Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: A systematic review and meta-analysis.

BACKGROUND: Cutaneous leishmaniasis is caused by different protozoan parasites of the genus Leishmania. Leishmania RNA virus (LRV) was identified as the first Leishmania infecting virus in 1998. Different studies showed the presence and role of the LRV in Leishmania parasites causing cutaneous leishmaniasis (CL). However, there is limited data on the pooled prevalence of LRV in Leishmania parasites causing CL. Therefore, the aim of this systematic review and meta-analysis was to determine the pooled prevalence of LRV in Leishmania parasite isolates and/or lesion biopsies in patients with CL from the available literature globally.
METHODOLOGY: We retrieved the studies from different electronic databases. The studies were screened and identified based on the inclusion and exclusion criteria. We excluded studies exclusively done in experimental animals and in vitro studies. The review was conducted in line with PRISMA guidelines. The meta-analysis was performed with Stata software version 14 with metan command. The forest plot with random-effect model was used to estimate the pooled prevalence with 95% confidence interval. Inverse variance index (I2) was used to assess the heterogeneity among the included articles. PRINCIPAL
FINDINGS: A total of 1215 samples from 25 studies were included. Of these, 40.1% (487/1215) were positive for LRV. The overall pooled prevalence of LRV globally was 37.22% (95% CI: 27.54% - 46.90%). The pooled prevalence of LRV in the New World (NW) and Old World (OW) regions was 34.18% and 45.77%, respectively. Leishmania guyanensis, L. braziliensis, L. major, and L. tropica were the most studied species for the detection of LRV. The prevalence of LRV from Leishmania isolates and lesion biopsies was 42.9% (349/813) and 34.3% (138/402), respectively.
CONCLUSION: This systematic study revealed that there is high prevalence of LRV in Leishmania parasites isolated from patients with CL. More comprehensive studies would be required to investigate the presence of the LRV in other Leishmania species such as L. aethiopica to fully understand the role of LRV in different clinical manifestations and disease pathology presented in CL patients.

1 Introduction

The different clinical manifestations of tegumentary leishmaniasis comprise localized (LCL) mucocutaneous (MCL) and diffuse cutaneous leishmaniasis (DCL); they are neglected tropical diseases affecting millions of people worldwide. Leishmania species probably co-evolved with mammals [1] and have been identified in humans from at least 4 millennia before present [2]. About 431 million of the world population living in CL endemic areas are at risk of CL with 0.7–1.2 million new cases annually [3,4]. The mortality and morbidity of CL is also increasing [5,6]. It impacts mostly on the poorest community [5]. Cutaneous leishmaniasis is caused by a protozoan parasite of the genus Leishmania [7,2]. It is transmitted through the bites of female phlebotomine sandfly vectors. The infected sand fly vectors harbouring the parasite inoculate the metacyclic promastigote stage into the skin of the host during blood meal [8]. There are more than 20 heterogenous Leishmania species known to cause CL. Old World CL, which occurs in Africa, Asia, and Europe, is predominantly caused by L. tropica, L. major, and L. aethiopica species. New World CL occurs in America and is mainly caused by L. braziliensis, L. mexicana and L. amazonensis [9,2].

While the majority of infected individuals remain asymptomatic, CL occurs in various clinical forms in symptomatic patients [10]. The clinical manifestation extends from a self-limited localized lesion to chronic and disfiguring mucocutaneous and diffused cutaneous lesions. These different clinical manifestations depend on several factors: the causative parasite, the immune response mounted by the host, and the sandfly factors [11,12]. Presence of Leishmania RNA virus in the parasite has also been reported to play a role in defining the clinical form of leishmaniasis [13,14]. However, there is no consensus on a defining role of LRV in disease progression, severity, metastasis, and treatment failure. Valencia et al., 2022 did not find clinical, parasitological or immunological evidence supporting the hypothesis that LRV1 is a significant determinant in the pathobiology of American Tegumentary Leishmaniasis [15]. Localised cutaneous leishmaniasis (LCL) is a benign form characterized by a single or a small number of lesions mostly around the face or extremities. It causes skin damage such as small ulcers and usually resolves spontaneously over time though the healing process is very slow [12]. However, the healed lesions generally result in permanent scars [16]. Mucocutaneous leishmaniasis (MCL) is the most severe form which leads to severe disfiguration and removal of the nose, mouth and/or the pharynx unless early treatment is initiated [17]. The pathophysiology of the ulcerative destruction is poorly understood, and relapse is common even after treatment and clinical cure [18,19]. Diffused cutaneous leishmaniasis (DCL) is a rare form of CL characterized by multiple non-ulcerative nodular lesions with large numbers of parasites in the lesions [20]. New World CL rarely evolves to self-cure; the lesions in most of the cases do not self-heal and are chronic, progressive and non-responsive to most anti-leishmanial drugs [18, 21]. Relapse is common even after successful treatment of MCL patients [22] and MCL often develops in more susceptible individuals after clinically healed LCL [18]. The distinct clinical manifestations are associated with different species, but there is substantial variation within species and it is poorly understood why Leishmania parasites cause different clinical manifestations.

