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Simple Summary: The aim of this study was to gain new insights into the genetic diversity of the parasite Leishmania infantum, which causes leishmaniasis disease in the Mediterranean basin. Twenty-six DNA samples of L. infantum obtained from ten hospital patients in Barcelona and five dogs from Aragon in north-east Spain were analyzed to learn more about how the parasite behaves and spreads. The use of two techniques revealed several genetic variations, some of them previously unreported. Single-nucleotide polymorphism analysis identified genotype G13 as the most common, whereas genotype B was the most frequent according to restriction fragment length polymorphism analysis. Both methods indicated that several genotypes were present in both human and dog samples. By highlighting the genetic diversity of this parasite, these results may help to improve tracking and management of the disease. Increasing knowledge of the parasite will allow scientists to develop better strategies to control its spread and protect both humans and animals from infection. Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*–G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring. [ABSTRACT FROM AUTHOR]

Abstract Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia’s diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed. [ABSTRACT FROM AUTHOR]

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*–G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring.

Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei , and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the APS. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) is a vector-borne disease widely distributed in tropical and subtropical areas of North and South America, Europe, Asia, and Africa. Considering the increasing number of CL cases in recent years and the fact that no study has been conducted to identify CL fauna and vectors in Alborz province, this study was carried out to identify sand flies and CL vectors in this region. Sand flies were collected from August to October 2021 from plain and mountainous indoor and outdoor areas of the region using sticky paper traps and were detected morphologically. DNA was extracted from the midguts of female sand flies. In this study, 1157 sand flies were collected and identified. The number of sand flies caught from indoor and outdoor places was 367 (31.72%) and 790 (68.28%), respectively. Overall, six species of flies were of the genus Phlebotomus (Raynal, 1937), including Phlebotomus papatasi (P. papatasi, 695 [60.07%]; Scopoli, 1786), P. kandelakii (13 [1.12%]; Shchurenkova, 1926), P. sergenti (232 [20.05%]; Parrot, 1917), P. major (14 [1.21%]; Annandale, 1910), P. caucasicus (4 [0.35%]; Marzinowsky, 1917), P. alexandri (18 [1.56%]; Alexandri Sinton, 1920), and four were of the genus Sergentomyia (Artemiev, 1978), including Sergentomyia tiberiadis (109 [9.42%]; Adler, Theodor & Lourie, 1930), Sergentomyia baghdadis (53 [4.58%]), Sergentomyia sintoni (14 [1.21%]; Sintoni Pringle, 1933), Sergentomyia clydei (5 [0.43%]). P. papatasi spp. were dominant in indoor and outdoor places, with a prevalence of 695 (60.07%). The Leishmania major (L. major) gene was identified in five samples of P. papatasi spp. This suggests that P. papatasi is the potential vector spp. in the study area. Moreover, L. major was confirmed as the aetiological agent of CL cases in Alborz province. The identification of vectors and parasite spp. is very important for the treatment and operational planning of disease vectors. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a protozoal and vector-borne disease. World health organization has considered the disease as a neglected tropical disease. Phlebotomus and Lutzumyia species (order: Diptera, family: Psychodidae) are human leishmaniasis vectors in new and old worlds. Sergentomyia spp. (Diptera, Psychodidae) are proven vectors of lizard leishmaniasis. Although some studies have identified human Leishmania parasites in Sergentomyia, their role in parasite circulation is unknown yet. Hence, the parasitological and molecular methods were used to study the possible Leishmania infection of Sergentomyia spp., in the human and canine visceral leishmaniasis endemic area in North West of Iran. Even though Sergentomyia specimens were caught in a dominant number compared to Phlebotomus spp., no Leishmania promastigote or DNA was detected in live-caught or sticky trap-caught specimens, respectively. Sergentomyia spp. are proven vectors of sauroleishmaniasis, and despite several global reports of Leishmania infection in Sergentomyia spp., such findings should be carefully interpreted to avoid false vector incriminations. [ABSTRACT FROM AUTHOR]

