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Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis

Open AccessPublished:December 01, 2021DOI:https://doi.org/10.1016/j.ijid.2021.11.029

      Highlights

      • Visceral leishmaniasis can occur due to co-infection with several Leishmania spp.
      • Co-infection in visceral leishmaniasis changes our understanding of the disease.
      • Several trypanosomatids can be identified per sample using this protocol.
      • Amplicon-based next-generation sequencing is a cost-effective method in molecular epidemiological studies.

      ABSTRACT

      Visceral leishmaniasis (VL) is a mammalian protozoal disease propagated in the Americas by female phlebotomine sandflies, mainly caused by Leishmania infantum. However, in recent years, cases of VL caused by different Leishmania species, such as L. amazonensis and L. colombiensis, have been reported in the continent. This study used an amplicon-based next-generation sequencing approach to identify VL aetiologic species using high-depth sequencing targeting a region on the Heat Shock Protein 70 gene. In this first approach, six samples from five patients diagnosed with VL were selected and analysed to identify DNA of Leishmania spp. All samples harboured DNA of L. infantum; five samples were found to be co-infected with other Leishmania spp. or with Trypanosoma cruzi, and just one sample was mono-infected with L. infantum. This study demonstrates the usefulness of this methodology to identify trypanosomatid co-infections in clinical samples, which presents an interesting study panorama considering their biological, clinical and epidemiological implications.

      Graphical abstract

      Keywords

      Visceral leishmaniasis (VL) is a neglected tropical disease affecting at least 57 countries around the tropical and subtropical equatorial belt. VL is associated with Leishmania donovani species complex (
      • Franssen SU
      • Durrant C
      • Stark O
      • Moser B
      • Downing T
      • Imamura H
      • et al.
      Global genome diversity of the Leishmania donovani complex.
      ) and, to a lesser extent, Leishmania enriettii (
      • Paranaiba LF
      • Pinheiro LJ
      • Torrecilhas AC
      • Macedo DH
      • Menezes-Neto A
      • Tafuri WL
      • et al.
      Leishmania enriettii (Muniz & Medina, 1948): a highly diverse parasite is here to stay.
      ). In addition, sporadic cases of human VL have been associated with Leishmania colombiensis (
      • Rodriguez-Bonfante C
      • Bonfante-Garrido R
      • Grimaldi G
      • Momen H
      • Cupolillo E.
      Genotypically distinct Leishmania colombiensis isolates from Venezuela cause both cutaneous and visceral leishmaniasis in humans.
      ), Leishmania amazonensis (
      • Barral A
      • Badaró R
      • Barral-Netto M
      • Grimaldi G
      • Momem H
      • Carvalho EM.
      Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis.
      ;
      • Aleixo JA
      • Nascimento ET
      • Monteiro GR
      • Fernandes MZ
      • Ramos AMO
      • Wilson ME
      • et al.
      Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy.
      ), Leishmania major and Leishmania tropica (
      • Fakhar M
      • Pouladfar GR
      • Alborzi A
      • Kadivar MR
      • Hatam GR
      • Motazedian MH.
      Isolation of Leishmania tropica from a patient with visceral leishmaniasis and disseminated cutaneous leishmaniasis, Southern Iran.
      ;
      • Al Balushi A
      • Khamis F
      • Klaassen CHW
      • Gangneux J-P
      • Van Hellemond JJ
      • Petersen E.
      Double infection with Leishmania tropica and L. major in an HIV patient controlled with high doses of amphotericin B.
      ).
      Recently, trypanosomatid co-infections have been reported from asymptomatic patients in areas endemic for both leishmaniasis and trypanosomiasis in Brazil (
      • Mendes DG
      • Lauria-Pires L
      • Nitz N
      • Lozzi SP
      • Nascimento RJ
      • Monteiro PS
      • et al.
      Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon: human exposure to mixed kinetoplastid infections.
