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.

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.

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.

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.

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.

Neosomes of tungid fleas on wild and domestic animals.

Tunga is the most specialized genus among the Siphonaptera because adult females penetrate into the skin of their hosts and, after mating and fertilization, undergo hypertrophy, forming an enlarged structure known as the neosome. In humans and other warm-blooded animals, neosomes cause tungiasis, which arises due to the action of opportunistic agents. Although its effects on humans and domestic animals are well described in the literature, little is known about the impact of tungiasis on wild animals. This review focuses on the morphology, taxonomy, geographical distribution, hosts, prevalence, sites of attachment, and impact of tungid neosomes on wild and domestic animals. Because neosomes are the most characteristic form of the genus Tunga and also the form most frequently found in hosts, they are here differentiated and illustrated to aid in the identification of the 13 currently known species. Perspectives for future studies regarding the possibility of discovering other sand flea species, adaptation to new hosts, and the transfer of tungids between hosts in natural and modified habitats are also presented.

Establishment of Tunga trimamillata (Siphonaptera: Tungidae) in Brazil.

Tunga trimamillata is a species of sand flea occurring in Ecuador and Peru parasitizing cattle, goat, sheep, swine, and man. This is the first report of this species in Brazil, having been found on the hooves of cows in Barretos, São Paulo State, and Felixlândia, Minas Gerais State, and previously misidentified as Tunga penetrans. A previous report concerning Hydrochoerus hydrochaeris from Rio Novo, Minas Gerais State, may also be attributed to that species of sand flea, a possible the primary host. Given the large geographical distribution of T. trimamillata, the vast cattle population in Brazil, and the high number of individuals subject to the risk of tungiasis, the number of cases attributed to this sand flea will most likely increase over time.

Morphology and growth characteristics of cultured Leptomonas ctenocephali from Ctenocephalides felis felis (Siphonaptera: Pulicidae) of dogs in Brazil.

To confirm the taxonomic identification of a trypanosomatid found in the hindgut, rectum and Malpighian tubules of dog fleas captured in Belo Horizonte, Minas Gerais, Brazil, between April and November of 2005, 910 specimens of Ctenocephalides felis felis were removed from street dogs and dissected, and isolates from their digestive tracts were cultivated in NNN-alpha-MEM medium. Four different morphological forms were observed in culture: long, slender, twisted promastigotes with a long flagellum; short, stubby, non-twisted promastigotes; rounded amastigotes; and cyst-like bodies. Twisted and non-twisted promastigotes were frequently seen forming rosettes, and these two forms presented significant differences (P<0.01) in terms of their morphological characteristics. Unlike the promastigote forms observed throughout the culture period, rounded amastigotes were seen only in the lag phase, and the cyst-like bodies were only seen in the decline phase. The trypanosomatid DNA obtained from the culture was analyzed by the polymerase chain reaction (PCR) method and found to be negative for Leishmania infantum chagasi. Based on the growth pattern, morphological parameters and molecular analysis, the flagellates were confirmed to be Leptomonas ctenocephali. The significance of this infection for animals is also commented.