ORIGINAL PAPER

On opportunist infections by Trypanosoma lewisiin humans and its differential diagnosis from T. cruziand T. rangeli

Maria Auxiliadora de Sousa

Received: 31 July 2014 /Accepted: 29 August 2014 /Published online: 14 September 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Trypanosoma lewisi is a cosmopolitan species orig-inally found inRattus spp., being nonpathogenic, host-restrict-ed, and transmitted by rat fleas. This species has been recordedas an opportunist blood parasite of human beings mainly inAsia, with a case in Africa. In Brazil, this species was recentlyrecorded in captive monkeys. As T. lewisi can share vertebratehosts both with Trypanosoma rangeli and Trypanosoma cruzi,some markers for the differential diagnosis of these specieswere examined and discussed herein. The identification ofT. lewisi was based on morphological features of bloodstreamstages at the initial phase of infection in mammals, isoenzymeelectrophoresis at the MDH locus, and PCR products ofkinetoplast DNA (kDNA) minicircles using the primersTC121/TC122.

Keywords Trypanosoma lewisi . Trypanosoma cruzi .

Trypanosoma rangeli . Isoenzymes . PCR products .

Morphology

Introduction

In Latin American countries, Trypanosoma cruzi (the etiolog-ic agent of Chagas’ disease) and Trypanosoma rangeli havebeen found in the same invertebrate and vertebrate and hosts,including humans, sometimes in mixed infections (e.g., Hoare1972; D’Alessandro 1976; Miles et al. 1983; Cuba-Cuba1998; Sousa et al. 2008). These species can be differentiatedby classical parasitological techniques (e.g., Deane 1964;Hoare 1972; Miles et al. 1983; Holguín et al. 1987; Sousa1999; Sousa et al. 2005, 2008) besides several biochemical

and molecular approaches (e.g., Miles et al. 1983; Holguínet al. 1987; Steindel et al. 1994; Zingales et al. 1998; Guhlet al. 2002; Maia-da-Silva et al. 2004, 2008; Sousa et al. 2008;Ortiz et al. 2009; Hamilton et al. 2011). Trypanosoma lewisi isa cosmopolitan species originally found in Rattus spp., beingusually host-restricted, nonpathogenic, and transmitted by ratfleas (Hoare 1972). Using biological and morphological pa-rameters, the distinction between this species and T. cruzi canbe very easy (Hoare 1972; Sousa 1999; Sousa et al. 2008).However, the distinction between T. lewisi from T. rangeli iscomplicated. The bloodstream trypomastigotes of T. rangeli(usually very scarce) and the “adult” forms of T. lewisi aresimilar, as well as their stages in axenic cultures (Hoare 1972;D’Alessandro 1976).

The first case of T. lewisi in humans was recorded inan Asian premature child (Johnson 1933; Weinman1970) who lived in a rat-infested dwelling. Other humaninfections caused by this parasite (or T. lewisi-like) weredescribed mainly in Asia (Shrivastava and Shrivastava1974; Kaur et al. 2007; Sarataphan et al. 2007; Vermaet al. 2011), including a fatal case (apud Verma et al.2011), and also in Africa (Howie et al. 2006). In Brazil,T. lewisi was recently reported in a few captive mon-keys, also as opportunist infections (Maia-da-Silva et al.2010), and as far as I know, this species was notdescribed in humans from the Americas. In the presentwork, I examined some morphological, biochemical, andmolecular markers for the differential diagnosis of theabove-cited trypanosomes.

Materials and methods

All trypanosome stock analyzed in the present workproceeded from the Trypanosomatid Collection at OswaldoCruz Institute, whose number codes are referred as CT-IOC.