Objective: As a global health challenge, especially in Iran, cutaneous leishmaniasis is one of the major health and medical problems in Sistan and Baluchestan Province and plays a significant role in the inhibition of socioeconomic growth of this deprived province. The aim of our study was molecular identification of cutaneous leishmaniasis species using kinetoplast DNA (kDNA) gene in in a small part (Mirjaveh and Reg-e Malek) of Sistan and Baluchestan region of Iran. Methods: This descriptive cross-sectional study was carried out on patients suspected of cutaneous leishmaniasis with epidemiologics information. Samples of the skin lesions and ulcers suspected of cutaneous leishmaniasis were collected from patients with active ulcers, and the collected samples were diagnosed by of parasitologic (direct smears of the lesions and staining them using Giemsa Staining Method) and molecular methods. The specific Polymerase Chain Reaction (PCR) method was applied to the kinetoplast DNA's isolated from the direct smears to identify the genus and species of Leishmania. Finally, the resulting information was analyzed in SPSS software. Results: The microscopic test results of 43 (57.9%) patients of the 76 patients suspected of cutaneous leishmaniasis were positive. In a PCR assay, the Leishmania kDNA fragment was identified in 50 patients (65.8%). Besides, 44 samples were diagnosed with Leishmania major and 6 samples were diagnosed with Leishmania tropica using species-specific primers. The agreement between the results of the molecular method and the parasitology method in the samples in Mirjaveh and Reg-e Malek was (Kappa=0.8) compelling agreement. There was a significant difference between the results of molecular with the parasitologic results obtained in the samples of cutaneous leishmaniasis in the city of Mirjaveh and the Reg-e Malek (Pv=0.05). Conclusion: According to the present research, cutaneous leishmaniasis prevalance caused by Leishmania major in Mirjaveh City in Sistan and Baluchestan Province in Iran was high. Our findings also confirmed the necessity of carrying out direct molecular tests (including direct smears) on the clinical samples of patients for negative results and species identification. [ABSTRACT FROM AUTHOR]

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10-2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels. [ABSTRACT FROM AUTHOR]

Purpose: Surra is an economically important livestock disease in many low- and middle-income countries, including those of Northern Africa. The disease is caused by the biting fly-transmitted subspecies Trypanosoma brucei evansi, which is very closely related to the tsetse-transmitted subspecies T. b. brucei and the sexually transmitted subspecies T. b. equiperdum. At least two phylogenetically distinct groups of T. b. evansi can be distinguished, called type A and type B. These evolved from T. b. brucei independently. The close relationships between the T. brucei subspecies and the multiple evolutionary origins of T. b. evansi pose diagnostic challenges. Methods: Here we use previously established and newly developed PCR assays based on nuclear and mitochondrial genetic markers to type the causative agent of recent trypanosome infections of camels in Southern Algeria. Results/conclusion: We confirm that these infections have been caused by T. b. evansi type A. We also report a newly designed PCR assay specific for T. b. evansi type A that we expect will be of diagnostic use for the community. [ABSTRACT FROM AUTHOR]

Background: Ethiopia has a high burden of visceral leishmaniasis. Recently, there was a significant increase in cases in the South Omo Zone. This study aims to assess the prevalence of Leishmania donovani infection and its associated factors., Methods: A household-based cross-sectional study was carried out in January 2023 in the South Omo Zone in Ethiopia. Dried blood spot samples were collected from 382 randomly selected study participants. Direct agglutination test (DAT) and kinetoplast DNA real-time PCR tests were performed to detect L. donovani infection. Participants' sociodemographic, clinical and risk factors for L. donovani infection data were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyze the data. Febrile cases were checked for malaria with a multiplex PCR assay., Results: Overall prevalence of L. donovani infection among the sampled population was 32.5% (n=124), of which 41.1% (n=51) was detected by PCR, 33.9% (n=42) by DAT and 25.0% (n=31) by both tests. The majority of the positives were from the Logira (28.2%; n=35) and Dilbayne (29.0%; n=36) villages. Participants residing in Logira (adjusted OR [AOR]: 5.80; 95% CI 1.85 to 18.15) and Dilbayne (AOR: 3.38; 95% CI 1.15 to 9.96) villages and owning cows (AOR: 2.31; 95% CI 1.03 to 5.15) showed an association with Leishmania infection. Plasmodium falciparum was detected in 3.4% (n=2) of 59 febrile participants., Conclusions: The prevalence of L. donovani infection in the South Omo Zone is high. Further research on the role of cows in the transmission cycle is needed to design the best strategy to control Leishmania infection in the South Omo Zone. Such interventions should focus on the Logira and Dilbayne villages, where most of the infections were identified., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named "non-JPC like", may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability. [ABSTRACT FROM AUTHOR]

Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. To assess the frequency and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania infection were examined for CL by a nested polymerase chain reaction (nPCR). Seven (25.9%) out of 27 human patients scored positive for Leishmania spp. (i.e., L. major in five patients from Riyadh and L. tropica in two patients from Al-Qaseem). Out of 311 dogs, five (1.6%) were infected by L. tropica. Data herein presented demonstrate the occurrence of L. tropica in dogs and humans in Saudi Arabia, as well as the occurrence of L. major in humans. [ABSTRACT FROM AUTHOR]

Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces , inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani , but much less so against Trypanosoma evansi . Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%-50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei , and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs., Competing Interests: The authors declare no conflict of interest.

The recently developed ultrastructure expansion microscopy (U-ExM) technique allows us to increase the spatial resolution within a cell or tissue for microscopic imaging through the physical expansion of the sample. In this study, we validate the use of U-ExM in Trypanosoma brucei measuring the expansion factors of several different compartments/organelles and thus verify the isotropic expansion of the cell. We furthermore demonstrate the use of this sample preparation protocol for future studies by visualizing the nucleus and kDNA, as well as proteins of the cytoskeleton, the basal body, the mitochondrion and the endoplasmic reticulum. Lastly, we discuss the challenges and opportunities of U-ExM.

Background: Cutaneous Leishmaniasis, is endemically observed in different parts of Iran in two forms of anthroponotic and zoonotic. The identification of both species and the type of disease are beneficial for treatment and prevention. Microscopic identification of Leishmania species has not provided promising efficacy. The aim of this study was to determine the Leishmania species that are responsible for cutaneous leishmaniasis in Zahedan/ Iran by using PCR and PCR-RFLP techniques. Method: Direct smears were obtained from cutaneous leishmaniasis suspected individuals with low parasitemia in cutaneous lesions referred to Zahedan health centers. Eventually, the DNA was extracted from smears using DNA extraction kit. PCR was used to amplify both Leishmania kinetoplastic DNA (kDNA) and ITS1 locus of ribosomal DNA. Additionally, PCR-RFLP on ITS1 products was conducted to determine parasite species. Results: PCR-RFLP test (detecting ITS1 locus) on all positive samples in microscopic analysis led to the identification of Leishmania major in 52 samples (54.7%), and 43 cases were detected to have Leishmania tropica (45.3%). On the other hand, kDNA-PCR results indicated a frequency of 68 (55.7%) for L. major and 54 (44.3%) for L. tropica. Conclusion: Due to the high frequency of kDNA in parasitic genome, PCR-kDNA compared to PCR-RFLP shows a higher efficiency and accuracy not only in identifying infection, but also in determining parasite species, especially among the patients with fewer lesions. This study also indicates that both L. tropica and L. major could be found in Zahedan, with a greater tropical leishmaniasis endemicity.

Cutaneous leishmaniasis (CL) is a major health problem in Iran, with a heavy burden on human health and society. There is little knowledge about the molecular epidemiology of the disease, as well as phylogenetic relationship of causative agents in south‐eastern Iran. The aim of the present study was to investigate the molecular aspects of CL, especially atypical CL in the Bam district, Kerman province, south‐eastern Iran, as an endemic region of CL in Iran. The smears were collected from lesion samples of 353 patients clinically suspected to CL, who attended local health centres in the Bam district during 2016–2017. Direct smears were examined for Leishmania parasites using the Giemsa staining technique. Amplification of kinetoplast DNA (kDNA) and the ribosomal internal transcribed spacer 1(ITS‐1) gene were carried out using polymerase chain reaction (PCR). Then, the ITS1‐PCR products were sequenced for phylogenetic analysis. Overall, 278 cases were confirmed as CL by microscopic examination of Giemsa‐stained slides. Clinical presentation of the lesions was basically of two types: (a) typical lesions and (b) atypical including lupoid ulcers, sporotrichoid, nodular and exudative lesions. The PCR assay on all specimens of skin lesions proved L. tropica as the main pathogenic agent. Phylogenic analysis revealed high similarity among isolates from the Bam district in the south‐east with isolates from Birjand in eastern Iran, as well as with isolates from Herat province in western Afghanistan. The study provided valuable information concerning the genetic diversity of the parasite as one of the factors influencing the clinical manifestations in CL in south‐eastern Iran, which could be the basis for planning future control strategies. [ABSTRACT FROM AUTHOR]

