Identification of biological factors of Lutzomyia longipalpis using MALDI-TOF peptide/protein profiles.

Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis, by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 µL solution was deposited over 1 µL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics' impact on peptide/protein profiles in sand fly species identification.

Pharmacokinetics, Dose-Proportionality, and Tolerability of Intravenous Tanespimycin (17-AAG) in Single and Multiple Doses in Dogs: A Potential Novel Treatment for Canine Visceral Leishmaniasis.

In the New World, dogs are considered the main reservoir of visceral leishmaniasis (VL). Due to inefficacies in existing treatments and the lack of an efficient vaccine, dog culling is one of the main strategies used to control disease, making the development of new therapeutic interventions mandatory. We previously showed that Tanespimycin (17-AAG), a Hsp90 inhibitor, demonstrated potential for use in leishmaniasis treatment. The present study aimed to test the safety of 17-AAG in dogs by evaluating plasma pharmacokinetics, dose-proportionality, and the tolerability of 17-AAG in response to a dose-escalation protocol and multiple administrations at a single dose in healthy dogs. Two protocols were used: Study A: four dogs received variable intravenous (IV) doses (50, 100, 150, 200, or 250 mg/m2) of 17-AAG or a placebo (n = 4/dose level), using a cross-over design with a 7-day "wash-out" period; Study B: nine dogs received three IV doses of 150 mg/m2 of 17-AAG administered at 48 h intervals. 17-AAG concentrations were determined by a validated high-performance liquid chromatographic (HPLC) method: linearity (R2 = 0.9964), intra-day precision with a coefficient of variation (CV) ≤ 8%, inter-day precision (CV ≤ 20%), and detection and quantification limits of 12.5 and 25 ng/mL, respectively. In Study A, 17-AAG was generally well tolerated. However, increased levels of liver enzymes-alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)-and bloody diarrhea were observed in all four dogs receiving the highest dosage of 250 mg/m2. After single doses of 17-AAG (50-250 mg/m2), maximum plasma concentrations (Cmax) ranged between 1405 ± 686 and 9439 ± 991 ng/mL, and the area under the curve (AUC) plotting plasma concentration against time ranged between 1483 ± 694 and 11,902 ± 1962 AUC 0-8 h µg/mL × h, respectively. Cmax and AUC parameters were dose-proportionate between the 50 and 200 mg/m2 doses. Regarding Study B, 17-AAG was found to be well tolerated at multiple doses of 150 mg/m2. Increased levels of liver enzymes-ALT (28.57 ± 4.29 to 173.33 ± 49.56 U/L), AST (27.85 ± 3.80 to 248.20 ± 85.80 U/L), and GGT (1.60 ± 0.06 to 12.70 ± 0.50 U/L)-and bloody diarrhea were observed in only 3/9 of these dogs. After the administration of multiple doses, Cmax and AUC 0-48 h were 5254 ± 2784 µg/mL and 6850 ± 469 µg/mL × h in plasma and 736 ± 294 µg/mL and 7382 ± 1357 µg/mL × h in tissue transudate, respectively. In conclusion, our results demonstrate the potential of 17-AAG in the treatment of CVL, using a regimen of three doses at 150 mg/m2, since it presents the maintenance of high concentrations in subcutaneous interstitial fluid, low toxicity, and reversible hepatotoxicity.

Elucidating the role played by bone marrow in visceral leishmaniasis.

Leishmaniasis is a widespread group of infectious diseases that significantly impact global health. Despite high prevalence, leishmaniasis often receives inadequate attention in the prioritization of measures targeting tropical diseases. The causative agents of leishmaniasis are protozoan parasites of the Leishmania genus, which give rise to a diverse range of clinical manifestations, including cutaneous and visceral forms. Visceral leishmaniasis (VL), the most severe form, can be life-threatening if left untreated. Parasites can spread systemically within the body, infecting a range of organs, such as the liver, spleen, bone marrow and lymph nodes. Natural reservoirs for these protozoa include rodents, dogs, foxes, jackals, and wolves, with dogs serving as the primary urban reservoir for Leishmania infantum. Dogs exhibit clinical and pathological similarities to human VL and are valuable models for studying disease progression. Both human and canine VL provoke clinical symptoms, such as organ enlargement, fever, weight loss and abnormal gamma globulin levels. Hematologic abnormalities have also been observed, including anemia, leukopenia with lymphocytosis, neutropenia, and thrombocytopenia. Studies in dogs have linked these hematologic changes in peripheral blood to alterations in the bone marrow. Mouse models of VL have also contributed significantly to our understanding of the mechanisms underlying these hematologic and bone marrow abnormalities. This review consolidates information on hematological and immunological changes in the bone marrow of humans, dogs, and mice infected with Leishmania species causing VL. It includes findings on the role of bone marrow as a source of parasite persistence in internal organs and VL development. Highlighting gaps in current knowledge, the review emphasizes the need for future research to enhance our understanding of VL and identify potential targets for novel diagnostic and therapeutic approaches.