The role of endosymbiotic LRV has been documented in the development of different clinical manifestations and disease severity of Leishmania infection [23,24] and it has been reported that it leads to the development of severe and disfiguring MCL [21]. The LRV is a double-stranded RNA virus belonging to the Totiviridae family [24]. Its genome has two open reading frames that encode for the capsid protein and the RNA polymerase [25-28]. The virus particle is composed of a capsid protein of about 40 nm in diameter and its genome has 5,280 nucleotides [25].

Research has focused on investigating different viruses infecting the Leishmania parasite and other unicellular eukaryotes since the discovery of the first protozoan virus in Entamoeba histolytica in 1960 [26]. Leishmania RNA virus was identified in 1998 as the first virus infecting Leishmania parasites [29]. Various studies, most of which are in the New World, have been conducted on the prevalence and role of the LRV in Leishmania parasites. However, there is limited comprehensive data scientifically analysed. Therefore, the aim of this systematic review and meta-analysis was to determine pooled prevalence of LRV in Leishmania parasite isolates and/or lesions in patients with cutaneous leishmaniasis. In our study the term tegumentary comprises localized, mucocutaneous and diffuse cutaneous leishmaniasis.

2. Methods

2.1. Search strategy and eligibility criteria

2.1. Search strategy and eligibility criteria

We first searched the articles using keywords and Medical Subject Headings (MeSH) in the following database: PubMed, Web of Science, Scopus, and Google Scholar; using the following key words:

Leishmania RNA virus AND “cutaneous leishmaniasis”;

Leishmania RNA virus AND “localized cutaneous leishmaniasis”

Leishmania RNA virus AND “mucocutaneous leishmaniasis”;

Leishmania RNA virus AND “diffuse leishmaniasis”;

Leishmania RNA virus AND “cutaneous leishmaniasis” AND “prevalence”;

Leishmania RNA virus AND “localized cutaneous leishmaniasis” AND prevalence”

Leishmania RNA virus AND “mucocutaneous leishmaniasis” AND “prevalence”;

Leishmania RNA virus AND “diffuse leishmaniasis” AND “prevalence”;

Only peer-reviewed original articles published in English were searched and no restriction was set by publication date, sample size, parasite species, methods used and study settings during article searching. We also had searched from the reference lists of all searched articles to further search similar studies and references.

2.2. Eligibility criteria and study selection

We included articles reporting primary data that aimed to determine the presence of LRV in Leishmania parasite isolates and/or lesion biopsies from patients with CL and MCL. We excluded studies exclusively done in experimental animals and in vitro studies. Review articles, non-CL based studies, and prevalence studies of other forms of leishmaniasis than CL were excluded. The review was conducted in line with Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines (S1 Table) [30].

The searched articles were selected in a two-step process. First, we read each title of study, downloaded, and checked whether it was related with the review question. If it was found to be relevant, the abstracts were read and then the whole paper read, and all necessary data were extracted. The articles were independently reviewed by two investigators (EY, EN). In the second stage, the selected full-text articles were again reviewed for eligibility through detailed reading. The reasons for exclusion (studies focused on experimental model, method evaluation, immunology, case reports, non-CL based studies and studies using previously isolated parasites) were registered and reported when full-text articles were excluded. Finally, disagreements between the two investigators were resolved by discussions with all authors of this study until consensus was reached.

2.3. Data extraction and quality assessment

A data extraction form was prepared and used to extract the basic qualitative and quantitative data from each included article (S1 Data). Data from the included articles were extracted in parallel by two authors (EY, EN) independently and checked with each other when they had finished. The data included basic information from retrieved articles (the first author and year of publication), geographical region, method used to detect LRV, number and type of Leishmania species tested, and type of identified Leishmania RNA virus. The clinical manifestation of the patients and the sample type used for detection of LRV were also extracted. The extracted data was entered into Microsoft Excel sheets. The quality of included studies was assessed by Joana Brigg’s Institute (JBI) critical appraisal checklist for studies reporting prevalence data [31]. Articles of low quality that did not meet the JBI critical appraisal checklist were excluded.