M. A. de Sousa (*)Coleção de Tripanossomatídeos, Instituto Oswaldo Cruz, Fiocruz,Avenida Brasil 4365, Rio de Janeiro, RJ, Brazile-mail: msousaa@ioc.fiocruz.br

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The stocks of T. rangeli (KP1+ and KP1−) used were:Macias (CT-IOC 273), Choachi (CT-IOC 271), APS56(CT-IOC 546), SC-58 (CT-IOC 270), and SC-61 (CT-IOC 272). Those of T. cruzi were Dm28c (CT-IOC 10),Y (CT-IOC 106), and CL Brener clone (CT-IOC 005).A T. lewisi isolate obtained by M. P. Deane (Deanestrain) was used throughout this study and proceededeither from the blood of experimentally infected rats(CT-IOC 009) or axenic cultures (CT-IOC 095) in liverinfusion-tryptose (LIT) medium supplemented with20 % fetal calf serum (FCS). The morphological fea-tures of this T. lewisi strain were analyzed in cameralucida drawings of Giemsa-stained smears from parasitestages found in the blood of experimentally infectedrats, as well as in axenic cultures (optical microscope;×1000).

For biochemical and molecular analyses, the parasitestocks were grown in LIT medium (T. cruzi) or in LITsupplemented with 10–20 % FCS (T. rangeli andT. lewisi). The trypanosomes under study were com-pared by multilocus enzyme electrophoresis on agarosegels according to standard procedures described byCupolillo et al. (1994). The stocks were tested foractivity of four enzymatic loci: malate dehydrogenase(electron capture (EC) 1.1.1.37; MDH), malic enzyme(EC 1.1.1.40; ME), phosphoglucomutase (EC 2.7.5.1;PGM), and glucose phosphate isomerase (EC 5.3.1.9;GPI).

For analysis of polymerase chain reaction (PCR)products, the DNA from each species was extracted bythe DNAzol method (Chomczynski et al. 1997) and theamplifications performed with the primers TC121 (5′-AAATAATGTACGGG(T/G)GAGATGCATGA-3′) andTC122 (5′-GGTTCGATTGGGGTTGGTGTAATATA-3′)(Wincker et al. 1994).

Results and discussion

During the acute infection in rats, the bloodstreamstages of T. lewisi (Deane strain) were highly polymor-phic, their multiplication mainly occurring by binaryand multiple fission of epimastigotes (Fig. 1), a pecu-liarity of T. lewisi which is neither found in T. cruzi norin T. rangeli (Hoare 1972; Sousa 1999; Maia-da-Silvaet al. 2010). Accordingly, a rapid diagnosis (at low cost)of a T. lewisi infection can be achieved by microscopicexamination of bloodstream forms in Giemsa-stainedsmears at the beginning of an infection in the mammalhosts. Moreover, the bloodstream forms of T. lewisi

(reproductive and “adult” forms) typically presentedsmall kinetoplasts, usually rod-like shaped (Fig. 1),which markedly differed from those of T. cruzi bloodtrypomastigotes (Hoare 1972; Cuba-Cuba 1998; Sousa

Fig. 1 Representative bloodstream stages of T. lewisi (Deane strain)found in experimentally infected rat. a “Adult” trypomastigotes, b typicaldividing stages as epimastigotes, c unusual dividing form, and d–epolymorphic trypomastigotes (only found in the multiplicative phase).Camera lucida drawings from Giemsa-stained smears (optical micro-scope; ×1000)

Fig. 2 Representative stages of T. lewisi (Deane strain) from axenicculture in LIT medium supplemented with 20 % FCS. a Epimastigotes,b trypomastigotes, c–d transitions to spheromastigotes, d tadpole-likeform, and e spheromastigote. Camera lucida drawings from Giemsa-stained smears (optical microscope; ×1000)

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1999). The culture stages of T. lewisi Deane strain(Fig. 2), including their kinetoplasts, presented closesimilarity with those of T. rangeli, but differed enoughfrom those of T. cruzi (Hoare 1972; Sousa et al. 2005,2008; Madeira et al. 2009). Actually, the microscopicexamination of Giemsa-stained smears (×1000) is a use-ful approach for distinguishing T. lewisi from T. cruzi,as well as from Salivarian trypanosomes, and this ap-proach has been routinely used by several authors(Shrivastava and Shrivastava 1974; Sousa 1999;Desquesnes et al. 2002; Howie et al. 2006; Kaur et al.2007; Verma et al. 2011).