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.

Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.

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.

Immune response dynamics and Lutzomyia longipalpis exposure characterize a biosignature of visceral leishmaniasis susceptibility in a canine cohort.

BACKGROUND: Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators' interactions, and vector exposure intensity associated with CVL resistance and susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11-2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. CONCLUSIONS/SIGNIFICANCE: The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way.

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.

Seroprevalence of Trypanosoma cruzi infection among blood donors in the state of Bahia, Brazil.

INTRODUCTION: Chagas disease (CD) affects 5.7-7.0 million individuals worldwide, and its prevalence reached 25.1% in the state of Bahia, Brazil. There is an association between the prevalence of CD, the socioeconomic status of the population, and the risk of re-emergence due to non-vectorial transmission, such as blood transfusion. This study determined the seroprevalence of T. cruzi infection among blood donors in the state of Bahia, located in northeastern Brazil, and their epidemiological profile during a 10-year period. METHODS: We performed a descriptive cross-sectional study involving a database review. Data were collected from patients with non-negative results for T. cruzi infection during a 10-year period. RESULTS: A total of 3,084 (0.62%) samples were non-negative for T. cruzi infection in an initial serological screening, and 810 (0.16%) samples were non-negative in the second screening. The correlation between infection and age (30 years or older) and between infection and lower educational level (12 years or less) in the first and second screening was statistically significant. The seroprevalence of T. cruzi infection was higher in men in the first screening. In addition, 99.52% of the municipalities of Bahia had at least one case of CD. Livramento de Nossa Senhora and Salvador presented the highest disease prevalence and recurrence, respectively. CONCLUSIONS: The seroprevalence of T. cruzi infection in these populations was lower than that found in other studies in Brazil but was comparatively higher in densely-populated areas. The demographic characteristics of our population agreed with previous studies.

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4-rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4-rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4-rLci1A and E4-rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Seroprevalence of trypanosoma cruzi infection among blood donors in the state of Bahia, Brazil

Abstract INTRODUCTION: Chagas disease (CD) affects 5.7-7.0 million individuals worldwide, and its prevalence reached 25.1% in the state of Bahia, Brazil. There is an association between the prevalence of CD, the socioeconomic status of the population, and the risk of re-emergence due to non-vectorial transmission, such as blood transfusion. This study determined the seroprevalence of T. cruzi infection among blood donors in the state of Bahia, located in northeastern Brazil, and their epidemiological profile during a 10-year period. METHODS: We performed a descriptive cross-sectional study involving a database review. Data were collected from patients with non-negative results for T. cruzi infection during a 10-year period. RESULTS: A total of 3,084 (0.62%) samples were non-negative for T. cruzi infection in an initial serological screening, and 810 (0.16%) samples were non-negative in the second screening. The correlation between infection and age (30 years or older) and between infection and lower educational level (12 years or less) in the first and second screening was statistically significant. The seroprevalence of T. cruzi infection was higher in men in the first screening. In addition, 99.52% of the municipalities of Bahia had at least one case of CD. Livramento de Nossa Senhora and Salvador presented the highest disease prevalence and recurrence, respectively. CONCLUSIONS: The seroprevalence of T. cruzi infection in these populations was lower than that found in other studies in Brazil but was comparatively higher in densely-populated areas. The demographic characteristics of our population agreed with previous studies.

Alterations in the lipid profiles and circulating liver enzymes in individuals infected by Schistosoma mansoni.