2.4. Data analysis

The basic information of the included articles was summarized and presented in tables. The meta-analysis was done with Stata software (version 14, STATA Corp College Station, TX) with metan command. The extracted data was imported into the Stata software. The forest plot was used to estimate the combined prevalence and the effect of each study with their respective 95% confidence interval (CI).

Inverse variance index (I2) was used to assess the extent of heterogeneity among the included articles in this systematic review and meta-analysis. The I2 value ranges from 0 to 100%. I2 values above 50%, between 25–50% and below 25% were considered as high, medium and low heterogeneity respectively [32]. P-value <0.05 was considered to conclude the presence of heterogeneity among the studies not by chance. Due to the presence of high heterogeneity about the prevalence among the included studies, we used random effect model at 95% CI to determine the pooled prevalence. We conducted subgroup analysis and meta-regression analysis was used to assess the possible causes for the observed heterogeneity. Publication bias across the included studies was assessed with Egger’s funnel plot symmetry (qualitatively).

3. Results

3.1. Search results and the selection process

Initially, we retrieved a total of 3176 published articles from the preliminary searching of the English language electronic databases and manual searching. 206 duplicate articles were then removed from the total retrieved articles. After removing the duplicates, we screened the titles and abstracts of 2970 articles, and excluded 2882 articles (Fig 1). The full-text articles of the remaining 108 were assessed for eligibility. A further 83 articles were excluded after a detailed assessment considering all the inclusion and exclusion criteria: studies focused on experimental models, method evaluation, immunology, case reports, non-CL based studies and studies using previously isolated Leishmania parasites. The exclusion criteria are detailed in Fig 1.

Fig 1

PRISMA flow diagram describing the strategy for article selection for the prevalence of LRV in Leishmania parasites in patients with cutaneous leishmaniasis, 2021.

Finally, 25 articles were found to be eligible and included in the systematic review and meta-analysis. The searched articles were identified following the preferred reporting items for systematic reviews and meta-analyses (PRIMSA) diagram (Fig 1).

3.2. Description of the included studies

The retrieved studies included in this systematic review and meta-analysis were published between 1998 and January 2022 with 36% published in 2019–2022. Most of the included studies (72%, 18/25) were conducted in New World regions. The presence of endosymbiotic LRV was detected in different Leishmania species causing CL. Leishmania braziliensis, L. guyanensis, L. major, L. aethiopica, L. tropica, L. amazonensis and L. panamensis were Leishmania species used in most of the studies. Different Leishmania species were diagnosed, isolated and used for the detection of LRV in some of the included studies.

Detection of the LRV was performed on Leishmania isolates and/or lesion biopsies. Regarding the diagnostic methods in the included articles, the presence of the virus was detected from parasite isolates and/or lesion biopsies by the polymerase chain reaction (PCR) in most of the studies. Electrophoresis, immunofluorescence microscopic techniques, and sequencing technologies were also used to detect LRV in some of the studies. Some of the included studies used different methods to detect LRV. LRV1 was the type of LRV reported in most of the studies in New World regions and LRV2 was reported from the Old World studies. Few studies reported both LRV1 and LRV2 in a single study. The LRV detected in Ethiopia from L. aethiopica was reported as “LRV-Lae”. On the other hand, all the included articles were published as original articles except the study conducted by Valencia et al., 2014 [33] which was published as a conference abstract. The profiles of included articles are summarized in Table 1.

Table 1

Overview of the studies describing the presence of LRV in Leishmania parasites and/or lesions in patients with CL, 2021.

