Developments in Leishmaniasis diagnosis: A patent landscape from 2010 to 2022.

The current study aims to contribute to the understanding of leishmaniasis diagnosis by providing an overview of patent filings in this field and analyzing whether the methods revealed are consistent with the needs described by the scientific community, in special the main gaps detected by the World Health Organization's 2021-2030 Roadmap for Neglected Tropical Diseases. To this aim, a patent search was carried out focusing on documents disclosing leishmaniasis diagnostic methods supported by experimental evidence and with earliest priority date from 2010 onwards. Our results show that patenting activity is low and patent families are often formed by individual filings. Most R&D activity occurs in Brazil, which is also the main market of protection. Brazilian academic institutions are the main patent drivers, and collaboration between different institutions is rare. Most patent families describe immunological methods based on ELISA assays, using antibodies directed to K39 and homologues. kDNA is the primary gene for molecular testing. Experimental evidence of test performance in fulfilling critical diagnostic gaps is usually absent. The patent scenario suggests that leishmaniasis diagnostic gaps need to be more closely addressed to drive innovation directed to the control and/or elimination of leishmaniasis. From the public policy point of view, the following strategies are suggested: (i) strengthening collaborative networks, (ii) enhancing the participation of the private sector, and (iii) increasing funding, with special focus on the remaining diagnostic gaps.

Accuracy of serological tests in diagnosing mucosal leishmaniasis.

In this serum panel-based study, we evaluated the accuracy of serological tests originally developed for visceral leishmaniasis (VL), for diagnosis of mucosal leishmaniasis (ML). A total of five tests were evaluated, four of which are registered at the National Agency of Sanitary Surveillance (Agência Nacional de Vigilância Sanitária-ANVISA) (RIDASCREEN® Leishmania Ab from R-Biopharm AG., Leishmania ELISA IgG + IgM from Vircell S.L., IFI Leishmaniose Humana-BioManguinhos, and IT-LEISH® from Bio-Rad Laboratories, Inc.), and the other a direct agglutination test (DAT-LPC) prototype kit developed at Fiocruz. The panel was composed of 40 serum samples from patients with confirmed ML and 20 from patients with mucosal involvement and negative parasitological/molecular tests for leishmaniasis and confirmation of another etiology. All cases were treated from 2009 to 2016 in a referral center for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil (Instituto René Rachou, Fiocruz). Diagnostic accuracy, based on the cut-off point for VL diagnosis, was 86.2% with RIDASCREEN® Leishmania Ab, 73.3% with Leishmania ELISA IgG + IgM, and 66.7% with IFI Leishmaniose Humana, while IT-LEISH® and DAT-LPC had the lowest accuracy (38.3%), despite high specificity (100% and 95%, respectively). New cut-off points defined with sera from ML patients improved accuracy from 86.2 to 89% (p = 0.64) and 73.3 to 88% (p = 0.04) for RIDASCREEN® Leishmania Ab and Leishmania ELISA IgG + IgM, respectively. Moreover, these tests presented greater sensitivity and immunoreactivity in patients with moderate/severe clinical ML forms. The data of this study suggest that ELISA assays can contribute to laboratory diagnosis, especially for patients with moderate or severe mucosal involvement.

Performance differences among commercially available antigen rapid tests for COVID-19 in Brazil.

A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT-qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer's recommendations and were compared with RT-qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT-PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT-qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission.

Diagnosis of visceral and cutaneous leishmaniasis using loop-mediated isothermal amplification (LAMP) protocols: a systematic review and meta-analysis.

