum is endemic in several countries of the Mediterranean Basin, Latin America, and Asia. Dogs are the main hosts and reservoirs of human infection. Thus, from a One Health perspective, early diagnosis of Leishmania infection in dogs is essential to control the dissemination of the parasite among other dogs and to humans. The aim of this study was to estimate the diagnosis accuracy of three serological tests to detect antibodies to Leishmania in dogs from two endemic settings using Bayesian latent class models (BLCM). METHODS: A total of 378 dogs from two Portuguese and Brazilian endemic areas of leishmaniosis (194 animals from Portugal and 184 from Brazil) were screened. Detection of anti-Leishmania antibodies was performed using two commercial ELISA (L. infantum IgG-ELISA® and EIE-LVC®) and a rapid immunochromatographic test (DPP-LVC®). Bayesian latent class models were used to estimate Leishmania infection prevalence, together with sensitivities and specificities of the three diagnostic tests, in the two dog populations simultaneously. Predictive values were also calculated. Credibility intervals (CI) were obtained, considering different types of prior information. RESULTS: A posterior median Leishmania seroprevalence of 13.4% (95% CI 9.0-18.7) and of 21.6% (15.0-28.3) was estimated to the Portuguese and Brazilian dog subpopulations, respectively. The Bayesian analysis indicated that all tests were highly specific (specificity above 90%), and that the DPP-LVC® was more sensitive (96.6%; 83.1-99.9) than both ELISAs in the Portuguese subpopulation, while in the Brazilian subpopulation, EIE-LVC® and L. infantum IgG-ELISA®, had the highest sensitivity (88.2%; 73.7-97.0) and specificity (98.7%; 95.1-99.9), respectively. CONCLUSIONS: In general, the levels of diagnosis accuracy of the three serological tests to detect Leishmania antibodies assessed by BLCM indicate their utility in canine epidemiological studies. The same approach should be used to assess the performance of these techniques in the clinical management of infected and sick dogs using representative samples from the wide spectrum of clinical situations, namely from subclinical infection to manifest disease. The low positive predictive value of the serological tests used in the current protocol of the Brazilian Ministry of Health suggests that they should not be used individually and may not be sufficient to target reservoir-based control interventions.

Diversity of Mosquitoes (Diptera: Culicidae) in an Atlantic Forest Urban Park, Salvador, Brazil.

We identified mosquito species (Diptera: Culicidae) in an Atlantic Forest fragment located in a large urban park in Salvador, Brazil, one year after a citywide epizootic of yellow fever virus (YFV). Between May 2 and August 2, 2018, adult mosquitoes were collected using the human attraction method, followed by trapping with hand-nets, and CO2-baited light traps placed at ground level and in the canopy. We collected a total of 11,914 mosquitoes, which belonged to three tribes, five genera, and at least seven species. The most abundant taxa captured by CO2-baited light traps were Culex quinquefasciatus (Say, Diptera: Culicidae) Limatus spp. (Diptera: Culicidae), and Wyeomyia spp. (Diptera: Culicidae), while by human attraction, Cx. quinquefasciatus, Wyeomyia spp., and Aedes albopictus (Skuse, Diptera: Culicidae) were captured most often. The diversity of mosquitoes by species was greater in the park area with restinga vegetation compared to the area with dense rainforest. Although vectors commonly associated with sylvatic YFV transmission were not captured, we collected several species capable of transmission of other arboviruses. Given the high likelihood of encounters between mosquitoes and human visitors in environments, such as the one studied, periodic entomological surveys to determine the risk of arbovirus transmission in these settings are warranted.