Author, Year (ref)	Leishmania species	Methods	Sample type	Parasites no	Pos. LRV (%)	Type of LRV	Region
Tarr et al., 1988 [29]	L. braziliensis, L. guyanensis	Electrophoresis	Isolates	12	2 (16.7)	LRV2	NW
Widmer et al., 1989 [35]	L. guyanensis	Electrophoresis	Isolates	10	2 (20)	LRV1	NW
Scheffter et al., 1995 [27]	L. major	Electrophoresis	Isolates	14	6 (42.8)	LRV2-1	OW
Zangger et al., 2014 [46]	L.aethiopica	IFM, dot plot, PCR	Isolates	8	4 (50)	LRV-Lae	OW
Saberi et al., 2020 [45]	L. major, L. tropica	PCR	Isolates	85	59 (69.4)	LRV2	OW
Abtahi et al., 2020 [39]	L. major	PCR	Isolates	30	9 (30)	LRV2	OW
de Carvalho et al., 2019 [42]	L. guyanensis	PCR	Isolates	49	19 (38.8)	LRV1	NW
Cantanhêde et al., 2015 [13]	L. braziliensis, L. amazonensis, L. guyanensis	PCR, Sequencing	Lesion	141	61 (39)	LRV1	NW
Nalçacı et al., 2019 [47]	L. tropica, L. major	PCR, Sequencing	Isolates	25	8 (32)	LRV2	OW
Kleschenko et al., 2019 [48]	L. major	Sequencing	Isolates	3	2 (66.7)	LRV2	OW
Kariyawasam et al., 2019 [37]	L. braziliensis, L. guyanensis, L. panamensis	PCR	Isolates	8	4 (50)	LNV1	NW
Kariyawasam et al., 2020 [55]	L. braziliensis	PCR	Isolates	78	21 (26.9)	LNV1	NW
Ito et al., 2015 [49]	L. braziliensis, L. guyanensis	PCR	Isolates	37	26 (70.3)	LNV1	NW
Bourreau et al., 2016 [41]	L. guyanensis	PCR	Lesion	75	44 (58.7)	LNV1	NW
Adaui et al., 2016 [40]	L. braziliensis	PCR, Sequencing	Isolates	97	32 (33)	LRV1	NW
Ginouvès et al., 2016 [36]	L. guyanensis, L. braziliensis	PCR	Isolates	129	96 (74.4)	LRV1	NW
Hartley et al., 2016 [56]	L. guyanensis	PCR	Isolates	78	30 (38.5)	LRV1	OW
Ogg et al., 2003 [57]	L. braziliensis	PCR	Lesion	36	12 (25.5)	LRV1	NW
Parra-Muñoz et al., 2021 [54]	L. braziliensis	PCR	Isolates & Lesion	47	15 (41.7)	LRV1	NW
Kariyawasamet al., 2017 [50]	L. braziliensis, L. panamensis	PCR	Isolates	5	3 (60)	LRV1	NW
Pereira et al., 2013 [52]	L. guyanensis	PCR	Lesion	48	2 (4.2)	LRV1	NW
Saiz et al., 1998 [53]	Leishmania species	PCR	Lesion	11	2 (18.2)	LRV1-1, LRV1-4	NW
Guilbride et al., 1992 [34]	L. braziliensis, L. guyanensis	Electrophoresis	Isolates	71	12 (16.9)	LRV1	NW
Valencia et al., 2014 [33]	L. braziliensis, L. guyanensis, L. peruviana	PCR	Isolates	56	6 (10.7)	LRV1	NW
Valencia et al., 2022 [15]	L. braziliensis, L. peruviana, L. guyanensis	PCR	Lesion	56	10 (17.8)	LRV1	NW

Abbreviations: IFM: Immunofluorescence Microscopy; L: Leishmania; PCR: Polymerase Chain Reaction; OW: Old World; NW: the New World

3.3. Synthesis of results

The forest plot was used to estimate the pooled prevalence with 95% confidence interval (CI). The heterogeneity among the studies included in this systematic review and meta-analysis was evaluated using inverse variance index (I2). There was high heterogeneity among the included studies (I2 = 92.6%), p = 0.000. Due to the presence of high heterogeneity about the prevalence among studies, we used random effect model at 95% CI for the analysis of the pooled prevalence of LRV in Leishmania isolates and/or lesions from patients with CL. Subgroup analysis and meta-regression analysis were used to assess the possible causes for the observed heterogeneity.

The data retrieved from the included articles was analysed by meta-analysis to determine the pooled prevalence of LRV in the Leishmania isolates and /or lesions of patients with CL. A total of 1215 samples (promastigote isolates and lesions) were tested for the presence of the virus, of which 40.1% (487/1215) were positive for LRV. Using random effect model analysis, the overall pooled prevalence was 37.22% with 95% CI (27.54–46.90%) (Fig 2).

Fig 2

Forest plot diagram of included studies depicting the pooled and weighted prevalence of LRV in Leishmania isolates and/or lesions from CL patients, 2021.