Sensitive, reliable and fast diagnostic tools that are applicable in low-resource settings, at the point of care (PoC), are seen as crucial in the fight against visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Addressing the need for a PoC test, several diagnostic tests, including serological and molecular methods, have been developed and evaluated in the past. One promising molecular method, already implemented for diagnosis of a range of diseases, is the loop-mediated isothermal amplification (LAMP) protocol. In this systematic review and meta-analysis, using a comprehensive search strategy, we focus on studies evaluating the performance of LAMP for the diagnosis of leishmaniasis in humans and other mammals such as dogs, compared with microscopy and/or any other molecular diagnostic method. A meta-analysis, pooling sensitivity and specificity rates and calculating areas under the curve (AUCs) in summary receiver operating characteristic (SROC) plots, was conducted on datasets extracted from studies, grouped by clinical condition and sample type. We found high sensitivity and specificity for LAMP when compared with microscopy and PCR using blood samples, with pooled estimate values of > 90% for all subgroups, corresponding to calculated AUC values > 0.96, except for LAMP compared to microscopy for diagnosis of CL. However, only a limited number of studies were truly comparable. Most of the observed heterogeneity is likely based on true differences between the studies rather than sampling error only. Due to simple readout methods and low laboratory equipment requirements for sample preparation compared to other molecular methods, LAMP is a promising candidate for a molecular (near-)PoC diagnostic method for VL and CL.

Impact assessment of different DNA extraction methods for non-invasive molecular diagnosis of tegumentary leishmaniasis.

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method - commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL.

Correlations between tissue parasite load and common clinical signs in dogs naturally infected by Leishmania infantum.

qPCR is being used for the quantification of parasite load in different tissues of dogs infected by Leishmania infantum with or without clinical manifestations. It may be employed in the diagnosis, monitoring of the infection during treatment, and clinical studies for validation of vaccines. Aimed at enhancing the molecular diagnosis and the subsequent monitoring of the infection, this study evaluated the parasite load in several tissues from dogs infected by Leishmania infantum, showing different clinical status. Thus, the qPCR was performed on skin, conjunctival swab, popliteal lymph node, and bone marrow puncture samples taken from 65 dogs naturally infected by L. infantum. Dogs were divided into three groups per clinical score: group 1 (n = 12), included animals with zero points and no clinical manifestations of the disease; group 2 (n = 35), included animals with a score ranging from 1 to 5 points and moderate clinical manifestations; and group 3 (n = 18), included dogs with a score ranging from 6 to 11 points and intensive clinical manifestations. Another analysis was performed classifying the animals into two groups, considering the presence of, or lack of clinical signs of the disease. Analyses of these results showed that the skin was the tissue with a higher parasite load, followed by popliteal lymph node and bone marrow punctures, and conjunctival swab samples having the lowest loads. Furthermore, the skin was also the tissue with the highest parasite load when evaluating the groups individually. Animals in group 3, with intensive clinical manifestations, showed a higher parasite load in different tissues when compared to animals from groups 1 and 2. Finally, animals with clinical manifestations of the disease showed a higher parasite load when compared to dogs with no manifestations. The importance of the dog as a reservoir of L. infantum in nature is reinforced by the demonstration of skin having the highest amount of parasites/µL in this study's analysis, as well as the fact that skin is the main point of access to the parasite vector. Also, a strong and positive correlation between the intensity of clinical manifestations and the increase of parasite load in the skin was observed. In conclusion, skin was the tissue that was demonstrated to be the best option for the molecular diagnosis of L. infantum infection in dogs with varying clinical statuses used in this study.

Anti-mitochondrial Tryparedoxin Peroxidase Monoclonal Antibody-Based Immunohistochemistry for Diagnosis of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) remains a globally spreading public health problem. Among Latin America countries, Brazil has the greatest number of recorded CL cases with several Leishmania species being associated with human cases. Laboratory diagnosis is one of the major challenges to disease control due to the low accuracy of parasitological techniques, the restricted use of molecular techniques, and the importance of differential diagnosis with regard to several dermatological and systemic diseases. In response, we have developed and validated an immunohistochemistry (IHC) technique for CL diagnosis using anti-mTXNPx monoclonal antibody (mAb). Recombinant Leishmania-mTXNPx was produced and used as an immunogen for mAb production through the somatic hybridization technique. The viability of mAb labeling of Leishmania amastigotes was tested by IHC performed with skin biopsies from hamsters experimentally infected with Leishmania amazonensis, Leishmania braziliensis, and Leishmania guyanensis. The enzymes horseradish peroxidase (IHC-HRP) and alkaline phosphatase (IHC-AP), both biotin-free polymer detection systems, were used in the standardization step. The IHC was further validated with skin biopsies from 49 CL patients diagnosed by clinical examination and quantitative real-time polymerase chain reaction and from 37 patients presenting other dermatological infectious diseases. Other parasitological techniques, such as direct examination and culture, were also performed for confirmed CL patients. Histopathology and IHC were performed for all included patients. Overall, the highest sensitivity was observed for IHC-AP (85.7%), followed by IHC-HRP (79.6%), direct examination (77.6%), histopathological examination (HE; 65.3%), and in vitro culture (49%). Only IHC and HE presented specificity over 90% and were able to detect CL patients regardless of parasite burden (odds ratio > 1.94; 95%CI: 0.34-11.23). A significant increase in positivity rates was observed when IHC-AP was combined with direct examination (95.9%) and HE (93.9%). The IHC techniques evaluated in here detected the main Leishmania species causing CL in Brazil and can support diagnostic strategies for controlling this neglected disease, especially if used in combination with other approaches for an integrative laboratorial diagnosis.

