Case Report: Cutaneous Leishmaniasis in a Rheumatoid Arthritis Patient Receiving Methotrexate

ABSTRACT.

The immunosuppressive effect of methotrexate has rarely been associated with reactivation of cutaneous leishmaniasis. Here we present a case of a cutaneous leishmaniasis patient with atypical clinical symptoms without splenomegaly but with cutaneous manifestations after treatment of rheumatoid arthritis with methotrexate and blood recovery of the parasite. Next-generation sequencing was used to identify Leishmania infantum chagasi in the patient’s blood sample.

INTRODUCTION

Human leishmaniasis is caused by intracellular Leishmania parasites that can cause visceral and tegumentary disease.¹ Tumor necrosis factor (TNF-α) plays an important role in host defense against Leishmania infection.² Therapy for rheumatoid arthritis (RA) involves immunosuppressive drugs, such as methotrexate (MTX), monoclonal antibodies (mAbs), and corticosteroids, which can disrupt granuloma formation associated with parasite control.³

Use of MTX, a competitive inhibitor of folic acid–dependent enzymes, leads to impaired synthesis of purines and pyrimidines, which inhibits lymphocyte proliferation.⁴ The immunosuppressive effect of MTX has rarely been associated with reactivation of leishmaniasis.^5–7 Here, we present an adult patient with cutaneous leishmaniasis associated with parasitemia after RA treatment with MTX. Next generation sequencing was used to identify the species responsible for the infection as Leishmania infantum chagasi.

CASE REPORT

A 61-year-old woman from Barueri, a city in São Paulo State, Brazil, but who lived in a rural area during childhood, presented with a cutaneous ulcer on the right lower limb (Figure 1A) 6 months before admission and was referred by a dermatologist to the infectious diseases division of a university hospital after treatment with clindamycin for 9 days and application of sulfadiazine ointment to the lesion for 1 month.

Figure 1.

(A) The lesion in the right lower limb before treatment. (B) The lesion healed after treatment with liposomal amphotericin. (C) The scar examination of methanol-fixed and Giemsa-stained smears of tissue lesion by optical microscopy (1,000× magnification) indicating the presence of the amastigote form of Leishmania sp. This figure appears in color at www.ajtmh.org.

In a follow-up session with a rheumatologist, the patient reported that she had been administering 7.5 mg of methotrexate (weekly) and 400 mg of hydroxychloroquine (daily) to control RA that was diagnosed approximately 20 years earlier. The patient reported severe constitutional symptoms, including long-term anemia with a hemoglobin level of 8 g/dL, nausea, weakness, malaise, lack of appetite, alopecia, worsening kidney function, intermittent fever, occasional night sweats, dry cough, and weight loss (10 kg over a period of 6 months). Blood tests showed hemoglobin at 7.5 g/L, leukocytes at 2,800/mm³, platelets at 85,000/mm³, and normal liver enzymes, creatinine, blood urea nitrogen, sodium, and potassium (Table 1). A bone marrow aspirate revealed dysmegakaryopoiesis, dyserythropoiesis, and increased macrophagic activity, not indicating primary hematologic diseases, such as myeloma and lymphoproliferative disease. On the basis of the epidemiology, pancytopenia, chronic cutaneous ulcers, and nonspecific symptoms, and absence of amastigotes in the bone marrow, cutaneous leishmaniasis was considered.

Table 1.

Laboratorial data of patient with cutaneous leishmaniasis after methotrexate use for rheumatoid arthritis

Laboratory data/date	6 months before admission	2 months before admission	1 month before	Hospital admission	1 month after admission	3 months after admission
Hemoglobin (g/L)	7.6	8.0	7.4	7.5	11.1	12.9
Hematocrit (%)	23.6	24.7	22.7	23.6	35.6	39.8
Leukocytes ×109/L	4.8	3.2	1.5	2.3	8.1	5.8
Neutrophils (%)	58%	56%	48%	47%	52%	50%
Linphocytes (%)	35%	34%	45%	47%	20%	39%
Monocytes (%)	2%	10%	5%	8%	8%	8%
Platelets ×109/L	132	150	56	85	234	143
Blood urea nitrogen	39	45	38	47	27	38
Creatinine	1.58	1.48	1.51	1.29	1.76	1.21
Sodium	–	138	134	135	–	140
Potassium	–	4.6	4.3	4.4	–	4.5

The patient received liposomal amphotericin treatment in the hospital.

During the investigation, 10 mL of peripheral blood was collected in a tube without anticoagulants for serological tests. Another tube, containing 3 mL of peripheral blood with EDTA anticoagulant, was centrifuged at 4,000 × g for 10 min at 22°C; the buffy coat was used for culture and DNA extraction, and a sample coded 247 was purified. Tissue samples from the peripheral edges of the lesions were obtained with a punch, from which three fragments were obtained (Leishmania culture, DNA extraction, and direct examination).

Blood and tissue culture samples were cultivated in Medium 199 (Sigma-Aldrich, San Luis, MI), incubated at 26°C for promastigote growth, and checked weekly up to 1 month. All cultures were found to be negative for Leishmania spp.

DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer’s instructions. Leishmania DNA was detected in the buffy coat of the peripheral blood and tissue samples after amplification of the conserved region of kDNA minicircles of Leishmania species. Polymerase chain reaction was performed using kDNA 20 (5′ GGG KAG GGG CGT TCT SCG AA 3′) and kDNA 22 (5′ SSS WCT ATW TTA CAC CAA CC 3′) (Invitrogen, Thermo Fisher Scientific Corporation, Waltham, MA), producing a 120-bp fragment. A direct microscopic search revealed the presence of a protist amastigote form (Figure 1C). The skin biopsy revealed a lymphoplasmocytic infiltration with rare granulomas. ELISA with serological samples and indirect immunofluorescence were negative for Leishmania sp.