3.3.1. Publication bias assessment

Egger’s funnel plot symmetry test for small study effects was used to check the publication bias among the included articles. The results showed that publication bias was not significant (p = 0.213) in the included studies (Fig 3)

Fig 3

Egger’s funnel plot indicates absence of publication bias across the included studies, 2021.

We included studies done in New World and Old World regions. The prevalence of LRV was 34.18% and 45.77% in the New World and Old World regions, respectively. Regarding the clinical manifestations of CL, 40.3% (381/946) of samples isolated from patients with localized cutaneous leishmaniasis were positive for LRV. Leishmania guyanensis, L. braziliensis, L. major, and L. tropica were the most common Leishmania species included in studies for the detection of LRV. Zangger et al., 2014 used L. aethiopica and 50% of samples were positive for LRV. Moreover, the prevalence of LRV from Leishmania isolates and lesion biopsies was 42.9% (349/813) and 34.3% (138/402), respectively (Table 2).

Table 2

Summarized data extracted from the included studies for sub-group analysis in the meta-analysis.

Category	No.	Positive for LRV	Prevalence (%)
Type of LRV
LRV1	1044	398	38.12
LRV2	163	85	52.1
LRV-Lae	8	4	50.0
Total	1215	487	40.1
Clinical Form
CL	946	381	40.3
MCL	237	99	41.8
DCL	32	7	21.9
Total	1159	477	41.1
Metastatic	269	106	39.4
Non-metastatic	946	381	40.3
Total	1215	487	40.1
Leishmania Species
Leishmania guyanensis	473	208	44.0
Leishmania braziliensis	518	179	34.5
Leishmania major	133	78	58.6
Leishmania aethiopica	8	4	50
Leishmania tropica	24	6	25
Leishmania species	56	12	21.4
Total	1215	487	40.1
Sample type
Isolates	813	349	42.9
Lesion	402	138	34.3
Total	1215	487	40.1

4. Discussion

This systematic review and meta-analysis aimed to determine the comprehensive prevalence of LRV in different Leishmania species causing human CL. The Leishmania parasite has been known to harbour the endosymbiotic LRV since 1988 [28] and the early 1990s [34,35]. Different studies have been conducted to determine the status of the LRV in different Leishmania species since the first evidence in L. guyanensis and L. braziliensis in 1988 by Tarr et al., 1988 [36]. The pooled LRV prevalence in parasite isolates and/or lesion biopsies from patients with cutaneous leishmaniasis was 37.22%, 95% CI (27.54%-46.90%). This reveals that a significant proportion of the Leishmania parasites causing human CL harbour the endosymbiotic LRV. This might be one of the reasons for the increasing burden, different clinical presentation and severity of CL [13,37]. Evidence shows that the severe forms of CL including disseminated and diffused leishmaniasis are emerging and increasing due to infection with different L. (viannia) braziliensis parasites [38]. The presence of LRV in the Leishmania parasite was also associated with failure to respond to antimonial treatment and to relapse of the symptomatic disease [39-41]. However, the role of LRV on the pathobiology of cutaneous leishmaniasis is still unclear and contradictory data showing lack of association of LRV positivity and treatment failure have been reported [15]. It has been indicated that in the presence of the LRV in the Leishmania parasite, the immune response favours parasite survival and results in severe disease. The dsRNA of the virus acts as potent innate immunogen recognized via toll like receptor-3 (TLR-3) [42]. According to the findings from studies conducted in L. guyanensis parasites, there is an association between LRV1 and antimonial treatment failure [40,41]. This might suggest that LRVs can be considered as a potential target for treatment and prevention. There is evidence that LRV1 can be found in exosome vesicles within the Leishmania parasite [43]. This would explain the role of the LRV virus in the development of severe leishmaniasis as the vesicle helps the virus to disseminate further. However, recent reports have shown that LRV has no role in the disease severity, determination of clinical forms, and treatment failure [15].

Articles included in this systematic review and meta-analysis used various methods to detect the presence of LRV in Leishmania parasite isolates and/or lesions of patients with CL. Most of the studies used PCR techniques of different types, summarised as PCR for simplicity of analysis. Some of the studies used more than one detection method to increase the sensitivity. The type of detection method was among the variables for the heterogeneity of the studies (Fig 4 below).

Fig 4

Forest plot showing the pooled LRV prevalence estimate by detection method in Leishmania parasites and/or lesions of patients with CL, 2021.