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

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Leishmaniasis has an important impact on global public health, and the common form of the disease is cutaneous form as well in Iran. Different species of Leishmania parasite make variable clinical manifestations, so prompt diagnosis and recognition at the species level are important due to their impact on the treatment and outcome of the disease. We aimed to examine the potential existence of the Leishmania parasite genome in the exudate materials derived from lesions of the cutaneous leishmaniasis suspected patients referred to Varamin Health Center Laboratory, that were reported negative microscopically. Regarding the object of the study, kDNA-Nested-PCR was used. A 570 bp band equal to what expected for Leishmania major was amplified in 18 out of 29 tested samples (62%). Findings indicate the effectivness of kDNA as a high copy number gene to avoid false-negative results. [ABSTRACT FROM AUTHOR]

Aims: Cutaneous leishmaniasis (CL) is a skin infection that causes various forms of ulcers and also remains scars even after treatment. This disease is prevalent in many countries of Middle East including Iran. Since determining the species of the parasite is important for prevention and control programs, this study was conducted to identify Leishmania species in Aran and Bidgol, Isfahan province, center of Iran. Materials and Methods: This cross-sectional study was carried out on 112 CL suspected patients who referred to health centers of Aran and Bidgol. Serosity of the wound was collected, and amastigote form was detected by microscopic method. After extraction of DNA from serosity, kDNA-polymerase chain reaction (PCR) was used to identify Leishmania species. Results: Fifty-four of all suspected CL samples (48.2%) were positive microscopically, while 55 (49.1%) were positive using kDNA-PCR. The results of PCR revealed that 51 isolates (92.7%) were Leishmania major and 4 (7.3%) Leishmania tropica, respectively. The most lesion form caused by L. major was papular or volcanic-like, while all of wounds caused by L. tropica were papular/nodular forms. Conclusion: The results of the study indicated that the predominant species was L. major and zoonotic CL is more prevalent in this region.

Cutaneous leishmaniasis (CL) is a widespread health problem and considered one of the endemic diseases in Iraq. The dermal lesion occurs due to an obligate intracellular Leishmania parasite, which transmits by the bite of the infected female sandfly. This study aims to identify Leishmania species in Thi-Qar province/ South of Iraq and detect IL-17 level in serum of infected patients with L. tropica. The study was conducted in three local locations, Al-Hussein Teaching, Al-Suq Al-Shyokh General, and Al-Shatrah General Hospitals in the province for the period from the beginning of November 2018 to the end of October 2019. After clinical diagnosis, eighty out of two hundred forty-seven samples were selected for molecular examination by nested-PCR technique, where the lesion edge was injected by normal saline and pulled again to obtain the parasite DNA. Also, a measure of the IL-17 concentration level in serum of the patients with ELISA. The findings of the electrophoresis of the kinetoplast minicircle DNA gene showed that 65 samples were positive for cutaneous leishmaniasis, and observed two species of Leishmania spp. in the study area, 46 (57.5%) samples were L. tropica at 750bp and 19 (23.75%) samples were L. major. Serum IL-17 concentration recorded a significant increase among patients infected with L. tropica at different infection stages than control samples. Generally, the Nested-PCR technique is an accurate method for diagnosing clinical samples and molecular determination of Leishmania parasites. L. tropica is the dominant specie that caused CL in Thi-Qar province, while L. major recorded a low incidence. [ABSTRACT FROM AUTHOR]

