Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia

Abstract

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

Keywords: L. braziliensis; RNA viruses; Totivirus; Viannia; antimony drug treatment; drug resistance; drug treatment failure; leishmaniasis.

Figure 1.

Geographical distribution of LRV1-positive Leishmania braziliensis isolates from Peru and Bolivia. The origins of L. braziliensis lines summarized in Table 1 are displayed on a map of Peru and Bolivia, created using the software package Quantum GIS, version 2.0.1 (available at: http://www.qgis.org/en/site/forusers/download.html), and the latitude and longitude coordinates of each locality. Both LRV-positive (star) and LRV-negative (circle) isolates occurred in the same geographic areas; in Peru, mostly in the jungle. Most Bolivian L. braziliensis isolates (33 of 35) originated from the Indigenous Territory and National Park Isiboro Sécure (municipality of Villa Tunari), and 2 isolates (CUM67 and CUM68) originated from the town of Shinahota (municipality of Tiraque), all located in the department of Cochabamba.

Figure 2.

Reverse transcription–polymerase chain reaction (PCR) detection of LRV1 in Leishmania braziliensis. Agarose gel electrophoresis of PCR products obtained using the LRV universal primers SMB4647 and SMB4648 with randomly primed complementary DNA derived from RNA from the species/strains is shown, as described in “Materials and Methods” section. M, double-stranded DNA (dsDNA) molecular weight marker (1 kb plus; Life Technologies, CA). Lanes 1–11: L. braziliensis isolates LC2143 (lane 1), LC2147 (lane 2), LC2176 (lane 3), LC2177 (lane 4), LC2284 (lane 5), LC2289 (lane 6), LC2321 (lane 7), LC2353 (lane 8), LC2367 (lane 9), LC2398 (lane 10), and LC2318 (lane 11). Lanes 12 and 13, L. guyanensis isolates: Lg17 (LRV1 negative; lane 12) and Lg5313 (LRV1 positive; lane 13).

Figure 3.

Treatment failure versus LRV1 prevalence among Leishmania isolates. The number of cures (open bars) and treatment failures (closed bars) following chemotherapy is shown for the complete data set (n = 54). Within each grouping, the number of isolates positive or negative for LRV1 are shown. Data are taken from Table 1.

Figure 4.

LRV1 molecular phylogeny and drug treatment outcomes. The figure shows a molecular tree based on comparisons of a 299-nucleotide region of LRV1 (described in “Materials and Methods” section). When known for a given isolate, the clinical outcome of pentavalent antimonial therapy is shown (no patients treated with amphotericin B yielded strains bearing LRV1). The tree was constructed using the neighbor-joining algorithm, based on the uncorrected number of nucleotide differences and uniform rate assumptions. The scale shows a branch length of 5-nucleotide differences. Bootstrap values calculated from 5000 replicas are shown at each node. Cure, open boxes; treatment failure, dark boxes (see Table 1 for data and classification).

Figure 5.

LRV1 relationships for 2 sympatric populations of Leishmania braziliensis in Peru and Bolivia. The figure associates the LRV1 sequence relationships depicted in Figure 4 with the geographical relationships shown in Figure 1. The fine distribution of LRV1 genotypes is shown in the insets for the district of Pilcopata at Paucartambo, Cusco, Peru; Isiboro Sécure National Park; and the municipality of Shinahota at Cochabamba, Bolivia. From this and the data in Figure 1, it can be seen that both LRV1-positive and LRV1-negative lines occur within both populations (insets), including LRV1s whose sequences differ considerably.