The isoenzyme locus presenting lower intraspecificvariability (MDH) promptly distinguished T. lewisi,T. rangeli, and T. cruzi (Fig. 3). The former speciesshowed similar banding patterns at the ME locus (witha single band), differently from T. cruzi (with twobands) (Fig. 4). The analysis at PGM and GPI lociconfirmed the variability intraspecific of T. cruzi andT. rangeli (Miles et al. 1980; Tibayrenc and Ayala1988; Steindel et al. 1994), but they were regarded lessuseful for distinction of T. lewisi (Fig. 4)

The PCR products from kinetoplast DNA (kDNA)minicircles of the trypanosome species studied herein areshown in Fig. 5. T. lewisi presented 400- and 700-bp products,being distinct from those of T. cruzi (330 bp) and T. rangeli(760 bp and others around 330 bp). The primers used in thepresent study (TC121/TC122) were described asT. cruzi-specific, but they can also amplify kDNA minicircle

sequences from other trypanosome species (Sabbadini et al.2002; Sousa et al. 2008; Madeira et al. 2009). The presentstudy shows their usefulness for T. lewisi identification aswell.

It is interesting to mention that PCR assays based oninternal spacer 1 (ITS1) of ribosomal DNA (rDNA) andsequence analyses have been used for sensitive detection ofT. lewisi (or T. lewisi-like) trypanosomes and Salivarian try-panosomes in humans, monkeys, and rodents (Desquesneset al. 2002; Howie et al. 2006; Kaur et al. 2007; Sarataphanet al. 2007; Verma et al. 2011). However, although the PCRamplification based on ITS1 rDNA could also identifyT. cruzi, it could not distinguish T. lewisi from T. rangeli(Maia-da-Silva et al. 2010).

Fig. 4 Diagrammatic representation of isoenzyme patterns at three locidisplayed by T. lewisi, T. rangeli, and T. cruzi. Stocks of T. rangeli: a SC-58, b SC-61, c Choachi, and d Macias. Stocks of T. cruzi: f Dm28c, g Y,and h CL Brener

Fig. 3 Isoenzyme profiles at MDH locus presented by T. lewisi, T. cruzi,and T. rangeli. T. cruzi stocks: a CL Brener, b Y, and c Dm28c

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Acknowledgments I am thankful to Barbara Neves dos Santos,Cristina Carvalhal, Sheila Medeiros dos Santos Pereira, and Tatiana daSilva Fonseca for collaborating in this work.

References

Chomczynski P, Mackeey K, Drews R, Willfinger W (1997) A reagentfor the rapid isolation of genomic DNA. Biotechniques 22:550–553

Cuba-Cuba CA (1998) Revisión de los aspectos biológicos ydiagnósticos del Trypanosoma (Herpetosoma) rangeli. Rev SocBras Med Trop 31:207–220

Cupolillo E, Grimaldi G Jr, Momen H (1994) A general classification ofNew World Leishmania using numerical zymotaxonomy. Am JTrop Med Hyg 50:296–311

D’Alessandro A (1976) Biology of Trypanosoma (Herpetosoma) rangeliTejera, 1920. In: Lumsden WHR, Evans DA (eds) The biology ofthe kinetoplastida, vol I. Academic, New York, pp 327–403

Deane LM (1964) Tripanosomídeos de mamíferos da RegiãoAmazônica. III. Hemoscopia e xenodiagnóstico de animaissilvestres dos arredores de Belém, Pará. Rev Inst Med TropSao Paulo 6:225–232

Desquesnes M, Ravel S, Cuny G (2002) PCR identification ofTrypanosoma lewisi, a common parasite of laboratory rats.Kinetopl Biol Dis 2:1–6