The patient was hospitalized to undergo treatment for leishmaniasis, and bronchial alveolar lavage was performed to diagnose tuberculosis, once culture and acid-fast bacilli smear were negative. Xpert MTB/RIF PCR test was performed on the bronchial alveolar lavage. Computed tomography showed active tuberculosis. The patient, who weighed 53 kg, was given 20 mg/kg liposomal amphotericin to a total accumulated dose of 1,050 mg without detectable acute kidney injury. Tuberculosis was treated with rifampin, isoniazid, pyrazinamide, and ethambutol. The patient was discharged 10 days after successful treatment and continued with MTX and hydroxychloroquine (Figure 1B). The following 6 months of laboratorial tests are detailed in the Table 1.

To identify the Leishmania species in the patient’s samples, we amplified a 234-bp region of hsp70,⁸ the only target that allows identification of up to six Leishmania species of clinical importance in Brazil, and sequenced the region using MinION technology.⁹ Utilizing the sequencing data, the neighbor-joining method¹⁰ was used to perform a phylogenetic analysis (Figure 2) using MEGA v. 10.2.6. Topology robustness was assessed with 1,000 replicates using the bootstrap method. The patient’s samples were determined to be most similar to L. infantum chagasi reference strains in the phylogenetic tree.

Figure 2.

A phylogenetic tree of the patient’s buffy coat, sample 247 purified, tissue, sample 261, sample 261 purified, and reference strains. Reference strains consisted of Leishmania amazonensis IFLA/BR/1967/PH8, Leishmania infantum chagasi MHOM/BR/1974/PP75, Leishmania lainsoni MHOM/BR/M6426, Leishmania naiffi MDAS/BR/1979/M2903, Leishmania braziliensis MHOM/BR/1975M2903, Leishmania guyanensis MHOM/BR/1975/M4147, and Leishmania shawi MCEB/BR/1984/M8408.

DISCUSSION

Visceral and cutaneous leishmaniasis has been spreading in Brazil—particularly in the southeastern part of the country—since the 1980s due to increasing rates of urbanization, deforestation, and human migration.¹¹ Parasite proliferation in the reticuloendothelial system causes prolonged fever, anemia, hepatosplenomegaly, and weight loss. However, not all leishmanial infections lead to overt clinical disease, making diagnosis difficult, especially in immunosuppressed individuals. This is because the protozoan can persist in scars of ancient lesions for years, where it is sustained by a persistent, well-modulated Th1 local immune response pattern even after treatment.^2,12,13 Reactivation of leishmaniasis is associated with dysregulation of certain immune responses, specifically reduced production of inflammatory cytokines (interferon-γ and TNF-α) and an increase in cytokines associated with the Th2 immune response (interleukin-4 and -10).^14,15

The patient was living in a city without leishmaniasis reports and denied recent travel to endemic area. Considering that the patient lived in a rural area during childhood, the probable diagnosis was a reactivation of cutaneous leishmaniasis. The picture of the ulcer suggests a typical case of cutaneous leishmaniasis. Skin biopsy was performed, and the inflammatory process suggested the diagnosis, unfortunately no parasite was found in the histology, only in the smear. However, the PCR performed in the tissue recovered Leishmania DNA. An atypical feature of this case is the cutaneous form with L. infantum in Latin America,¹⁶ which is commonly caused by L. (V) brasiliensis, L. (L) amazonensis, and L. (V) guyanensis.¹⁷ The treatment with liposomal amphotericin was successful using the dose previously described in the literature.¹⁸

RA affects 0.5% to 1% of the world’s population. Patients are generally treated with MTX, but despite being effective at controlling disease, its use is often accompanied by serious adverse effects, including hematotoxicity, which can develop in up to 15% of RA patients.^19,20 The identification of Leishmania DNA in blood sample indicates parasite dissemination.²¹ Diagnostic tests for the visceral leishmaniasis form must be highly sensitive and exact because of the high risk of mortality associated with the disease.²² Leishmania parasites circulate in the peripheral blood; in 53% of the HIV-infected patients, the parasite was detected by its appearance in the buffy coat.²³ In the present case, Leishmania DNA detection in the buffy coat showed elevated sensitivity compared with that in the peripheral blood. However, we cannot assume the diagnosis of visceral leishmaniasis because the parasite was not identified in visceral tissue, and the final diagnosis was cutaneous leishmaniasis with possible blood dissemination, probably due to immunosuppressive therapy. DNA identification in blood of patients with cutaneous leishmaniasis has been described in some cases.^21,24 The clinical form of the disease is cutaneous leishmaniasis, but some atypical aspects of the disease that may be related to L. infantum chagasi as the causal agents of the disease and the possibility that parasite may have disseminated to other sites such as liver, spleen, and bowel despite of parasites have not been found in bone narrow.

The results of serological tests, indirect immunofluorescence, and ELISA were negative; antibodies may be undetectable or present at low levels in individuals with immunosupressive drugs, including MTX.^25,26 False-negative test results limit the utility of serological assays. DNA-based tests should be performed if other diagnostic tests are negative, as in vitro isolation remains the primary reference method for unequivocally identifying the parasite to species; however, it is not practical in clinical practice because of the extended length of time required to obtain a definitive result.²⁷ In the present case, for instance, small differences were observed between the sequences of the patient’s sample and the cultured reference of L. infantum chagasi in the phylogenetic tree (Figure 2).

In conclusion, our analysis suggests that chronic use of methotrexate to treat RA led to an opportunistic and unusual reactivation of L. infantum chagasi with cutaneous manifestations.