Wild mammals are susceptible to infection by Leishmania parasites. Although canine leishmaniasis is widely distributed in mainland Portugal, the sylvatic cycle of the parasite remains poorly understood. In this study, the occurrence of L. infantum in wild carnivores from Portugal was assessed by molecular screening of 132 hunted or accidentally road-killed animals. Spleen samples from Egyptian mongoose, red fox, stone marten, common genet and European badger were tested by amplification of Leishmania kinetoplastid DNA and ITS1. Five egyptian mongoose were confirmed Leishmania DNA-positive by kDNA-PCR. Phylogenetic analysis of a kDNA amplicon sequence clustered the strain with L. infantum sequences from Portugal. These results may suggest that L. infantum strains circulating in wild animals are genetically related with strains from more humanized settings. Exposure of wild carnivores to Leishmania infantum emphasizes the need of systematic studies to clarify the role of several taxa in the eco-epidemiology of leishmaniasis in Portugal, particularly in areas of carnivore species synanthropy and wherein disease control in the domestic population is inefficient or insufficient. Image 1 • Leishmania sp. occurrence in wild carnivores (n = 132) from Portugal was assessed by PCR. • Egyptian mongoose, red fox, stone marten, common genet and European badger were tested. • Five Egyptian mongoose were confirmed Leishmania positive by kDNA-PCR. • The 402-nt long sequence of mongoose kDNA amplicon displayed high similarity (>97,51%) with L. infantum from dogs and humans. • Phylogenetic analysis by Maximum Likelihood clustered wild mongoose strain with urban L. infantum from Portugal. [ABSTRACT FROM AUTHOR]

Leishmania infantum produces an endemic disease in the Mediterranean Basin that affects humans and domestic and wild mammals, which can act as reservoir or minor host. In this study, we analyzed the presence of the parasite in wild American minks, an invasive species in Spain. We screened for L. infantum DNA by PCR using five primer pairs: Two targeting kinetoplast DNA (kDNA), and the rest targeting the ITS1 region, the small subunit of ribosomal RNA (SSU) and a repetitive sequence (Repeat region). The detection limit was determined for each method using a strain of L. infantum and a bone marrow sample from an infected dog. PCR approaches employing the Repeat region and kDNA (RV1/RV2 primers) showed higher sensitivity than the other PCR methods when control samples were employed. However, only PCR of the Repeat region and nested PCR of SSU (LnSSU) detected the parasite in the samples, while the other three were unable to do so. The majority of the analyzed animals (90.1%) tested positive. American mink may act as an incidental host of the disease for other mammals and should be further investigated, not only for their negative impact on the local fauna, but also as carriers of zoonotic diseases.

Abstract Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. Results The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts. Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum. Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used. Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ∼100 parasite mL−1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing. Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.

Background: Dogs can act as reservoirs of canine leishmaniasis, caused by Leishmania species. The aims of this study were to determine the prevalence of canine leishmaniasis using a PCR technique among stray dogs living in three provinces of Saudi Arabia, Riyadh, Al-Ahsa Oasis and Al-Qaseem, where the disease is endemic; and to identify and document different Leishmania to species levels Methods: This cross-sectional investigation was conducted, from Mar 2016 to Apr 2018, in three parts of Saudi Arabia: Central province (Riyadh), Eastern province (Al-Ahsa Oasis) and Al-Qaseem province. Blood samples were collected from 526 dogs; 40 presented cutaneous nodules so were suspected clinically of cutaneous leishmaniasis. Biopsy tissue collections and parasite cultures were performed. A generic kDNA was performed using different primers for Leishmania differentiation. Results: All blood samples were negative for Leishmania infantum infection by molecular analysis, though forty dogs had thick cutaneous lesions in different parts of their body. Four dogs’ skin lesions were associated with dermatitis, splenomegaly and lymphadenomegaly. Parasite culture was used to diagnose cutaneous leishmaniasis, identifying 31/40 (77.5%) positive samples. Overall, of 526 samples, the prevalence of L. major and L. tropica was found to be 4% and 1.9%, respectively. Gender and age had a significant effect on Leishmania prevalence: (P=0.0212 and 0.0357), respectively. Conclusion: This was the first molecular study of dog leishmaniasis from Saudi Arabia of dogs confirmed to have cutaneous leishmaniasis. Further epidemiological and molecular investigations of domestic and wild canine infections with L. major, L. tropica and L. infantum in endemic and nonendemic areas of Saudi Arabia are required, for leishmaniasis control.