      ); from symptomatic patients in Bolivia (
      • Bastrenta B
      • Mita N
      • Buitrago R
      • Vargas F
      • Flores M
      • Machane M
      • et al.
      Human mixed infections of Leishmania spp. and Leishmania–Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme.
      ); and from patients with cutaneous leishmaniasis in Bolivia (
      • Bastrenta B
      • Mita N
      • Buitrago R
      • Vargas F
      • Flores M
      • Machane M
      • et al.
      Human mixed infections of Leishmania spp. and Leishmania–Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme.
      ), Peru (
      • Veland N
      • Adaui V
      • Llanos-Cuentas A
      • Arevalo J
      • Boggild AK
      • Valencia BM
      • et al.
      Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis.
      ) and Mexico (
      • Monroy-Ostria A
      • Nasereddin A
      • Monteon VM
      • Guzmán-Bracho C
      • Jaffe CL.
      ITS1 PCR-RFLP diagnosis and characterization of leishmania in clinical samples and strains from cases of human cutaneous leishmaniasis in states of the Mexican Southeast.
      ). As there are increasing reports of co-infections with Leishmania species, and capitalizing on the advantages of a metagenomics approach for detection of a wide range of pathogens, the objective of this study was to demonstrate co-infections in patients with VL using amplicon-based next-generation sequencing (NGS).
      Three serum and three bone marrow aspirate smears were selected from five patients with parasitological or serological diagnosis of VL (Table 1). DNA extracted from each sample was used for Leishmania spp. identification by Sanger sequencing and HSP70-amplicon-based NGS as per protocol (
      • Patiño LH
      • Castillo-Castañeda AC
      • Muñoz M
      • Jaimes JE
      • Luna-Niño N
      • Hernández C
      • et al.
      Development of an amplicon-based next-generation sequencing protocol to identify Leishmania species and other trypanosomatids in leishmaniasis endemic areas.
      ). Leishmania spp. were identified in all samples (Table S1, see online supplementary material). As expected, considering the nature of each sequencing method (
      • Voelkerding KV
      • Dames SA
      • Durtschi JD.
      Next-generation sequencing: from basic research to diagnostics.
      ), Sanger sequencing detected one species per sample: Leishmania infantum or L. amazonensis. In contrast, with amplicon-based NGS, only one sample was found to be solely infected with L. infantum, and the remaining five samples were co-infected by two or more species (Figure 1), including L. infantum, L. amazonensis, Leishmania braziliensis, Leishmania panamensis, Leishmania lindenbergi, Leishmania naiffi and Trypanosoma cruzi.
      Table 1Demographic and clinical characteristics of patients with visceral leishmaniasis selected for the study.
      Sample IDSample typeDepartment of occurrenceMunicipality of occurrenceRural zoneSexAgeWeightComorbiditiesEvolution time (days)SignsTreatment
      H78BMA smearSucreOvejasAlmagraMale1 year10 kg15Fever, splenomegaly, anaemia, thrombocytopeniaN-methyl glucamine
      H83BMA smearCesarLa Jagua de IbiricoFemale10 years25 kg40Fever, splenomegaly, anaemia, leukopenia.Amphotericin B
      H79BMA smearTolimaChaparralRisaralda-CalarmaMale37 years45 kgHIV positive, on antiretroviral treatment since 20115Skin lesions on upper limbs, hepatomegaly, splenomegaly, anaemia, pancytopeniaN-methyl glucamine
      H85Serum
      H86SerumSantanderGirónPalogordoFemale25 years23.3 kgImmunocompromised secondary to inspection of intestinal malabsorption syndrome76Hepatomegaly, splenomegaly, anaemia, pancytopenia, 1-year history of chronic diarrhoea, cachexia, depressionAmphotericin B
      H720SerumVenezuela, Aragua
      Attended Herasmo Meoz Hospital, reported by Public Health Laboratory of Norte de Santander (Colombia).
      MaracayFemale11 months8 kgDifferential diagnosis with Chagas disease. Total antigens (TestELISA Chagas Grupo Bios III), recombinant antigens (Vircell), IIF T. cruzi NR 1/16

