LEISHMANIA

The genus Leishmania is a protozoan parasite of the family Trypanosomatidae of the order Kinetoplastida. It is the causative agent of leishmaniasis in mammals in the tropical countries (Alvar et al. Reference Alvar, Velez, Bern, Herrero, Desjeux, Cano, Jannin, den Boer and Team2012). The three major forms of leishmaniases are visceral (VL), cutaneous (CS) and mucocutaneous (MCS) leishmaniases. Of these, VL is fatal if not treated. The typical symptoms of VL are chronic fever and enlargement of spleen and liver due to the parasite's survival and replication in these organs. Due to its endemicity more than 90% of deaths occur in the Indian sub-continent and in Sudan (Africa). About 10–15% and >50% of the treated populations in India and Sudan, respectively, develop PKDL. The migration of the residual parasites from infected visceral organs to the dermal site causes PKDL with macular and papular type of lesions (Kumar et al. Reference Kumar, Ramesh, Verma, Ramam and Salotra2009). PKDL patients are considered to be as human reservoirs to the continuing VL in these regions. It is transmitted by insect vector sandfly. The parasite is digenic with flagellated promastigote form that multiplies in the vector gut and the other non-motile amastigote form that replicates in the mammalian macrophages. Medications for leishmaniasis are available but expensive, take long time to cure and are threatened by development of drug resistance due to prolonged use (Sundar and Chatterjee, Reference Sundar and Chatterjee2006). Although few licensed canine VL vaccines are in use, no vaccine is yet available for human leishmaniasis (Selvapandiyan et al. Reference Selvapandiyan, Dey, Gannavaram, Solanki, Salotra and Nakhasi2014; Jain and Jain, Reference Jain and Jain2015).

LEPTOMONAS

Leptomonas is another Trypanosomatidae member known mostly as a monogenic form (promastigotes) living in the gut of certain insects (Yurchenko et al. Reference Yurchenko, Lukes, Jirku, Zeledon and Maslov2006). Unlike Leishmania, Leptomonas was not a known habitant in vertebrates and hence not considered economically important. Its co-infection in the VL and PKDL cases in India through recent reports is a major concern. Presence of flagellated non-Leishmania Trypanosomatid parasites, most likely Leptomonas, has also been reported in the sandfly (vector in leishmaniasis) population in certain regions in Nepal (Bhattarai et al. Reference Bhattarai, Das, Rijal, van der Auwera, Picado, Khanal, Roy, Speybroeck, Berkvens, Davies, Coosemans, Boelaert and Dujardin2009).

COMPARISON BETWEEN THE PARASITES

Except for minor differences, Leishmania and Leptomonas share many features like, most of the genome sequences, physiology and antigenicity (Tyzzer and Walker, Reference Tyzzer and Walker1919; Bacchi et al. Reference Bacchi, Lambros, Ellenbogen, Penkovsky, Sullivan, Eyinna and Hutner1975; Singh et al. Reference Singh, Chikara and Sundar2013; Ferreira et al. Reference Ferreira, Kesper, Teixeira, Laurenti, Barbieri, Lindoso and Umezawa2014). In their promastigote stages, both the parasites have a single set of organelles, viz., flagellum, nucleus, kinetoplast (mitochondrium), Golgi apparatus and basal bodies etc. placed in the cells in identical polarity (McGhee and Cosgrove, Reference McGhee and Cosgrove1980). Leptomonas promastigotes show optimal growth, gene transfection and clonal selection in Leishmania specific culture conditions in vitro (Bellofatto et al. Reference Bellofatto, Torres-Munoz and Cross1991; Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015). Susceptibility of Leptomonas is known to many established anti-trypanosomatid agents. (Bacchi et al. Reference Bacchi, Lambros, Goldberg, Hutner and de Carvalho1974; Goldberg et al. Reference Goldberg, Lambros, Bacchi and Hutner1974). The absence of drugs that can selectively eliminate Leptomonas renders it impossible to get rid of Leptomonas from the mixed cultures with Leishmania. This makes more challenging to develop drugs or vaccines against VL (Singh et al. Reference Singh, Chikara and Sundar2013). Although in vivo in mammals the multiplication rate of Leptomonas has not been ascertained, in the in vitro cultures, this parasite's promastigote stage grows faster than that of L. donovani (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010; Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015). Hence it became necessary for us to recognize the presence of Leptomonas in the L. donovani cultures originating from the clinical samples and eliminate it to have pure populations of Leishmania for downstream immunological and genetic studies. We and others after careful observation of the cultured Leishmania and Leptomonas promastigotes under bright field microscope reported the minor differences in their morphology (Tyzzer and Walker, Reference Tyzzer and Walker1919; Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015). In the in vitro cultures, Leishmania promastigotes were seen as heteromorphic cell population (rounded, ovoid or tadpole shaped), whereas Leishmania seymouri promastigotes were all uniform in shape (long ovoid). In the in vitro culture, L. seymouri promastigotes displayed faster (3·4-fold more) mobility than L. donovani promastigotes (Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015). We have also recently devised a simple procedure to selectively eliminate Leptomonas from the in vitro co-culture with Leishmania based on its differential growth at 37 °C (Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015). At this temperature optimal growth of axenic amastigotes and reduced growth of promastigotes of Leishmania and a complete growth inhibition and death of Leptomonas were observed. This corroborates with the earlier observation by others where promastigotes of Leptomonas costoris infecting cultured macrophages were found to differentiate into amastigote like structures but failed to further replicate. Leishmania donovani on the other hand thrives in cultured macrophages and is even able to inhibit their capacity to digest this parasite (Kutish and Janovy, Reference Kutish and Janovy1981). One report has also revealed the isolation of Leptomonas from the spleen of mice previously infected with Leptomonas spp. (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010). These observations suggest the possible existence of Leptomonas in vivo in the macrophages even though it did not replicate in the vertebrate host free macrophages in culture. Both the parasites' promastigotes otherwise are grown optimally at 26 °C in culture. The differences between these two parasites have been briefly summarized in Fig. 1.