Diagnostic performance of commercially available COVID-19 serology tests in Brazil.

Timely and accurate laboratory testing is essential for managing the global COVID-19 pandemic. Reverse transcription polymerase chain reaction remains the gold-standard for SARS-CoV-2 diagnosis, but several practical issues limit the test's use. Immunoassays have been indicated as an alternative for individual and mass testing. OBJECTIVES: To access the performance of 12 serological tests for COVID-19 diagnosis. METHODS: We conducted a blind evaluation of six lateral-flow immunoassays (LFIAs) and six enzyme-linked immunosorbent assays (ELISAs) commercially available in Brazil for detecting anti-SARS-CoV-2 antibodies. RESULTS: Considering patients with seven or more days of symptoms, the sensitivity ranged from 59.5% to 83.1% for LFIAs and from 50.7% to 92.6% for ELISAs. For both methods, the sensitivity increased with clinical severity and days of symptoms. The agreement among LFIAs performed with digital blood and serum was moderate. Specificity was, in general, higher for LFIAs than for ELISAs. Infectious diseases prevalent in the tropics, such as HIV, leishmaniasis, arboviruses, and malaria, represent conditions with the potential to cause false-positive results with these tests, which significantly compromises their specificity. CONCLUSION: The performance of immunoassays was only moderate, affected by the duration and clinical severity of the disease. Absence of discriminatory power between IgM/IgA and IgG has also been demonstrated, which prevents the use of acute-phase antibodies for decisions on social isolation.

Canine visceral leishmaniasis in area with recent Leishmania transmission: prevalence, diagnosis, and molecular identification of the infecting species.

INTRODUCTION: Canine visceral leishmaniasis (CVL) is an endemic disease in Brazil, and integrated control actions have been adopted by the Brazilian Ministry of Health to control its spread. However, the transmission profile is unknown in areas with recent CVL cases, including Itaúna, located in the Brazilian state of Minas Gerais, where the present study was carried out. METHODS: A total of 2,302 dogs from 12 neighborhoods were serologically tested for canine VL using the current diagnostic protocol adopted by the Brazilian Ministry of Health. Test positivity rate (TPR) and CVL prevalence were determined for each neighborhood. The presence of Leishmania was assessed in 60 seropositive dogs which had been recommended for euthanasia. Twenty-two of them (37%) were asymptomatic, and 38 (63%) were symptomatic for CVL. Parasitological (myeloculture and smear/imprint) and molecular (PCR) methods were employed for Leishmania detection in bone marrow, spleen, mesenteric lymph nodes, and ear skin. The infecting Leishmania species was identified by DNA sequencing. RESULTS: CVL prevalence (per 1,000 dogs) varied from 0.0-166.67, depending on the neighborhood, with a mean of 68.96 (SD 51.38). Leishmania DNA was detected in at least one tissue from all seropositive dogs, with comparable TPR among tissues. Leishmania parasites were identified in most (54/60) seropositive dogs, and the infecting parasite was identified as Leishmania infantum in all of these. CONCLUSIONS: Prevalence of CVL is a contributor to the spread of visceral leishmaniasis in Itaúna.

