Bibliometric Analysis of Chemotherapy of Canine Leishmaniasis (2000-2017)

Background Zoonotic visceral leishmaniasis by Leishmania infantum is a rst order pathology in veterinary clinics of dogs in endemic areas. Moreover, canine infections (CanL) are considered the main reservoir for human disease. Despite the importance of CanL in the control of the diseases within a One Health approach no scientometric study has been published. Aims of the study included the impact of CanL on scientic literature, drugs or combinations used, trends in the period from 2000-2017 and ecacy criteria employed. Methods A Web of Science (WOS) based analysis of publications on CanL and chemotherapy of the disease in the period 2000-2017 was carried out using a stepwise methodology. Data were analyzed by year, geographical origin, chemical groups and drugs and ecacy criteria. Results Reports on CanL represented a 20% of all publications on leishmaniasis and from these ca. 14% were related to chemotherapy. Publications records on CanL followed the distribution of the infection in endemic areas although an overrepresentation of Mediterranean countries was evident. Published reports on the main antileishmanial drugs used in clinical practice showed a sustained tendency in the period analyzed. Sb V , alone or in combination with allopurinol, represented over 50% of all publications on chemotherapy of CanL despite the availability of more recently marketed drugs. Conclusions Chemotherapy of CanL still relies on Sb V and combinations. Reports on chemotherapy are scarce, mostly publicly funded and the variability of experimental conditions make extraordinarily dicult the comparison of ecacy. The vast majority of reports on ecacy do not include any information on supportive therapy; this reduces the actual value of the experiments if intended for the practical management of the disease. Complete reports of the treatments (etiological + symptomatic) would add value to the assays performed. The parasite in most studies carried out with allopurinol, alone in with MIL, MIL and AmB Reported ecacy of Sb V was high and in 17 out of 19 papers treatment with antimonials reduced/eliminated clinical signs and elicited a reduction of antibody levels. Combination of Sb V with allopurinol elicited comparable results and was considered as the most ecacious treatment against CanL. Allopurinol reportedly reduced the clinical signs in all cases and, in most publications, a reduction of specic antibodies was observed. However, its inferior chemotherapeutic value, administered alone, compared to combinations with MIL or Sb V was highlighted. MIL, alone or in combination, was ecacious to reduce signs and to improve the clinical conditions of treated dogs; results on its effect on antibodies were variable or not reported. Results with AmB were positive, with disappearance of clinical signs and reduction in antibodies in all cases. The number of publications related to other drugs was scarce although marbooxacin, aminosidine, pentamidine and oleilphosphocholine were reported as ecacious whereas metronidazole, combined with spiramicine o enrooxacin, was not; dioxacin + metronidazole reduced the clinical signs and diminished the antibody levels. Several papers involved the use of immunomodulators (e.g. domperidone, magnesium ammonium phospholinoleate anhydride). It is noteworthy to indicate that in no case supportive therapies, besides the etiological treatment, were indicated in the published reports.


Abstract
Background Zoonotic visceral leishmaniasis by Leishmania infantum is a rst order pathology in veterinary clinics of dogs in endemic areas. Moreover, canine infections (CanL) are considered the main reservoir for human disease. Despite the importance of CanL in the control of the diseases within a One Health approach no scientometric study has been published. Aims of the study included the impact of CanL on scienti c literature, drugs or combinations used, trends in the period from 2000-2017 and e cacy criteria employed. Methods A Web of Science (WOS) based analysis of publications on CanL and chemotherapy of the disease in the period 2000-2017 was carried out using a stepwise methodology. Data were analyzed by year, geographical origin, chemical groups and drugs and e cacy criteria. Results Reports on CanL represented a 20% of all publications on leishmaniasis and from these ca. 14% were related to chemotherapy. Publications records on CanL followed the distribution of the infection in endemic areas although an overrepresentation of Mediterranean countries was evident. Published reports on the main antileishmanial drugs used in clinical practice showed a sustained tendency in the period analyzed. Sb V , alone or in combination with allopurinol, represented over 50% of all publications on chemotherapy of CanL despite the availability of more recently marketed drugs. Conclusions Chemotherapy of CanL still relies on Sb V and combinations. Reports on chemotherapy are scarce, mostly publicly funded and the variability of experimental conditions make extraordinarily di cult the comparison of e cacy. The vast majority of reports on e cacy do not include any information on supportive therapy; this reduces the actual value of the experiments if intended for the practical management of the disease. Complete reports of the treatments (etiological + symptomatic) would add value to the assays performed.