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis , the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization. • Rbp38 prior described as a mitochondrial protein, in Leishmania sp. is also found in the nucleus. • L. amazonensis Rbp38 (LaRbp38) dual subcellular localization is cell cycle-dependent. • LaRbp38 has a monopartite non-classical NLS that binds to importin-alpha. • LaRbp38 binds telomeric G-rich DNA and kDNA. • LaRbp38 DNA-binding region is located between amino acid residues 95–235. [ABSTRACT FROM AUTHOR]

Background: Dogs can act as reservoirs of canine leishmaniasis, caused by Leishmania species. The aims of this study were to determine the prevalence of canine leishmaniasis using a PCR technique among stray dogs living in three provinces of Saudi Arabia, Riyadh, Al-Ahsa Oasis and Al-Qaseem, where the disease is endemic; and to identify and document different Leishmania to species levels. Methods: This cross-sectional investigation was conducted, from Mar 2016 to Apr 2018, in three parts of Saudi Arabia: Central province (Riyadh), Eastern province (Al-Ahsa Oasis) and Al-Qaseem province. Blood samples were collected from 526 dogs; 40 presented cutaneous nodules so were suspected clinically of cutaneous leishmaniasis. Biopsy tissue collections and parasite cultures were performed. A generic kDNA was performed using different primers for Leishmania differentiation. Results: All blood samples were negative for Leishmania infantum infection by molecular analysis, though forty dogs had thick cutaneous lesions in different parts of their body. Four dogs' skin lesions were associated with dermatitis, splenomegaly and lymphadenomegaly. Parasite culture was used to diagnose cutaneous leishmaniasis, identifying 31/40 (77.5%) positive samples. Overall, of 526 samples, the prevalence of L. major and L. tropica was found to be 4% and 1.9%, respectively. Gender and age had a significant effect on Leishmania prevalence: (P=0.0212 and 0.0357), respectively. Conclusion: This was the first molecular study of dog leishmaniasis from Saudi Arabia of dogs confirmed to have cutaneous leishmaniasis. Further epidemiological and molecular investigations of domestic and wild canine infections with L. major, L. tropica and L. infantum in endemic and nonendemic areas of Saudi Arabia are required, for leishmaniasis control. [ABSTRACT FROM AUTHOR]

Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes – kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts. Highlights Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential. Many proteins involved in segregation of kinetoplasts have been identified. A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC). A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation. Subcomplexes of TACAPs that form in vivo have been described. Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast. New kinetoplast-associated proteins have been identified. We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable. [ABSTRACT FROM AUTHOR]

Background: Cutaneous Leishmaniasis, is endemically observed in different parts of Iran in two forms of anthroponotic and zoonotic. The identification of both species and the type of disease are beneficial for treatment and prevention. Microscopic identification of Leishmania species has not provided promising efficacy. The aim of this study was to determine the Leishmania species that are responsible for cutaneous leishmaniasis in Zahedan/ Iran by using PCR and PCR-RFLP techniques. Method: Direct smears were obtained from cutaneous leishmaniasis suspected individuals with low parasitemia in cutaneous lesions referred to Zahedan health centers. Eventually, the DNA was extracted from smears using DNA extraction kit. PCR was used to amplify both Leishmania kinetoplastic DNA (kDNA) and ITS1 locus of ribosomal DNA. Additionally, PCR-RFLP on ITS1 products was conducted to determine parasite species. Results: PCR-RFLP test (detecting ITS1 locus) on all positive samples in microscopic analysis led to the identification of Leishmania major in 52 samples (54.7%), and 43 cases were detected to have Leishmania tropica (45.3%). On the other hand, kDNA-PCR results indicated a frequency of 68 (55.7%) for L. major and 54 (44.3%) for L. tropica. Conclusion: Due to the high frequency of kDNA in parasitic genome, PCR-kDNA compared to PCR-RFLP shows a higher efficiency and accuracy not only in identifying infection, but also in determining parasite species, especially among the patients with fewer lesions. This study also indicates that both L. tropica and L. major could be found in Zahedan, with a greater tropical leishmaniasis endemicity. [ABSTRACT FROM AUTHOR]

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10−4–1 × 10−6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

Background: Visceral leishmaniasis (VL), so-called Kala-azar is a life threating parasitic infectious disease caused by Leishmania spp. L. infantum is the main causative agent for Mediterranean form of Kala-azar which is endemic in northeastern Iran. This study attempted to investigate existence of canine visceral leishmaniasis (CVL) in Khorasan Razavi. Methods: Between 2014 and 2016, tissue samples collected from spleen and liver of 192 stray dogs were examined to investigate existence of L. infantum. Kinetoplast DNA (k-DNA) PCR was performed to identify the species of parasites. The positive PCR products were sequenced in both directions to confirm the kDNA PCR results Results: Among samples obtained from 192 dogs, kinetoplast DNA of L. infantum was detected in two female dogs. L. infantum was confirmed by sequence analysis of PCR products. Conclusion: Our data confirm stray dogs play as potential reservoirs for VL in this province. Further investigation will be necessary to clear role of stray dogs in the transmission of L. infantum to human and domestic dogs.