Canine visceral leishmaniasis in area with recent Leishmania transmission: prevalence, diagnosis, and molecular identification of the infecting species

Abstract INTRODUCTION: Canine visceral leishmaniasis (CVL) is an endemic disease in Brazil, and integrated control actions have been adopted by the Brazilian Ministry of Health to control its spread. However, the transmission profile is unknown in areas with recent CVL cases, including Itaúna, located in the Brazilian state of Minas Gerais, where the present study was carried out. METHODS: A total of 2,302 dogs from 12 neighborhoods were serologically tested for canine VL using the current diagnostic protocol adopted by the Brazilian Ministry of Health. Test positivity rate (TPR) and CVL prevalence were determined for each neighborhood. The presence of Leishmania was assessed in 60 seropositive dogs which had been recommended for euthanasia. Twenty-two of them (37%) were asymptomatic, and 38 (63%) were symptomatic for CVL. Parasitological (myeloculture and smear/imprint) and molecular (PCR) methods were employed for Leishmania detection in bone marrow, spleen, mesenteric lymph nodes, and ear skin. The infecting Leishmania species was identified by DNA sequencing. RESULTS: CVL prevalence (per 1,000 dogs) varied from 0.0-166.67, depending on the neighborhood, with a mean of 68.96 (SD 51.38). Leishmania DNA was detected in at least one tissue from all seropositive dogs, with comparable TPR among tissues. Leishmania parasites were identified in most (54/60) seropositive dogs, and the infecting parasite was identified as Leishmania infantum in all of these. CONCLUSIONS: Prevalence of CVL is a contributor to the spread of visceral leishmaniasis in Itaúna.

Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil.

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Evaluation of a new brand of immunochromatographic test for visceral leishmaniasis in Brazil made available from 2018.

Immunochromatographic tests based on the recombinant antigen K39 represent a major advance in diagnosing visceral leishmaniasis (VL) in recent years. Some performance variations are expected and have occurred in the use of several commercial rapid tests, especially in different geographical settings. This is the first evaluation in the Americas of the test recently provided by the public health system in Brazil for the diagnostic of VL, the OnSite™ Leishmania IgG/IgM Combo. In this first clinical test evaluation, 113 VL-positive patient samples and 73 negative controls were tested and a sensitivity of 91.2% and specificity of 94.5% were observed. These results indicate the need for further analysis and comparisons with the performance of other available commercial tests in order to define the impact of this new test on the quality of VL diagnosis in Brazil.

Meglumine antimoniate intralesional infiltration for localised cutaneous leishmaniasis: a single arm, open label, phase II clinical trial.

BACKGROUND: Cutaneous leishmaniasis (CL) is a world-wide health problem which currently lacks effective, affordable and easy to use therapy. Recently, the meglumine antimoniate (MA) intralesional infiltration was included among the acceptable therapies for New World leishmaniasis. While this approach is attractive, there is currently little evidence to support its use in Americas. OBJECTIVES: The aim of this study was to provide information about effectiveness and safety of a standardised MA intralesional infiltration technique for the treatment of CL. METHODS: It is a single-arm phase II clinical trial conducted at a Brazilian referral centre. CL cases with parasitological confirmation presenting a maximum of three CL-compatible skin lesions were treated with weekly MA intralesional infiltration by using a validated technique, up to a maximum of eight infiltrations. RESULTS: A total of 53 patients (62 lesions) were included. Overall, patients received a median of seven infiltrations (IQR25-75% 5-8) over a median treatment period of 43 days (IQR25-75% 28-52 days). The definitive cure rate at D180 was 87% (95% CI:77-96%). The majority of adverse events were local, with mild or moderate intensity. Bacterial secondary infection of the lesion site was observed in 13% of the treated patients, beside two intensity-three adverse events (hypersensitivity reactions).