Currently, LRV has been detected in Leishmania parasites isolated from both New World (in Central and South America regions) and Old World (Central, Western and Eastern Europe, Far and Middle East, and Africa regions) patients with CL. Most of the studies were focused on L. guyanensis, L. braziliensis, L. panamensis species of the New World region and reported LRV1. Recent evidence showed that LRV2 has also been detected in L. major and L. aethiopica, Leishmania species of the Old World [44]. Sub-group analysis indicates that the prevalence of LRV in the Old World was higher (45.77%) than the New World (34.18%) (Fig 5). This might be because more samples per study were included in studies conducted in the Old World regions than the New World regions. The variation in the level of endemicity of LRV in the two geographic areas might also explain the difference. Saberi et al., 2020 [45] tested 85 samples of L. major and L. tropica isolated from patients with CL from Iran. Out of these, 59 samples were LRV2 positive. There is limited study in the Old World regions regarding LRV in Leishmania parasite isolates and/or samples from patients with CL. However, a study conducted in L. aethiopica parasites in Ethiopia reported the presence of LRV2 in 5 out of 11 L. aethiopica isolates [46]. In addition, a study conducted in Turkey indicated 7 out of the 24 L. tropica and 3 out of the 3 L. major isolates were LRV2 positive [47]. Moreover, LRV2 was detected in two out of 3 L. major isolates from human patients with CL in south Uzbekistan [48].

Fig 5

Forest plot showing the pooled LRV prevalence estimate by geographical region in Leishmania parasites and/or lesions of patients with cutaneous leishmaniasis, 2021.

Leishmania RNA Virus has been isolated from different Leishmania isolates (Fig 6). Higher LRV prevalence was detected in Leishmania guyanensis and Leishmania braziliensis. Similar findings have been reported by Saberi et al, 2019 [44]. They reviewed different studies aimed to detect LRV in Leishmania parasite including Leishmania infantum. They indicated that the high prevalence of LRV among causative agents of New World Leishmania isolated from the metastatic clinical forms suggests potential association of LRV with metastatic clinical forms in New World endemic regions. However, Valencia et al, 2022 showed that the presence of LRV is not correlated with different clinical forms, treatment failure and disease severity [15]. Our study as well as other publications [15,44] illustrate that there is a gap of ​​knowledge and more work need to be carried out to reach consensus on the role of LRV in CL.

Fig 6

Forest plot showing the pooled LRV prevalence estimate in different Leishmania species from patients with cutaneous leishmaniasis, 2021.

Considering the clinical presentations, LRV has been reported from all clinical manifestations of CL with higher prevalence in metastatic form (MCL and DCL) than non-metastatic one (LCL). This is in line with different reports that have described that LRV results in disease severity and leads to the metastatic and disfiguring form of CL [33,47,48,49]. Ives et al., 2011 showed that metastasizing parasites have a high Leishmania RNA virus–1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) of immune cells to induce proinflammatory cytokines and chemokines [14]. This intense proinflammatory condition leads to tissue destruction and disfiguring [46,50]. Besides, the majority of the LRVs were detected in L. braziliensis (n = 518) and L. guyanensis (n = 473) parasites that are the causative agents for MCL [51]. On the other hand, it has been indicated that the presence of LRV might not be the only factor explaining the evolvement of the disease to severe forms [52].

It has also been described that there is variation in the intensity of the viral load and sequence divergence of LRV among different Leishmania parasites [33,46]. Different Leishmania parasites contain related viruses with distinct regions of sequence conservation and divergence in their genome. The presence of the virus and the intensity of the viral load can also be detected in lesion biopsies [53-57]. The detection of the LRV directly from the lesion biopsies would be much easier than detecting it from cultured parasites since it does not require parasite culturing and would help to run more samples in a short period of time in areas with poor laboratory setup. This might also avoid the effect of the culturing and processing procedures on the LRV status. The culturing environment of the Leishmania parasite may not be favourable for the endosymbiotic virus. Despite limited data available on the area to reach more reliable results, our study is relevant for the community since it shows the prevalence of LRV in isolates of different species of Leishmania.

5. Limitations

The main limitation of our study is the scarcity of published data on LRV in localized cutaneous, mucocutaneous and diffuse cutaneous leishmaniasis. The sample size used in most of the available data was limited too. There is not enough information on the role of LRV in the different clinical manifestation of cutaneous leishmaniasis. The mechanism by which the presence of LRV affects the disease pathogenesis, disease severity, treatment failure and relapse has not been identified and is not addressed in most of the included studies. We also did not show the impact of the viral load and sequence divergence of LRV among different Leishmania parasites on the disease outcome.