      IIF L. infantum R 1/256
      15Fever, anaemia, pancytopenia, hepatomegaly, splenomegaly, coughN-methyl glucamine
      BMA, bone marrow aspirate; IIF, indirect immunofluorescence; NR, non-reactive; R, reactive; T. cruzi, Trypanosoma cruzi; L. infantum, Leishmania infantum.
      a Attended Herasmo Meoz Hospital, reported by Public Health Laboratory of Norte de Santander (Colombia).
      Figure 1
      Figure 1Relative abundance of Leishmania spp. identified in patients with visceral leishmaniasis. Relative abundance of Leishmania spp. and Trypanosoma cruzi found in the analysed samples based on HSP70 gene amplicon-based next-generation sequencing. Bar colours represent the Leishmania and Trypanosoma species detected.
      These findings reflect the epidemiological complexities of Leishmania spp. infection. Samples H78 and H83 presented the highest abundance of Leishmania spp. (Table S1, see online supplementary material), highlighting the presence of L. naiffi in both samples, as well as L. lindenbergi in Sample H78. Interestingly, Sample H78 was from a child from Almagra, a rural zone where the proximity with domestic ecotope is an ecological niche, availability of vectors (
      • Paternina LE
      • Verbel-Vergara D
      • Romero-Ricardo L
      • Pérez-Doria A
      • Paternina-Gómez M
      • Martínez L
      • et al.
      Evidence for anthropophily in five species of phlebotomine sand flies (Diptera: Psychodidae) from northern Colombia, revealed by molecular identification of bloodmeals.
      ;
      • González C
      • León C
      • Paz A
      • López M
      • Molina G
      • Toro D
      • et al.
      Diversity patterns, leishmania DNA detection, and bloodmeal identification of Phlebotominae sand flies in villages in northern Colombia.
      ) and reservoirs (
      • López M
      • Erazo D
      • Hoyos J
      • León C
      • Fuya P
      • Lugo L
      • et al.
      Measuring spatial co-occurrences of species potentially involved in leishmania transmission cycles through a predictive and fieldwork approach.
      ;
      • Medkour H
      • Davoust B
      • Dulieu F
      • Maurizi L
      • Lamour T
      • Marié J-L
      • et al.
      Potential animal reservoirs (dogs and bats) of human visceral leishmaniasis due to Leishmania infantum in French Guiana.
      ;
      • Roque ALR
      • Jansen AM.
      Wild and synanthropic reservoirs of Leishmania species in the Americas.
      ) play an important role in the VL ecoepidemiology. Furthermore, this is a historical armed conflict zone, so the high flow of militaries, illegal groups and displacement due to violence may have generated an unusual pattern in the distribution of Leishmania spp. related to sylvatic and forest niches (
      • Berry I
      • Berrang-Ford L.
      Leishmaniasis, conflict, and political terror: a spatio-temporal analysis.
      ;
      • Patiño LH
      • Mendez C
      • Rodriguez O
      • Romero Y
      • Velandia D
      • Alvarado M
      • et al.
      Spatial distribution, Leishmania species and clinical traits of cutaneous leishmaniasis cases in the Colombian army.
      ). As such, one can hypothesize that the diversity of Leishmania spp. in Colombia is underestimated.
      L. infantum–L. amazonensis co-infection was recorded in two immunocompromised patients (Figure 1) – one with human immunodeficiency virus (HIV, Sample H85) and one with an unspecified malabsorption syndrome (Sample H86) (see Table 1). L. infantum alone was identified in the serum sample (Sample H75) from the same patient with HIV. In patients with HIV, the reduction of CD4+ T lymphocytes favours replication, spread and visceralization of Leishmania spp., and the co-existence of this pathogen accelerates progress towards acquired immunodeficiency syndrome (
      • Okwor I
      • Uzonna JE.
      The immunology of leishmania/HIV co-infection.
      ), therapeutic failure and relapse (
      • Lindoso J
      • Moreira C
      • Cunha M
      • Queiroz IT.
      Visceral leishmaniasis and HIV coinfection: current perspectives.
      ). Therefore, the implementation of NGS diagnostic approaches facilitates understanding the prognosis of the disease.
      An important finding was the co-infection with Leishmania and Trypanosoma spp. in one patient (Sample H720). Despite having a negative serological diagnosis for Chagas disease (Table 1), determinant sequences were retrieved for both T. cruzi (74%) and L. infantum (26%) (Table S1, see online supplementary material). From a clinical standpoint, it is hypothesized that initial infection and establishment of L. infantum in bone marrow affected haematopoietic stem cells (
      • McCall L-I
      • Zhang W-W
      • Matlashewski G.
      Determinants for the development of visceral leishmaniasis disease.
      ). In theory, a subsequent infection with T. cruzi could have a false-negative serological diagnosis associated with a short period of evolution and a limited immune response. In this clinical context, therapy with pentavalent antimonials could potentially exacerbate Chagasic cardiomyopathy in symptomatic patients due to their well-known cardiotoxicity, potentially leading to death (
      • Bastrenta B
      • Mita N
      • Buitrago R
      • Vargas F
      • Flores M
      • Machane M
      • et al.
      Human mixed infections of Leishmania spp. and Leishmania–Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme.
      ).
      As such, the clinical utility of metagenomics as depicted in these scenarios is relevant for being advantageous beyond mere species identification from direct clinical samples, but also to characterize virulence determinants and drug susceptibility profiles, giving information about co-infection with multiple Leishmania spp. in VL, particularly in endemic regions. These findings recalibrate the classical understanding of VL, in which mono-infection by any viscerotropic Leishmania sp. (L. donovani complex) is the standard description in disease pathogenesis.

      Acknowledgements

      The authors wish to thank Dirección de Investigación e Innovación from Universidad del Rosario for supporting open access of this manuscript.

      Conflict of interest statement

      None declared.

      Funding

      This work was supported by Dirección de Investigación e Innovación from Universidad del Rosario.

      Ethical approval

      The patient samples analysed in this study were authorized for use by, and belong to, the cryo-bank of Grupo de Parasitología, Instituto Nacional de Salud-INS (National Reference Laboratory in Colombia, charged with confirming cases of VL and holding custody of collected samples). The samples were de-identified to protect the identity of patients. The Colombian National Institute of Health (INS) is defined as the reference laboratory in Colombia. When a public health emergency occurs, such as VL, national law 9–1979, decrees 786–1990 and 2323–2006, authorizes the INS to use biological material and associated epidemiological information without informed consent, including the anonymous disclosure of results. This study was performed in accordance with the Declaration of Helsinki and its later amendments. Patient data were anonymized.

      Appendix. Supplementary materials

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