Background: The fatal form of leishmaniasis is visceral form (VL), found in some of the countries in the world. Visceral leishmaniasis has been reported sporadically from all provinces in Iran, including Lorestan. This study aimed to characterize parasite species in DAT positive and some of the DAT negative human blood samples of Delfan district, Lorestan Province, central Iran. Methods: Blood samples were collected from different geographical areas of Delfan. Serum was used for DAT test and remained part of molecular study. DNA was extracted by using DNG-plus extracted kit (Cinagen, Iran). Poly­merase chain reaction amplification of Leishmania kDNA and PCR-RFLP of ITS1 was done to identify Leishmania species. Some amplicons were sequenced, submitted to GenBank and analyzed by BLASTn. Results: Expected band of kDNA for L. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-Leishmania antibody at different titers or were negative serologically. Using BLASTn, 93% similarity with L. infantum has been shown. The rDNA-ITS1 was amplified only in 9 samples (4.7%). RFLP pattern was similar to what expected for L. infantum. Conclusion: A new emerging hypo-endemic focus, caused by L. infantum, is going to be established in Delphan District, Lorestan Province. Further studies on vector and reservoirs are necessary for the region and other parts of Lorestan Province.

Aims: Cutaneous leishmaniasis (CL) is a skin infection that causes various forms of ulcers and also remains scars even after treatment. This disease is prevalent in many countries of Middle East including Iran. Since determining the species of the parasite is important for prevention and control programs, this study was conducted to identify Leishmania species in Aran and Bidgol, Isfahan province, center of Iran. Materials and Methods: This cross-sectional study was carried out on 112 CL suspected patients who referred to health centers of Aran and Bidgol. Serosity of the wound was collected, and amastigote form was detected by microscopic method. After extraction of DNA from serosity, kDNA-polymerase chain reaction (PCR) was used to identify Leishmania species. Results: Fifty-four of all suspected CL samples (48.2%) were positive microscopically, while 55 (49.1%) were positive using kDNA-PCR. The results of PCR revealed that 51 isolates (92.7%) were Leishmania major and 4 (7.3%) Leishmania tropica, respectively. The most lesion form caused by L. major was papular or volcanic-like, while all of wounds caused by L. tropica were papular/nodular forms. Conclusion: The results of the study indicated that the predominant species was L. major and zoonotic CL is more prevalent in this region. [ABSTRACT FROM AUTHOR]

Background: The fatal form of leishmaniasis is visceral form (VL), found in some of the countries in the world. Visceral leishmaniasis has been reported sporadically from all provinces in Iran, including Lorestan. This study aimed to characterize parasite species in DAT positive and some of the DAT negative human blood samples of Delfan district, Lorestan Province, central Iran. Methods: Blood samples were collected from different geographical areas of Delfan. Serum was used for DAT test and remained part of molecular study. DNA was extracted by using DNG-plus extracted kit (Cinagen, Iran). Polymerase chain reaction amplification of Leishmania kDNA and PCR-RFLP of ITS1 was done to identify Leishmania species. Some amplicons were sequenced, submitted to GenBank and analyzed by BLASTn. Results: Expected band of kDNA for L. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-Leishmania antibody at different titers or were negative serologically. Using BLASTn, 93% similarity with L. infantum has been shown. The rDNA-ITS1 was amplified only in 9 samples (4.7%). RFLP pattern was similar to what expected for L. infantum. Conclusion: A new emerging hypo-endemic focus, caused by L. infantum, is going to be established in Delphan District, Lorestan Province. Further studies on vector and reservoirs are necessary for the region and other parts of Lorestan Province. [ABSTRACT FROM AUTHOR]

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation. [ABSTRACT FROM AUTHOR]