Background
Leishmaniasis is a widely distributed group of vector-borne parasitic diseases caused by kinetoplastids from the genus Leishmania. Clinical course relates to the Leishmania species involved and the functionality of the immune system of the hosts. Visceral leishmaniasis (VL), provoked by L.donovani and L.infantum, is the most severe condition; it is prevalent in Asia, South America and southern Europe [1][2][3] and its expansion to northern latitudes has been reported [4,5]. Moreover, new transmission patterns have been identi ed linked to immunocompromised patients, non-vectorial transmission and solid organs transplant recipients [6,7]. Dogs can be infected by several Leishmania sp [8] and are considered the main reservoir for human infections by L. infantum [9]. Canine leishmaniasis (CanL) is very frequent reaching over 30% prevalence in some "hot spots" [10] and constitutes a rst order veterinary pathology in endemic areas. Control of CanL has important shortcomings since marketed vaccines have limitations [11,12], environmental control is unfeasible; culling of infected dogs is debatable and its actual impact has been challenged [13][14][15]. Therefore, control of CanL largely relies on the use of repellents and, mainly, on chemotherapy of infected dogs. Therapeutic arsenal includes the same compounds used for the treatment of human leishmaniasis: Antimonials (Sb V ), Amphotericin (AmB), Miltefosine (MIL), Allopurinol, Paromomycin, among other drugs and combinations [16][17][18]. Treatment schedules are highly variable among veterinarians, and some efforts to harmonize chemotherapy schemes have been made [19,20]; insofar, variability is the rule.
Despite the importance of CanL by L.infantum in both dog clinics and public health by its zoonotic transmission -a clear example of the need of the One Health approach-few scientometric contributions on leishmaniasis are available and none focused on CanL [21][22][23]. On these grounds the aim of our study was the analysis of anti-leishmanial drugs employed in the treatment of CanL, using Web of Science (WOS) as data source, in the 21st century. Time window (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017) was selected by the growing awareness of the need of controlling the infection in the main reservoir, the launch of MIL, the only available antileishmanial oral drug, and the publication of several guidelines for CanL chemotherapy.

Documents and selection criteria
Analysis followed a stepwise methodology [24] using WOS as data source. Documents containing the terms "leishmaniosis" or "leishmaniasis" in their titles were selected. Reports were further restricted to CanL (Leishmaniosis OR leishmaniasis + dogs OR canine) and, nally, those directly focused on chemotherapy (leishmaniosis OR leishmaniasis + dogs OR canine + treatment analyzed considering year of publication; geographical origin (i.e. senior author's a liation); drugs, combinations and treatment schedule when available; e cacy criteria: improvement of clinical status and lesions, normalization of biochemical and hematological parameters; reduction of parasite burden; reduction of speci c antibodies.

Analysis of data
Comparison of series (documents/year) was carried out with normalized values (z) using the formula: where x are individual values, µ is the mean value of the series and σ, the standard deviation of the series. Tendencies of the number of documents related to the identi ed drugs or groups of drugs along the sampled period were calculated with the polynomial function: f(x) = a n *x n + ... + a 3 *x 3 + a 2 *x 2 + a 1 *x + a 0 , where a 0 ... a n are constants and n is the polynomial order. Data on dog populations and dog householders were obtained from www.worldatlas.com, www.statista.com and www.moag.gov.il. No data on Iran dog population could be obtained. Analysis of results and gures were carried out with GraphPad Prism 6.0. Level of signi cance was set at P<0.05