Fig. 1. Differences between Leishmania donovani and Leptomonas seymouri.

There are several reports comparing the genomes of these two parasites. Complete genome sequence of several species of Leishmania (at: tritrypdb.org) including for L. donovani and L. seymouri (at: sanger.ac.uk) is available. Both Leishmania and Leptomonas reside in the common Trypanosomatid family cluster along with Crithidia and Trypanosoma in the phylogenetic analysis comparing several kinetoplast-bearing organisms based either on 18S rRNA or small subunit rRNA sequences (Moreira et al. Reference Moreira, Lopez-Garcia and Vickerman2004; Lukes et al. Reference Lukes, Skalicky, Tyc, Votypka and Yurchenko2014). While comparing the DNA sequences of both ITS1 and GP63 of Leishmania infantum, L. donovani (both cause VL) and Leptomonas, the latter was found to be closer to L. donovani than L. infantum via unweighted pair group method with arithmetic mean (UPGMA) based phylogenetic analysis (Singh et al. Reference Singh, Chikara and Sundar2013). However, close monitoring of DNA sequences of heat shock protein-70 (HSP70) and internal transcribed spacer 1 (ITS1) genes of these parasites yielded genus specific polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) patterns, by which researchers now could differentiate between Leishmania and Leptomonas in their cultures (Fig. 1) (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010; Ghosh et al. Reference Ghosh, Banerjee, Sarkar, Datta and Chatterjee2012; Singh et al. Reference Singh, Chikara and Sundar2013). Leptomonas miniexon 1gene targeting Taqman real-time PCR was also suggested to specifically identify Leptomonas spp. in the clinical samples (Weirather et al. Reference Weirather, Jeronimo, Gautam, Sundar, Kang, Kurtz, Haque, Schriefer, Talhari, Carvalho, Donelson and Wilson2011). All such DNA based and microscopic methods are now useful tools to recognize Leptomonas when it appears as a contaminant with other intracellular pathogens in clinical samples.

The general perception among researchers is that the individuals once cured of leishmaniasis are protected thereafter. This is due to cellular (and humoral) immunological memory response generated against the primary infection of parasite (Selvapandiyan et al. Reference Selvapandiyan, Dey, Gannavaram, Lakhal-Naouar, Duncan, Salotra and Nakhasi2012). Similarly immunity generated by either freeze-thaw-killed Leptomonas collosoma or live cultured Leptomonas pessoai cells was shown to protect the animals (including albino mice) from challenge against Trypanosoma cruzi (Johnson et al. Reference Johnson, Neal and Gall1963; Souza Mdo et al. Reference Souza Mdo, Reis, Da Silva and Brener1974). These studies confirm that both the species of Leptomonas, which were not parasitic in vertebrates displayed stronger protective antigenicity against other trypanosomatids. In addition, comparison of cross-reactive epitopes of 6 different genera of Trypanosomatidae confirmed that the antigenicity of Leptomonas seymouri was similar to Leishmania chagasi (Ferreira et al. Reference Ferreira, Kesper, Teixeira, Laurenti, Barbieri, Lindoso and Umezawa2014). Otherwise detailed immune responses by either resistant or susceptible host during infection with different species of Leishmania in the animal models as well as in humans have been studied extensively (Selvapandiyan et al. Reference Selvapandiyan, Dey, Nylen, Duncan, Sacks and Nakhasi2009; Nylen and Gautam, Reference Nylen and Gautam2010; Kedzierski and Evans, Reference Kedzierski and Evans2014). To our knowledge detailed immune responses due to Leptomonas infections have so far not been studied. With occurrence of Leptomonas co-infection in leishmaniasis, it becomes necessary now to know individually and in co-infection condition the responses generated by Leptomonas in the host during infection and how that will affect the disease outcome.

