PLoS Negl Trop Dis. 2016 Dec 1;10(12):e0005100.

Atypical manifestations of cutaneous leishmaniasis in a region endemic for Leishmania braziliensis: clinical, immunological and parasitological aspects.

Luiz Henrique Guimarães1, 2, 3, Adriano Queiroz1, Juliana A. Silva1, Silvana C. Silva1, Viviane Magalhães1, Ednaldo L. Lago1, 2, Paulo Roberto L. Machado1, 2, Olívia Bacellar1, 2, Mary E. Wilson4, Stephen M. Beverley5, Edgar M. Carvalho1, 2, 6, Albert Schriefer1, 2, 7.

1 Serviço de Imunologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Brazil;
2 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT), Brazil;
3 Universidade Federal do Sul da Bahia, Brazil;
4 Departments of Internal Medicine and Microbiology, University of Iowa and the VA Medical Center, Iowa City, IA, USA;
5 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA;
6 Centro de Pesquisas Gonçalo Moniz, Brazil;
7 Departamento de Ciências da Biointeração, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Brazil.

*Corresponding Author:

E-mail: nab.schriefer@gmail.com. (AS)

 

BACKGROUND

Tegumentary leishmaniasis is a vector transmitted disease that is caused by protozoa of the genus Leishmania. Approximately 1.5 million new cases of the disease occur in tropical and subtropical countries per year. Brazil is one of ten countries that concentrate three quarters of all cases of tegumentary leishmaniasis in the world [1]. Leishmania braziliensis (or L. braziliensis) is the species of the parasite that causes most cases of the disease in the Americas, and in Brazil as well [2].

So far, four different forms of disease have been described in patients infected with L. braziliensis: localized cutaneous leishmaniasis (CL) [3], Mucocutaneous or mucosal leishmaniasis [4, 5], disseminated leishmaniasis [6-8], and atypical cutaneous leishmaniasis (ACL) [9]. All these forms of disease present very well described clinical pictures, with consistent features among different patients, except for ACL, which consists in a form of leishmaniasis caused by L. braziliensis that leads to varied clinical pictures in the patients that do not fit into any of the three disease categories above.

In our study, we examined whether these ACL patients presented a common profile in their immune responses to the parasite despite their varied clinical features, and whether strain (or line, or type) of L. braziliensis could be one reason for the patient to develop this difficult to treat form of leishmaniasis.

 

METHOD

We had made previous studies of the parasite genome that led us to describe a locus of 400 base-pairs starting at position 425,451 on chromosome 28 of L. braziliensis (we will refer to this locus as CHR28/425451 in the remainder of this text). This locus presents several variants (or alleles) that allow us to define different strains of the parasite in our study region [10]. As an example, we used CHR28/425451 alleles to show that some strains of L. braziliensis are associated with disseminated leishmaniasis, which is another severe and difficult to treat form of leishmaniasis in our region [10]. This region is located in the northeast of Brazil and is named Corte de Pedra. During current study we investigated whether strains of L. braziliensis were different between CL (CL is the mildest and most common form of tegumentary leishmaniasis) and ACL patients comparing the alleles most frequent in the locus CHR28/425451 of these parasites.

We also examined if the production of four proteins made by immune cells of the patients (those proteins are called cytokines) exposed to molecules of the parasite (the antigens) would differ between cells of ACL and CL individuals. We evaluated this exposing the cells of each patient to the parasite’s antigen in test tubes, and then comparing if on average the immune cells of ACL patients (test group) would produce more or produce less of the cytokines interferon gamma, tumor necrosis factor, interleukin 10 and interleukin 17 than cells of CL individuals (control group).

 

RESULTS

Fifty-one ACL and fifty-one CL patients participated in the study. The mean values of all four cytokines tested were statistically different (Mann Whitney’s test p<0.05) between the two groups of individuals. Immune cells from patients with ACL produced more interleukins 10 and 17 than cells from patients with CL when they were exposed in vitro (that is, in the test tubes) to antigen of L. brazliensis. On the other hand, cells from patients with CL produced more interferon gamma and tumor necrosis factor than cells from ACL during in vitro stimulation with parasite’s antigens.

We were capable of isolating and cultivating parasites from the lesions of sixteen ACL and thirty-eight CL patients. We obtained DNA from all cultured parasites and sequenced the locus CHR28/425451 in the L. brazilensis isolated from each of these individuals. Based on mutations that occur in nucleotides located within four positions of this locus (namely, positions 30, 254, 286 and 321) we could detect several alleles of CHR28/425451 in our sample. When we compared how frequent each of these alleles were present among parasites obtained from ACL (test group) versus CL patients (control group) we observed that the alleles CCCA, TATA, CACA and TCCA (each nucleotide in these alleles nomenclatures consist in the nucleotide found in positions 30, 254, 286 and 321 within CHR28/425451, respectively) were present only in parasites obtained from ACL individuals, occurring in 62% of them. This indicates that the ACL form of leishmaniasis is associated with and probably determined in part by the strain of L. braziliensis causing the infection in the patient.

 

CONCLUSION

Based on these findings, we conclude that although clinically heterogeneous the ACL patients correspond to a homogeneous group of individuals with similar immune responses to the L. braziliensis. This immune response differs from that observed in CL patients. We also conclude that this clinical presentation (that is, ACL) is in part determined by the parasite strain infecting the human being.

One possible link between these findings would be that the parasite strains found in ACL would lead to the specific immune response we detected by our limited panel of cytokines, triggering a pathogenesis mediated by the immune system’s cells and cytokines (or immunopathogenesis) that might ultimately result in the ‘atypical’ clinical pictures.

 

 

Figure 1. Example of a frequent presentation found among atypical cutaneous leishmaniasis (ACL) patients in Corte de Pedra region, Northeast Brazil. The patient gave permission to publish the picture after the photographing author (LHG) identified himself and explained the purpose of the photograph.

 

 

Figure 2. Photograph of Corte de Pedra region. Eight to fifteen hundred cases of tegumentary leishmaniasis occur in this region per year. Over 80% of these cases consist in localized cutaneous leishmaniasis (CL) patients, while close to 1.5% are atypical cutaneous leishmaniasis (ACL) patients. Corte de Pedra is composed of 20 municipalities in a rural area located in the southeastern region of the state of Bahia, in the northeast of Brazil. This region falls within the geographic coordinates (latitude South / longitude West) 14o/39o, 13o/39o, 14o/40o, 13o/40o.

 


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