Results And Discussion
Pattern of publications on CanL: Growing, aggregated and related to dog numbers and social awareness WOS covered articles/documents on leishmaniasis between 2000 and 2017 reached a total number of 15,173. Less than 20% (2,737), mostly written in English (> 88%), corresponded to CanL, with 390 documents including the term "treatment". Papers related to CanL displayed an increase in the analyzed period although the pattern of contributions on chemotherapy showed higher year-to-year variations (Fig. 1); variability is probably related to the signi cantly lower value compared to those from leishmaniasis and CanL.
Analysis of a liations showed that 88 countries were the origin of papers on CanL. Distribution showed an overdispersed pattern; hence, only three countries represented over 65% of all papers, with Brazil being the most productive (ca. 39%) (SI, Table 1). These countries were also those with a higher representation when therapeutics of CanL was considered although the values were lower and similar among them (21-24%). For comparative purposes data from "leishmaniasis" were analyzed in a similar way. Scienti c output on leishmaniasis and CanL, and between CanL and chemotherapy of the dog infection were correlated (R 2 = 0.7175, P < 0.0001 and R 2 = 0.6285, P < 0.0001, respectively) ( Fig. 2). Despite the correlation there were outliers in both cases. Exclusion of non-endemic areas for CanL (India, USA, Canada) among the most represented countries showed that the scienti c production on CanL from each country signi cantly correlated to its human (R 2 = 0.734, P = 0.00016) and dog (R 2 = 0.886, P < 0.0001) population.  The correlation found between papers published on CanL and the human and, particularly, dog population of the endemic countries is an evidence of the predominance of L.infantum as causative agent and the public health relevance of this zoonotic infection. This was clear in Mediterranean countries with low values of the ratio between the number of humans and dogs and the publication record. Similarly, endemicity of the disease and the number of dogs in Brazil (58% of households) [25] could account for its highest participation (ca. 40% of all papers on CanL). The low value found on CanL for India re ects the non-zoonotic transmission of L.donovani. By its part, the production by USA, with modest numbers of domestic human and CanL cases, possibly re ects global geostrategic interests. The low value of the ratio between the number of publications on leishmaniasis and CanL found in some European countries is related to the relatively low impact of the human disease [26] and the high prevalence of CanL in the region [27][28][29].
Correlation of CanL and chemotherapy of CanL although signi cant was weaker (R 2 = 0.6396, P = 0.0010) due to the comparatively lower values of Brazil in chemotherapy. Predominance of endemic countries in the publication record on chemotherapy of CanL was expected although no adequate explanation is presently available for the overrepresentation of Italy and Spain (combined 45%) except for the social and Antimonials have been, and still they are in many regions, the standard etiological treatment of CanL despite the con icting reports on toxicity of Sb V [16,17,36,37]; incomplete knowledge of its mechanism of action (MoA) [38] and resistance reports on the emergence of resistance phenomena in some canine isolates of L.infantum [39]. Over 50% of all publications on chemotherapy of CanL in the period deal with the combination of Sb V with allopurinol. This combined therapy has shown a growing tendency, particularly, up to 2012, and with new formulations [40][41][42]. AmB is an excellent leishmanicidal drug and formulations of lower toxicity (e.g. liposomes) are the rst choice treatment for human visceral leishmaniasis [43,44]. Therefore its use in dog leishmaniasis is not recommended to minimize cross-resistance between canine and human isolates of L.infantum. Main MoA of AmB, interference with ergosterol biosynthesis in Leishmania, probably makes the emergence of resistance to this antibiotic a rare event and it is widely used in human medicine as systemic antifungal [45]. However, resistant strains have been produced under laboratory conditions and several reports on resistance in humans have been published [18,46]. Therefore, the caveat against the medication of dogs with AmB seems reasonable when no new drugs are foreseen. Despite this, at least when bibliographic production is considered, AmB is used against CanLs and no tendency towards its reduction has been observed in the time window analyzed. Careful consideration of the possible cross resistance (dogs and human isolates) and the zoonotic nature of CanL create a scenario where the need of using different chemical families to treat human and CanL should be considered.
Reports on e cacy of anti-CanL drugs and combinations: scarce and highly variable Considering the evaluation of e cacy only 64 publications tted the objective in the sampled period. Sb V , alone or in combination, represented > 50% of all reports on chemotherapy of CanL In addition, in ca. 30% of all papers included in this section, antimonials were the only antileishmanial agent administered. Allopurinol was employed alone (14% of papers) or in combined therapy, particularly with Sb V (ca. 25% of the publications). By its part, MIL, administered alone or in combination with allopurinol, was reported by the 16% of publications.No clear conclusion can be drawn from the results obtained on the treatment of CanL with Sb V , alone (> 40% in France and Brazil) or in combination with allopurinol (> 35% in Spain and Italy). Further analyses considering the variation of L.infantum strains sensitivity, market issues, perception of veterinary clinicians, funding, role of veterinary associations, among other aspects could clarify these differences.
Criteria to determine the e cacy of treatments varied among published reports although clinical and biochemical parameters are predominant.
The reduction of parasite burden has been recorded in most studies carried out with allopurinol, alone or in combination with MIL, besides MIL and AmB (

Conclusions
We are aware of the limitations of this overview including the need of re nement of searching terms and the limitations of WOS as data source. However, from our analysis it is clear that publications on CanL are largely focused on L.infantum infections in European endemic areas and Brazil. Reports on chemotherapy of the dog disease are scarce and the variety of conditions (e.g. initial clinical status of the animals, dog breed and age, Leishmania isolate, follow up paramaters) makes comparison extraordinarily di cult. It is surprisingly low the number of published reports receiving private funding despite the obvious interest of the disease for Pharma companies. Whether or not this nding relates to the actual use of the drugs in veterinary clinics is not known. It is noteworthy to indicate that in the publications on chemotherapy only antileishmanial agents were considered. Thus, unless supportive therapy administered is clearly indicated, the value of these studies is limited and far from the prevailing practical conditions. We strongly advocate for a complete account of all medications received by the experimental dogs to add value to the assays performed. Our analysis has no market considerations but high contents trials (complete information, standard parameters determined) would be more helpful and would allow a fair comparison of e cacy.  Relationship between publications on leishmaniasis and canine leishmaniasis ( Fig. 2A) and canine leishmaniasis and chemotherapy of the dog infection (Fig. 2B). Each dot corresponds to a country.