Early detection and persistent positivity of anti-Leishmania antibodies using a recombinant protein-based ELISA in naturally infected dogs in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum, for which dogs constitute the main urban parasite reservoir. Control measures and the treatment of canine visceral leishmaniasis (CVL) are essential to reduce VL cases. Early and accurate detection of L. infantum-infected dogs is crucial to the success of VL control. To improve the serological detection of L. infantum-exposed dogs, we evaluated the early diagnosis capacity of a recombinant protein (rLci5) in an immunosorbent assay (ELISA) to detect naturally infected dogs. Additionally, we evaluated the persistence of the positive results obtained by rLci5 ELISA in comparison to other conventional diagnostic test methods. METHODS: Serum samples obtained from 48 L. infantum-infected dogs involved in a cohort study were evaluated using different diagnostic methods (qPCR, EIE-LVC, DPP-LVC and splenic culture). The results were compared to rLci5 ELISA to determine its capacity to diagnose L. infantum infection at earlier infection time points. The persistence of positive diagnostic test results was also compared for each dog evaluated. RESULTS: rLci5 ELISA presented higher rates of positive results at early time points compared to the other diagnostic tests employed in the cohort study, as early as 24 months prior to detection by other tests. rLci5 ELISA positivity was 52.1% (25/48) at baseline, while qPCR was 35.4% (17/48), DPP-LVC 27.1% (13/48), EIE-LVC 22.9% (11/48) and culture only 4.2% (2/48). In at least one of the time points of the 24-month cohort study, rLci5 ELISA was positive in 100% (48/48) of the dogs, versus 83% (40/48) for qPCR, 75% (36/48) for DPP-LVC, 65% (31/48) for EIE-LVC and 31% (15/48) for culture. Investigating clinical signs in association with diagnostic test positivity, rLci5 ELISA successfully detected CVL in 62.9% (95/151) of the clinical evaluations with a score of 0-3, 64.3% (45/70) with scores between 4 and 7, and 73.7% (14/19) with scores > 7, providing higher rates of positivity than all other methods evaluated. Moreover, rLci5 ELISA presented the greatest persistence with respect to test positivity: 45.8% of the dogs evaluated. CONCLUSION: Four diagnostic tests were compared to rLci5 ELISA, which presented earlier infection diagnosis and a greater persistence of positive test results. Accordingly, the use of the rLci5 ELISA can improve CVL diagnostic performance by detecting infected dogs sooner than other testing methods, with enhanced persistence of positive results over the course of the infection.

Correction: High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil-A Phase II study.

[This corrects the article DOI: 10.1371/journal.pntd.0006871.].

Natural infection by Leishmania infantum in the Lutzomyia longipalpis population of an endemic coastal area to visceral leishmaniasis in Brazil is not associated with bioclimatic factors.

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoan Leishmania infantum and in Brazil is transmitted mainly by the bite of Lutzomuyia longipalpis sand flies. Data about the presence, distribution, natural infection rate, seasonal and monthly dynamics of the vector population are important for optimizing the measures to control VL in endemic areas. This study aimed to identify sand fly fauna in an endemic area for VL to detect the prevalence of L. infantum infection in the Lu. longipalpis population and to elucidate the influence of bioclimatic factors on the monthly fluctuations of this vector. HP light traps were monthly set in the intradomicile and peridomicile of residences located in the central and beachfront areas of Camaçari, a VL endemic area. The sand fly collection was conducted in two periods: i) period 1-between December 2011 and November 2012 and ii) period 2-August 2014 and July 2015. Sand fly species were identified and detection of L. infantum infection by qPCR was performed in pools of female Lu. longipalpis. For the first time, the parasite load of positive pools was correlated with the number of Lu. longipalpis captured per month in both periods. Correlation analyses between the monthly fluctuation of the sand fly population and bioclimatic indices of the municipality in both collection periods were also performed. In both evaluated periods, more than 98% of the collected sand flies were Lu. longipalpis, confirming the predominance of this species in the region. It was captured mostly in the beachfront area in all months evaluated (99%). For the period 1, Leishmania DNA was detected in 81% of tested pools representing a minimal infection rate of 9.6%. In the period 2, 40% of the pools were positive with a minimal infection rate of 10.2%. Infected sand flies were only detected in the beachfront area in both periods. The parasite load was low and did not vary in the evaluated months despite the number of collected sand flies. No correlation was observed for climatic factors in both areas of Camaçari. These findings emphasize the high risk of Leishmania transmission in Camaçari regardless of the season and that other factors, aside from bioclimatic elements, are influencing the sand fly population monthly fluctuation.

High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil - A Phase II study.

BACKGROUND: Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85-99%) vs. 83% (IC 95%; 69-92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49-97%) vs. 54% (IC 95%; 33-74%) for asymptomatic dogs and 94% (IC 95%; 83-99%) vs, 88% (IC 95%; 76-95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81-91%) of sensitivity, 94% (IC 95%; 90-97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59-74%), specificity of 87% (IC 95%; 81-92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52-66%), specificity of 98% (IC 95%; 95-99%) and accuracy of 80% (IC 95%; 76-84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63-77%), specificity of 99% (IC 95%; 97-100%) and accuracy of 86% (IC 95%; 83-89%). CONCLUSION: Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors.

The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector.

The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs.

Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results.

Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis.

Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4-100) in splenic aspirate; 79.2% (CI 68-90.3) in lymph nodes; 77.3% (CI 64.5-90.1) in skin; 75% (CI 63.1-86.9) in blood; 50% (CI 30-70) in bone marrow; 37.5% (CI 24.2-50.8) in left-eye; and 29.2% (CI 16.7-41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7-79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5-96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.