Meglumine antimoniate intralesional infiltration for localised cutaneous leishmaniasis: a single arm, open label, phase II clinical trial

BACKGROUND Cutaneous leishmaniasis (CL) is a world-wide health problem which currently lacks effective, affordable and easy to use therapy. Recently, the meglumine antimoniate (MA) intralesional infiltration was included among the acceptable therapies for New World leishmaniasis. While this approach is attractive, there is currently little evidence to support its use in Americas. OBJECTIVES The aim of this study was to provide information about effectiveness and safety of a standardised MA intralesional infiltration technique for the treatment of CL. METHODS It is a single-arm phase II clinical trial conducted at a Brazilian referral centre. CL cases with parasitological confirmation presenting a maximum of three CL-compatible skin lesions were treated with weekly MA intralesional infiltration by using a validated technique, up to a maximum of eight infiltrations. RESULTS A total of 53 patients (62 lesions) were included. Overall, patients received a median of seven infiltrations (IQR25-75% 5-8) over a median treatment period of 43 days (IQR25-75% 28-52 days). The definitive cure rate at D180 was 87% (95% CI:77-96%). The majority of adverse events were local, with mild or moderate intensity. Bacterial secondary infection of the lesion site was observed in 13% of the treated patients, beside two intensity-three adverse events (hypersensitivity reactions).

Epidemiological aspects of vector, parasite, and domestic reservoir in areas of recent transmission and no reported human cases of visceral leishmaniasis in Brazil.

About 97% of the human cases of the American visceral leishmaniasis (VL) occur in Brazil. In the last few years, the disease expanded to medium- and large-sized cities, in which surveillance and control actions have been intensified, in an effort to control VL spreading. Our two-year study was conducted in Belo Horizonte, the sixth most populous city in Brazil, which is endemic for VL. We focused in two particular districts of recent transmission of the disease, with no reported human cases and submitted to minor surveillance and control actions. Our aim was to draw an epidemiological profile of the local situation concerning Lutzomyia vector, Leishmania parasites, and the main domestic reservoirs (dogs). Lutzomyia longipalpis comprised 96.5% of the total phlebotomine sand flies captured and displayed an expressive minimal infection rate by Leishmania infantum (16.7%). Positive correlations were found between the population densities of L. longipalpis, rainfall and temperature. L. infantum was also detected in the cortelezzii complex and, for the first time, in Lutzomyia lloydi. Leishmania braziliensis, an etiological agent of the American cutaneous leishmaniasis, was also identified in L. longipalpis. Among the 1408 dogs serologically tested by standard enzyme-linked and fluorescence immune assays (ELISA/IFA) 3.6% were positive for VL. L. infantum DNA and Leishmania parasites were identified in 100% and 72.5% of the seropositive dogs, respectively. The co-positivity of other diagnostic tests for VL-Leishmania-nested PCR, imprint and myeloculture-was compared to the standard serology. Both symptomatic or asymptomatic dogs displayed an equal average number of positive diagnostic tests for VL. The districts studied display favorable conditions for the rapid spreading of human infection, in terms of L. longipalpis population density, and presence of L. infantum in both vector and main reservoir.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS: Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react. .

Notes on the genus Tunga (Siphonaptera: Tungidae) II--neosomes, morphology, classification, and other taxonomic notes.

This review focuses on the neosomes, morphology, and taxonomy of adult species of the genus Tunga, complementing the previously published data on the phylogeny, ecology, and pathogenic role. Neosomes are structures formed after penetration of adult females into the skin of hosts resulting in significant enlargement, being the most characteristic and most frequently observed form in hosts. Neosomes can be differentiated by shape, measurements, and sites of attachment to principal hosts. The taxonomic value and morphometric data of the most widely used characteristics to separate species - such as frontal curvature, head chaetotaxy, preoral internal sclerotization, ventral and dorsal genal lobes, eyes, maxillary palps, fusion of pronotum and mesonotum, metacoxae, metatarsi chaetotaxy, spermatheca (females), manubrium, basimere, telomere, and phallosome (males) - are comparatively analyzed. The sexes, individual variations, undescribed species, higher taxa, as well as a proposal for division of the genus into two subgenera (Tunga and Brevidigita) are presented (as previously given by Wang). A key for females, males, and gravid females (neosomes) also is included for identifying the 13 known species. Data on host specificity and geographical distribution may also support the identification of Tunga species because some sand fleas and their hosts may have co-evolved.