INTRODUCTION: Portal hypertension and periportal fibrosis commonly occur in severe Schistosoma mansoni infection. Changes in lipid profile and elevated levels of circulating liver enzymes have also been described in infected individuals. The present study sought to assess the alterations in laboratory parameters associated with liver disorder in individuals infected by S. mansoni who visited a private routine laboratory service. Levels of circulating liver enzymes (gamma-glutamyl transferase [γ-GT], aspartate transaminase [AST], alanine transaminase [ALT], and alkaline phosphatase [ALP]) and a lipid panel (total cholesterol [COL], high-density lipoprotein [HDL], low-density lipoprotein [LDL], very low-density lipoprotein [VLDL], and triglycerides [TRI]) were evaluated in both infected and non-infected individuals and relative risk was used to measure associations. METHODS: Data were collected for analysis from a total of 1,078 cases identified in 379,600 individuals who submitted samples to the Datalab Laboratory (Salvador, Bahia) between 2004 and 2008. RESULTS: S. mansoni infection led to increased circulating levels of γ-GT in both women and men, AST (women), and ALP (men). S. mansoni infection was a protective factor against increased levels of TRI, CHO, and VLDL for individuals aged 19 years or older. The results of our analysis indicate that alterations in lipid metabolism and circulating liver enzymes in asymptomatic S. mansoni-infected individuals might be attributed to eggs lodged in the hepatic sinusoids. CONCLUSIONS: Parasitological testing for S. mansoni should be indicated in endemic areas when this pattern of alterations is detected.

Alterations in the lipid profiles and circulating liver enzymes in individuals infected by Schistosoma mansoni

Abstract INTRODUCTION Portal hypertension and periportal fibrosis commonly occur in severe Schistosoma mansoni infection. Changes in lipid profile and elevated levels of circulating liver enzymes have also been described in infected individuals. The present study sought to assess the alterations in laboratory parameters associated with liver disorder in individuals infected by S. mansoni who visited a private routine laboratory service. Levels of circulating liver enzymes (gamma-glutamyl transferase [γ-GT], aspartate transaminase [AST], alanine transaminase [ALT], and alkaline phosphatase [ALP]) and a lipid panel (total cholesterol [COL], high-density lipoprotein [HDL], low-density lipoprotein [LDL], very low-density lipoprotein [VLDL], and triglycerides [TRI]) were evaluated in both infected and non-infected individuals and relative risk was used to measure associations. METHODS Data were collected for analysis from a total of 1,078 cases identified in 379,600 individuals who submitted samples to the Datalab Laboratory (Salvador, Bahia) between 2004 and 2008. RESULTS S. mansoni infection led to increased circulating levels of γ-GT in both women and men, AST (women), and ALP (men). S. mansoni infection was a protective factor against increased levels of TRI, CHO, and VLDL for individuals aged 19 years or older. The results of our analysis indicate that alterations in lipid metabolism and circulating liver enzymes in asymptomatic S. mansoni-infected individuals might be attributed to eggs lodged in the hepatic sinusoids. CONCLUSIONS Parasitological testing for S. mansoni should be indicated in endemic areas when this pattern of alterations is detected.

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.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

The mass use of deltamethrin collars to control and prevent canine visceral leishmaniasis: A field effectiveness study in a highly endemic area.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonosis of great importance. Limitations in current VL control measures compromise efficacy, indicating the need to implement new strategies. The aim of this study was to evaluate the effectiveness of the mass use of deltamethrin-impregnated collars in dogs as a public health measure to control and prevent canine visceral leishmaniasis (CVL). METHODOLOGY: An interventional study was implemented in two endemic areas in the district of Monte Gordo (Bahia-Brazil): an intervention area, in which VL seronegative dogs were collared, and a control area in which only conventional CVL control measures were applied. At baseline, seropositive dogs were removed and seronegative dogs were included. Dogs were then reevaluated every 7-8 months for almost two years. At each time point, dogs in the intervention area that remained seronegative received new collars and newly identified seronegative dogs were included and collared. The local zoonosis control authorities were notified of any dogs that tested seropositive in both areas, which were subsequently marked for euthanasia as mandated by the Brazilian Ministry of Health. PRINCIPAL FINDINGS: In the first serological survey, seroprevalence was similar in both areas. At the second evaluation, significant reductions in seroprevalence were seen in both areas, while seroprevalence in the intervention area reduced to 6.0% during the final evaluation versus an increase of 11.0% in the control area. This significant increase and the estimated relative risk (RR = 0.55) indicated protection against CVL in the intervention area. Although CVL incidence did not differ significantly between the areas, an increased tendency was observed in the control area, which could be due to low seroconversion rates throughout the study or a high loss to follow-up. CONCLUSIONS/SIGNIFICANCE: Although our evaluation of the effectiveness of deltamethrin-impregnated collars as a community-wide public health control measure was inconclusive, this measure likely provides protection over time. In endemic areas of Brazil, this strategy represents an operational challenge for local zoonosis control authorities, indicating the need for adjustments, including improved collar design.

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis.

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease. In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen. Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61-0.80) very good (0.81-0.99) or perfect (1.00), for most of the parameters analyzed. Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.

A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator's hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.

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.