We recently described a new component (TAC102) of the mitochondrial genome segregation machinery (mtGSM) in the protozoan parasite Trypanosoma brucei. T. brucei belongs to a group of organisms that contain a single mitochondrial organelle with a single mitochondrial genome (mt-genome) per cell. The mt-genome consists of 5000 minicircles (1 kb) and 25 maxicircles (23 kb) that are catenated into a large network. After replication of the network its segregation is driven by the separating basal bodies, which are homologous structures to the centrioles organizing the spindle apparatus in many eukaryotes. The structure connecting the basal body to the mt-genome was named the Tripartite Attachment Complex (TAC) owing its name to the distribution across three areas in the cell including the two mitochondrial membranes.

Trypanosomatida parasites, such as Trypanosoma and Leishmania, are the cause of deadly diseases in many third world countries. The three dimensional structure of their mitochondrial DNA, known as kinetoplast DNA (kDNA), is unique since it is organized into several thousands of minicircles that are topologically linked. How and why the minicircles form such a network have remained unanswered questions. In our previous work we have presented a model of network formation that hypothesizes that the network is solely driven by the confinement of minicircles. Our model shows that upon confinement a percolation network forms. This network grows into a space filling network, called saturation network, upon further confinement of minicircles. Our model also shows, in agreement with experimental data, that the mean valence of the network (that is, the average number of minicircles topologically linked to any minicircle in the network) grows linearly with minicircle density. In our previous studies however we disregarded DNA flexibility and used rigid minicircles to model DNA, here we address this limitation by allowing minicircles to be flexible. Our numerical results show that the topological characteristics that describe the growth and topology of the minicircle networks have similar values to those observed in the case of rigid minicircles suggesting that these properties are robust and therefore a potentially adequate description of the networks observed in Trypanosomatid parasites.

Background: Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. Methods: DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. Results: The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. Conclusions: A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) is a vector-borne disease widely distributed in tropical and subtropical areas of North and South America, Europe, Asia, and Africa. Considering the increasing number of CL cases in recent years and the fact that no study has been conducted to identify CL fauna and vectors in Alborz province, this study was carried out to identify sand flies and CL vectors in this region. Sand flies were collected from August to October 2021 from plain and mountainous indoor and outdoor areas of the region using sticky paper traps and were detected morphologically. DNA was extracted from the midguts of female sand flies. In this study, 1157 sand flies were collected and identified. The number of sand flies caught from indoor and outdoor places was 367 (31.72%) and 790 (68.28%), respectively. Overall, six species of flies were of the genus Phlebotomus (Raynal, 1937), including Phlebotomus papatasi ( P. papatasi , 695 [60.07%]; Scopoli, 1786), P. kandelakii (13 [1.12%]; Shchurenkova, 1926), P. sergenti (232 [20.05%]; Parrot, 1917), P. major (14 [1.21%]; Annandale, 1910), P. caucasicus (4 [0.35%]; Marzinowsky, 1917), P. alexandri (18 [1.56%]; Alexandri Sinton, 1920), and four were of the genus Sergentomyia (Artemiev, 1978), including Sergentomyia tiberiadis (109 [9.42%]; Adler, Theodor & Lourie, 1930), Sergentomyia baghdadis (53 [4.58%]), Sergentomyia sintoni (14 [1.21%]; Sintoni Pringle, 1933), Sergentomyia clydei (5 [0.43%]). P. papatasi spp. were dominant in indoor and outdoor places, with a prevalence of 695 (60.07%). The Leishmania major ( L. major ) gene was identified in five samples of P. papatasi spp. This suggests that P. papatasi is the potential vector spp. in the study area. Moreover, L. major was confirmed as the aetiological agent of CL cases in Alborz province. The identification of vectors and parasite spp. is very important for the treatment and operational planning of disease vectors., Competing Interests: The authors declare that they have no conflict of interest.

The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ± 1 °C for satellite-DNA and 78.1 °C ± 1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10−3 parasite or 240 target copies, and for kDNA, 2 × 10−4 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines. • kDNA and satellite DNA Sybr Green real-time PCR were similar to T. cruzi detection. • kDNA qPCR achieved a better analytical sensitivity. • sat-qPCR gave better specificity results. [ABSTRACT FROM AUTHOR]

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.