CO-INFECTION OF LEPTOMONAS WITH LEISHMANIA IN LEISHMANIASIS

Co-infection with other pathogens in leishmaniasis is on the rise in India and other countries. This poses an additional challenge to control VL. Leishmania (VL/PKDL)-HIV co-infection is known (Shah et al. Reference Shah, Shah, Prajapati and Bilimoria2010; Diro et al. Reference Diro, Lynen, Ritmeijer, Boelaert, Hailu and van Griensven2014; Singh, Reference Singh2014). Several cases of Leishmania co-infection with malaria, chagas, schistosomiasis and tuberculosis were also reported (Griemberg et al. Reference Griemberg, Ferrarotti, Svibel, Ravelli, Taranto, Malchiodi and Pizzimenti2006; Rathnayake et al. Reference Rathnayake, Ranawake, Sirimanna, Siriwardhane, Karunaweera and De Silva2010; Gil et al. Reference Gil, Cimino, Lopez Quiroga, Cajal, Acosta, Juarez, Zacca, Orellana, Krolewiecki, Diosque and Nasser2011; Cota et al. Reference Cota, Gomes, Pinto, Santos-Oliveira, Da-Cruz, Pedrosa, Tafuri and Rabello2012; van den Bogaart et al. Reference van den Bogaart, Berkhout, Nour, Mens, Talha, Adams, Ahmed, Abdelrahman, Ritmeijer, Nour and Schallig2013; Vega Benedetti et al. Reference Vega Benedetti, Cimino, Cajal, Juarez Mdel, Villalpando, Gil, Marcipar, Krolewiecki and Nasser2013). Domestic cats and a dog showing co-infection with Leishmania spp. and Toxoplasma gondii were reported (Braga et al. Reference Braga, Correa, Camossi, Silva, Langoni and Lucheis2014; da Silva et al. Reference da Silva, Caffaro, Paula, Risseti, Langoni, Megid, Melanchauski, Souza, Takahira and Machado2015). Although Leptomonas has been thought to be inhabitant in insects, recent reports revealed unusual 7–17% cases of VL and PKDL with clinical isolates of L. donovani showing co-infection with Leptomonas in India (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010; Ghosh et al. Reference Ghosh, Banerjee, Sarkar, Datta and Chatterjee2012). The first such cases of VL were the splenic aspirates that yielded Leptomonas in addition to Leishmania in Bihar and Uttar Pradesh, India. The co-infection was confirmed by the sequencing of its HSP70 and 18S rRNA genes (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010). Further confirmation of such co-infection in the Indian leishmaniasis was observed from a report revealing the presence of L. seymouri in 4 out of 29 VL and 2 out of 7 PKDL cases by aberrant internal transcribed spacer 1 (ITS1) RFLP (Ghosh et al. Reference Ghosh, Banerjee, Sarkar, Datta and Chatterjee2012). Further a next generation sequencing of oligonucleotide ligation and detection (SOLID)^™ platform recognized the same non-Leishmania Trypanosomatid parasite during sequencing of clinical isolates of VL in India (Singh et al. Reference Singh, Chikara and Sundar2013). Leptomonas contamination in the clinical isolates of Leishmania from Nepal and Sri Lanka has also been observed recently. Sandfly population from distinct regions in Nepal harbouring Leptomonas like non-Leishmania parasites in their body has also been confirmed using rRNA gene targeted PCR (Bhattarai et al. Reference Bhattarai, Das, Rijal, van der Auwera, Picado, Khanal, Roy, Speybroeck, Berkvens, Davies, Coosemans, Boelaert and Dujardin2009). Such reports along with the clinical incidents of co-infection in VL in the Indian subcontinent indicate that the sandfly population itself might be harbouring Leptomonas. To its support a very recent report suggests that, L. seymouri stayed with L. donovani for several days in the Leishmania transmission vectors (sandflies), Phlebotomus spp. (Phlebotomus orientalis and Phlebotomus argentipes) under experimental condition (Kraeva et al. Reference Kraeva, Butenko, Hlavacova, Kostygov, Myskova, Grybchuk, Lestinova, Votypka, Volf, Opperdoes, Flegontov, Lukes and Yurchenko2015).