A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is a serious public health challenge in Brazil and dogs are considered to be the main urban reservoir of the causative agent. The culling of animals to control VL in some countries makes the accurate diagnosis of canine VL (CVL) essential. Recombinant antigens rLci1A and rLci2B were selected from a cDNA library of Leishmania infantum amastigotes due to their strong potential as candidates in diagnostic testing for CVL. The present multicentric study aimed to evaluate the sensitivity of a prototype test using these antigens (DPP rLci1A/rLci2B) against 154 sera obtained from symptomatic dogs within three endemic areas of VL in Brazil. The specificity was evaluated using 40 serum samples from negative dogs and dogs infected with other pathogens. Sensitivity and specificity rates of DPP rLci1A/rLci2B prototype were compared to rates from other diagnostic tests currently in use by the Brazilian Ministry of Health, including DPPLVC, EIELVC. FINDINGS: DPP rLci1A/rLci2B prototype offered similar performance to that offered by DPPLVC rapid test, as follows: sensitivity of 87% (CI 81-91) and 88% (CI 82-93) and specificity of 100% (CI 91-100) and 97% (CI 87-100), respectively for DPP rLci1A/rLci2B and DPPLVC. When results of these two tests were considered concomitantly, sensitivity increased to 93.5% (CI 89-96). CONCLUSIONS: The recombinant antigens rLci1A and rLci2B represent promising candidates for use in a multi-antigen rapid test for CVL. The inclusion of novel antigens to the DPP rLci1A/rLci2B prototype model could offer additionally enhanced sensitivity to detect animals infected by L. infantum.

Estudo da correlação entre a carga parasitária de cães com diferentes apresentações clínicas da leishmaniose visceral e a transmissão ao vetor da leishmania infantum / Study of correlation between parasite load of dogs with different clinical presentations of visceral leishmaniasis and transmission to the vector of Leishmania infantum

No Novo Mundo, a leishmaniose visceral (LV) é causada pela Leishmania infantum, que tem como vetor o inseto flebotomíneo Lutzomyia longipalpis. Os cães são considerados o principal reservatório urbano da infecção. Devido ao fato do controle da LV se basear, principalmente, no controle da leishmaniose visceral canina (LVC), é importante estudar o papel dos cães na transmissão da infecção. Foi demonstrado que cães apresentando diferentes apresentações clínicas da LV, inclusive os assintomáticos transmitem a infecção ao vetor flebotomíneo. Nenhum estudo sistemático avaliou a associação direta entre a carga parasitária em diferentes tecidos e a transmissão do parasito. A hipótese desse estudo é que cães com baixa carga parasitária na pele e no sangue não transmitem a infecção ao vetor flebotomíneo. O objetivo deste estudo foi analisar se há correlação entre a carga parasitária de cães com diferentes apresentações clínicas da LV e a transmissão ao vetor Lutzomyia longipalpis. Foram selecionados 35 cães de dois canis, locazidos em area endêmica (n=23) e não endêmica (n=12) para LV. Os animais foram classificados de acordo com o número de sinais clínicos em: assintomáticos (sem sinais; n=12), oligossintomáticos (1-3 sinais; n=15) e polissintomático (<3 sinais; n =8). Todos os 35 cães foram positivos em pelo menos um dos testes diagnósticos: ELISA (n=8), cultura de aspirado esplênico (n=9) e qPCR (n=35) dos tecidos avaliados. Diferentes tecidos (sangue periférico, aspirado esplênico e biópsia de pele) foram coletados para quantificação do DNA do parasito pela qPCR. Para avaliar a capacidade de transmissão dos cães, foi realizado xenodiagnóstico, seguido de determinação da carga parasitária em cada flebótomo utilizando qPCR. Finalmente, a capacidade de transmissão de Leishmania foi estimada pela determinação, após o xenodiagnóstico, da infectividade de cães ao flebótomo, da taxa de infecção de flebótomos, e da carga parasitária transmitida aos flebótomos. Baixa carga parasitária na pele e no sangue foi detectada em aproximadamente 85% dos cães assintomáticos. A infectividade de cães ao flebótomo variou de 60 a 90%, e foi similar entre animais apresentando diferentes números de sinais clínicos. Foi identificado que o maior percentual (51%) de cães transmite parasitos a um pequeno número de flebótomos (de 1 a 5 em 30 flebótomos utilizados no xenodiagnóstico). Entre os tecidos analisados, correlação positiva foi detectada entre a infectividade de cães ao vetor e a carga parasitária nas amostras de sangue (r = 0.50, p<0.01). Adicionalmente, foi observada, correlação positiva entre menor taxa de infecção dos flebótomos e baixa carga parasitária no sangue (r = 0.53, p<0.01). Em conjunto, estes dados mostram que cães com baixa carga parasitária são capazes de transmitir o parasito, porém a um pequeno número de flebótomos e com uma baixa carga parasitária.