Guhl F, Jaramillo C, Carranza JC, Vallejo GA (2002) Molecular charac-terization and diagnosis of Trypanosoma cruzi and T. rangeli. ArchMed Res 33:362–370

Hamilton PB, Lewis MD, Cruickshank C, Gaunt MW, YeoM, LlewellynMS, Valente SA, Maia-da-Silva F, Stevens JR, Miles MA, TeixeiraMMG (2011) Identification and lineage genotyping of SouthAmerican trypanosomes using fluorescent fragment lengthbarcoding. Infect Genet Evol 11:44–51

Hoare CA (1972) The trypanosomes of mammals. A zoological mono-graph. Blackwell Scientific Publications, Oxford, 722

Holguín AF, Saravia NG, D’Alessandro A (1987) Lack of enzymepolymorphism in Trypanosoma rangeli stocks from sylvatic anddomiciliary transmission cycles in Colombia. Am J Trop Med Hyg36:53–58

Howie S, Guy M, Fleming L, Bailey W, Noyes H, Faye JA, Pipkin J,Greenwood B, Wittle H, Molyneux D, Corrah T (2006) A Gambianinfant with fever and an unexpected blood film. PLoS Med 3(9):1508–1512, e355

Johnson PD (1933) A case of infection by Trypanosoma lewisi in a child.Trans R Soc Trop Med Hyg 26:467–468

Kaur R, Gupta VK, Dhariwal AC, Jain DC, Shiv L (2007) A rare case oftrypanosomiasis in a two month old infant in Mumbai, India. JCommun Dis 39:71–74

Madeira MF, Sousa MA, Barros JHS, Figueiredo FB, Fagundes A,Schubach A, De Paula CC, Faissal TS, Fonseca TS, Thoma HK,Marzochi MCA (2009) Trypanosoma caninum n. sp. (Protozoa:Kinetoplastida) isolated from intact skin of a domestic dog (Canis

Fig. 5 kDNA minicircle PCRproducts presented by T. lewisi(Deane strain), T. rangeli (SC-61strain), and T. cruzi (Y strain)using the primers TC121/TC122.M molecular marker (100-bpDNA ladder)

4474 Parasitol Res (2014) 113:4471–4475

familiaris) captured in Rio de Janeiro, Brazil. Parasitology 136:411–423

Maia-da-Silva F, Rodrigues AC, Campaner M, Takata CSA, BrigidoMC,Junqueira ACV, Coura JR, Takeda GF, Shaw JJ, Teixeira MMG(2004) Randomly amplified polymorphic DNA analysis ofTrypanosoma rangeli and allied species from human, monkeysand other sylvatic mammals of the Brazilian Amazon disclosed anew group and a species-specific marker. Parasitology 128:283–294

Maia-da-Silva F, Naiff RD, Marcilli A, Gordo M, D’Affonseca-Neto JA,Naiff MF, Franco AMR, Campaner M, Valente V, Valente AS,Camargo EP, Teixeira MMG, Miles MA (2008) Infection rates andgenotypes of Trypanosoma rangeli and T. cruzi infecting free-ranging Saguinus bicolor (Callitrichidae), a critically endangeredprimate of the Amazon rainforest. Acta Trop 107:168–173

Maia-da-Silva F, Marcili A, Ortiz PA, Epiphanio S, Campaner M, Catão-Dias JL, Shaw JJ, Camargo EP, TeixeiraMMG (2010) Phylogenetic,morphological and behavioural analyses support host switching ofTrypanosoma (Herpetosoma) lewisi from domestic rats to primates.Infect Genet Evol 10:522–529

Miles MA, Lanham SM, Souza AA, Póvoa M (1980) Further enzymiccharacters of Trypanosoma cruzi and their evaluation for strainidentification. Trans R Soc Trop Med Hyg 74:221–237