6. Conclusion

This systematic review and meta-analysis revealed that there is high prevalence of LRV in Leishmania isolates and/or lesion biopsies from patients with CL globally. Future studies using large numbers of isolates are needed to collect more information on the presence of the LRV in Leishmania parasites. Conducting experimental and clinical based studies will be key to better understand the role of the virus in clinical presentations of the disease, treatment failure and disease severity.

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6 Jan 2022

Dear Prof Shita

Thank you very much for submitting your manuscript "Prevalence of Leishmania RNA virus in Leishmania parasites in patients with cutaneous leishmaniasis: a systematic review and meta-analysis" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The manuscript was revised by three different reviewers. I strongly suggest taking into account the comments of the second and third reviewers. Despite little evidence in the literature, the data presented are of interest, but I suggest that there is a better explanation regarding the presence of LRV and the development of clinical forms of tegumentary leishmaniasis. The review cannot be based only on cutaneous leishmaniasis, since there are data associating mucosal leishmaniasis and the presence of LRV and, therefore, the word cutaneous should be replaced by tegumentary in the title of the manuscript.

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The manuscript was revised by three different reviewers. I strongly suggest taking into account the comments of the second and third reviewers. Despite little evidence in the literature, the data presented are of interest, but I suggest that there is a better explanation regarding the presence of LRV and the development of clinical forms of tegumentary leishmaniasis. The review cannot be based only on cutaneous leishmaniasis, since there are data associating mucosal leishmaniasis and the presence of LRV and, therefore, the word cutaneous should be replaced by tegumentary in the title of the manuscript.

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Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

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-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: see general comments below

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Answer: YES

-Is the study design appropriate to address the stated objectives?Answer: Yes, because the authors used the PRISMA guidelines

-Is the population clearly described and appropriate for the hypothesis being tested?Answer: No , because there are few published studies avialable to be analyzed

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?Answer: NO , because there are few published studies avialable to be analyzed

-Were correct statistical analysis used to support conclusions?Answer: YES

-Are there concerns about ethical or regulatory requirements being met?Answer: Yes

Reviewer #3: The aim of the study is clearly presented and the study design described thoroughly.

It is a review and meta-analysis MS and the selection criteria for the articles included for analysis are clearly presented.

In the introduction, some informations are not based in appropriate references such as case reports and visceral leishmaniasis paper (referring to asymptomatic cases). We notice that the authors are not aware that New World CL rarely evolves to self-cure; this information needs correction.

Among the articles included some were based in small number of parasite isolates and it is not clear wether in these articles the isolates were randomly included or they had any bias for the inclusion in the study.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: see general comments below

Reviewer #2: -Does the analysis presented match the analysis plan? Answer: YES

-Are the results clearly and completely presented?Answer:NO, not because the data extraction form is missing

-Are the figures (Tables, Images) of sufficient quality for clarity?Answer: No not because the prevalence could be separated by species

Reviewer #3: To estimate the prevalence of LRV, from the data presented in the Table 2, it seems that the presence of the virus does not relate to particular clinical presentation and the development of severe forms.

There is no other data that show correlation of the presence of the virus and worse disease development. It would be desirable the analysis of the data from articles showing high number of isolates where it would be possible to compare the development of the disease with parasites with and without the virus avoiding the confounding factors such as geographical area and Leishmania species diversity.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: see general comments below

Reviewer #2: -Are the conclusions supported by the data presented? Answer: no , Because some conclusions are not a consensus in the area

-Are the limitations of analysis clearly described?Answer:No , because it was not discussed that there is little bibliographical information available

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?Answer:YES but could better discuss

-Is public health relevance addressed?Answer:YES but could better discuss

Reviewer #3: Conclusions are not fully supported by the presented data. Limitations of the analysis are not clearly described.

The relevance of the study for the public health is not clearly addressed.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: could do with a native English speaker improving English in places

Reviewer #2: Introduction line 5 not necessarily leishmaniasis is prevalent in the poorest communities . paragraph 2 line 5

there is no consensus that LV can define the clinical form.paragraph 4 line 8 the authors could clarify whether the isolates were obtained from patients.

methods:

2.1 it was not clear the search strategy, it was not clear whether the keywords were used separately, together or concatenated.