CLINICAL IMPLICATIONS OF CO-INFECTION

The occurrence of co-infection of Leptomonas in the VL/PKDL cases has been inferred only after the observation of fast replicating Leishmania look alike Leptomonas in the culture of some of the clinical isolates. Such cultures were confirmed to be mixed cultures of Leishmania (slow moving) and Leptomonas [fast moving (Ahuja et al. Reference Ahuja, Arora, Khare and Selvapandiyan2015)] initially and after a few subcultures contained only Leptomonas. This was confirmed by RFLP analysis of either HSP70 or ITS1 genes with distinct patterns in Leptomonas and Leishmania (Srivastava et al. Reference Srivastava, Prajapati, Vanaerschot, Van der Auwera, Dujardin and Sundar2010; Ghosh et al. Reference Ghosh, Banerjee, Sarkar, Datta and Chatterjee2012).

Since such co-infection came into light only in recent years, its implications in the VL or PKDL disease severity, delay in the potential diagnostics and thereby increase in mortality in VL, when compared with patients infected only with L. donovani are yet to be determined. Since most of the leishmaniacides also kill Leptomonas (Singh et al. Reference Singh, Chikara and Sundar2013) appropriate common drug treatment that could eliminate both the parasites together need to be established. Sandflies carrying either of these two parasites have been reported (Bhattarai et al. Reference Bhattarai, Das, Rijal, van der Auwera, Picado, Khanal, Roy, Speybroeck, Berkvens, Davies, Coosemans, Boelaert and Dujardin2009) but there is no report as yet of a sandfly vector carrying both the parasites simultaneously, but such a scenario is possible. Hence at the moment it is not clear whether the entry and habitation of Leptomonas in humans is primary or secondary to Leishmania infection or it is a simultaneous infection due to bite by sandflies infected with both the parasites. An exclusive secondary infection due to immune suppressed state of VL/PKDL patients cannot be ruled out due to the fact that as an opportunistic infection Leptomonas amastigotes have also been noticed in the in vitro culture from bone marrow aspirate of a HIV patient (Pacheco et al. Reference Pacheco, Marzochi, Pires, Brito, Madeira Mde and Barbosa-Santos1998). This patient had VL, but Leishmania promastigotes were not detected in the cultures. It is also possible that Leishmania parasites were also indeed present initially in the in vitro culture from bone marrow, not noticed and subsequently got eliminated due to rapid growth of Leptomonas. Upon VL cure, presumed residual L. donovani parasites in the body causing PKDL in certain cases are known. The observed co-infection also in the PKDL cases leads to the question whether Leptomonas too coexisted as residual parasites in the body before participating in the PKDL. In other occasions presumed monoxenous non-Leishmania trypanosomatid was noticed in HIV patients (Dedet et al. Reference Dedet, Roche, Pratlong, Cales-Quist, Jouannelle, Benichou and Huerre1995; Jimenez et al. Reference Jimenez, Lopez-Velez, Molina, Canavate and Alvar1996; Morio et al. Reference Morio, Reynes, Dollet, Pratlong, Dedet and Ravel2008). Especially Dedet et al. (Reference Dedet, Roche, Pratlong, Cales-Quist, Jouannelle, Benichou and Huerre1995) revealed that such an apparent lower trypanosomatid member (Leptomonas?) isoenzymatically different from Leishmania also developed a diffuse cutaneous nodular syndrome in those HIV patients. These cases lead to speculation that those parasites were indeed Leptomonas, which can infect immune compromised humans independently without association with Leishmania. The observation of Leishmania-Leptomonas co-infection occurring so far only in the Indian subcontinent raises the question, whether it is due to new adaptation of Leptomonas into the sandflies that so far harboured only Leishmania in this region. In addition we do not know if there is an increase of VL disease outcome in case of such co-infection.

In conclusion recently discovered incidents of Leptomonas co-infection in VL in the Indian subcontinent are a cause of concern and require further investigation. Both Leishmania and Leptomonas are mostly similar in their structures and antigenicity. These two have also been found to be genetically very close to each other based on several phylogenetic analyses. The reason for the monoxenous (in single host) parasite Leptomonas in the insect invertebrates, shifting to dixenous (in two hosts) parasite like Leishmania living in both invertebrates (insects) and vertebrates (mammals), is yet to be understood. Is it because of Leptomonas's shifting its extracellular habitat into another insect, sandfly vector which also feeds on mammals is a moot point. In addition, it is not clear whether Leptomonas is seen as co-infectant only in recent years or it also existed in the past and was never explored. The reason for Leptomonas's inability to grow in the in vitro media at 37 °C and in the cultured macrophages, although showing growth in the spleen of VL and skin of PKDL patients is not clear. Moreover, whether such co-infection is also observed in other parts of the world, where VL is endemic; whether Leptomonas infection is primary, secondary or simultaneous infection with Leishmania; whether there is any sandfly population that harbours both Leishmania and Leptomonas together; whether Leptomonas is involved in leishmaniasis outcome or exasperating the disease along with Leishmania; whether Leptomonas has its own host immune modulation or shares the same with Leishmania are worth exploring. Such knowledge will help researchers and clinicians to develop appropriate therapeutic strategies for combined elimination of the co-infecting parasites.