Miles MA, Arias JR, Valente SAS, Naiff RD, Souza AA, Povoa MM,Lima JAN, Cedillos RA (1983) Vertebrate hosts and vectors ofTrypanosoma rangeli in the Amazon Basin of Brazil. Am J TropMed Hyg 32:1251–1259

Ortiz PA, Maia-da-Silva F, Cortez AP, Lima L, Campaner M, Pral EMF,Alfieri SC, Teixeira MMG (2009) Genes of cathepsin L-like prote-ases in Trypanosoma rangeli isolates: markers for diagnosis,genotyping and phylogenetic relationships. Acta Trop 112:249–259

Sabbadini PS, Felisbino KF, Gonçalves TCM, Junqueira ACV, SousaMA (2002) Trypanosoma cruzi and Trypanosoma conorhini: mor-phological biochemical and molecular analyses after co-cultivationand co-infection in Triatoma rubrofasciata. Rev Inst Med Trop SaoPaulo 44(Supl 12):93

Sarataphan N, Vongpakorn M, Nuansrichay B, Autarkool N,Keowkarnkah T, Rodtian P, Stich RW, Jittapalapong S (2007)Diagnosis of a Trypanosoma lewisi-like (Herpetosoma) infectionin a sick infant from Thailand. J Med Microbiol 56:1118–1121

Shrivastava KK, Shrivastava GP (1974) Two cases of Trypanosoma(Herpetosoma) species infection of man in India. Trans R SocTrop Med Hyg 68:143–144

Sousa MA (1999) Morphobiological characterization of Trypanosomacruzi Chagas, 1909 and its distinction from other trypanosomes.Mem Inst Oswaldo Cruz 94(Suppl I):205–210

Sousa MA, Santos BN, Santos-Pereira S, Sabbadini PS, Xavier-MonteiroC (2005) Caracteres morfobiológicos de diferentes amostras deTrypanosoma cruzi e T. rangelimantidas em culturas axênicas: valorpara o diagnóstico diferencial destas espécies. Rev Soc Bras MedTrop 38(Supl 1):404

Sousa MA, Fonseca TS, Santos BN, Santos-Pereira SM, Carvalhal C,Hasslocher-Moreno AM (2008) Trypanosoma rangeli Tejera, 1920,in chronic Chagas’ disease patients under ambulatory care at theEvandro Chagas Clinical Research Institute (IPEC-Fiocruz, Brazil).Parasitol Res 103:697–703

Steindel M, Dias-Neto E, Pinto CJC, Grisard EC, Menezes CLP, MurtaSMF, Simpson AJG, Romanha AJ (1994) Randomly amplifiedpolymorphic DNA (RAPD) and isoenzyme analysis ofTrypanosoma rangeli strains. J Eukariot Microbiol 41:261–267

Tibayrenc M, Ayala FJ (1988) Isozyme variability in Trypanosoma cruzi,the agent of Chagas’ disease: genetical, taxonomical, and epidemi-ological significance. Evolution 42:277–292

Verma A, Manchanda S, Kumar N, Sharma A, Goel M, Banerjee PS,Garg R, Singh BP, Balharbi F, Leon V, Deborggraeve S, Rana UVS,Puliyel J (2011) Case report: Trypanosoma lewisi and T. lewisi-likeinfection in a 37-day-old Indian infant. Am J Trop Med Hyg 85:221–224

Weinman D (1970) Trypanosomiasis in macaques and in man inMalaysia. Southeast Asian J Trop Med Pub Health 1:11–18

Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W,Morel CM (1994) Use o a simplified polymerase chainreaction procedure to detect Trypanosoma cruzi in bloodsamples from chronic chagasic patients in a rural endemicarea. Am J Trop Med Hyg 51:771–777

Zingales B, Souto RP, Mangia RH, Lisboa CV, Campbell DA, Coura JR,Jansen A, Fernandes O (1998) Molecular epidemiology ofAmerican trypanosomiasis in Brazil based on dimorphisms ofrRNA and mini-exon gene sequences. Int J Parasitol 28:105–112

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