2.2 why? all in vitro and animal model studies were eliminated, as these studies can be done with patient isolates.paragraph 2 lines 6 and 7 authors could provide the exclusion criteria made at this stage of the analysis

2.3 line 1 authors could provide the data extraction form and citeria.line 9 authors could make it clear if the "checklist-JBI" were used as exclusion criteria.

3.1line 4 the authors could indicate that these results are described in figure 1. line 5 the authors can describe how the 83 studies from the last 107 were excluded and indicate that the exclusion criteria is described in figure 1.

3.3 in line 4 what does "p=0.00." means? .

Discussion lines 9-10 there is no consensus that LV defines the clinical form of cutaneous leishmaniasis.lines 13-14 there is no consensus that LV causes therapeutic failure.line 15 separate leishmania from parasite.

Figure 4 was not shown in the results.

page 18-19 line 2 is not a consensus

Reviewer #3: Language revison and edition would be desirable.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Prevalence of Leishmania RNA virus in Leishmania parasites in patients with cutaneous leishmaniasis: a systematic review and meta-analysis.

This a useful and well performed study, and highlights areas relevant knowledge gaps and areas for future research.

My comments mostly relate to areas of text which could be clearer.

Principal findings abstract: ‘total of 1159 samples from 24 studies were tested’ makes it sound as if you tested them.

Author summary: ‘It needs further studies to well understand’ would read better as ‘It needs further studies to better understand…’

Your opening sentence is too vague ‘Cutaneous leishmaniasis (CL) is among neglected tropical diseases affecting millions of the world population since long years back [1, 2].’

I would suggest something like ‘Cutaneous leishmaniasis (CL) is a neglected tropical diseases affecting millions of people worldwide. Leishmania species probably co-evolved with mammals (Steveding et al 2017) and have been identified in humans from at least 4 millennia before present (Zink et al).’

multiplenon-ulcerative nodular lesionswith. should be : multiple non-ulcerative nodular lesions with lar

methods- can you be clearer about your search strategy. You write ‘combinations of search terms: “cutaneous leishmaniasis “AND “Leishmania RNA virus OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”

Was this combinations of search terms: “cutaneous leishmaniasis “AND (“Leishmania RNA virus OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”)?

Surely RNA virus was always in the search strategy? In which case I would have expectd Leishmania RNA virus AND (“cutaneous leishmaniasis “ “OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”)

Is this correct? Please clarify

‘The articles were independently reviewed by two investigators (EY, EN)’. What did you do if you disagreed?

Discussion: ‘The detection of the LRV from the lesion biopsies would be much easier and help to run more samples in short period of time in areas with poor laboratory setup.’ I don’t understand this sentence. Please clarify.

Reviewer #2: the authors can discuss the article titled "Global status of synchronizing Leishmania RNA virus in Leishmania parasites: A systematic review with meta‐analysis" because are similar and to discuss limitations of the present study, discuss some similar finding to, argue that more studies need to be carried out to reach more reliable results and at the same time to emphasize that there is a gap in this area of ​​knowledge.

Reviewer #3: The MS is relevant to show the prevalence of LRN in the isolates of different species of Leishmania. However, the main concern that is the correlation of the presence of the virus with the disease development is not clearly presented based on the supporting data.

--------------------

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Reviewer #1: Yes: Dr Richard Weller

Reviewer #2: No

Reviewer #3: No

Figure Files:

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

27 Feb 2022

Submitted filename: point-by point response.docx

Click here for additional data file.

22 Mar 2022

Dear Mr Shita,

Thank you very much for submitting your manuscript "Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: a systematic review and meta-analysis" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The manuscript was revised after some changes made by the authirs. The answers provided by the authors, in relation to the comments of the first review, covered all the questions raised. Only one additional suggestion, made by reviewer 2, should be taken into account. As described below:

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet , after text modifications; maybe a table with the word combination strategy used in the present work will be necessary.

Improvement oh this topic is very important for other groups would reproduce the work or use the same estrategy to other studies , this is important for reproducibility of the work and increases the chances of the work being cited.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

José Angelo Lauletta Lindoso

Guest Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

The manuscript was revised after some changes made by the authirs. The answers provided by the authors, in relation to the comments of the first review, covered all the questions raised. Only one additional suggestion, made by reviewer 2, should be taken into account. As described below:

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet , after text modifications; maybe a table with the word combination strategy used in the present work will be necessary.

Improvement oh this topic is very important for other groups would reproduce the work or use the same estrategy to other studies , this is important for reproducibility of the work and increases the chances of the work being cited.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Objectives, design and analysis all clearly described. Sample size is a function of the relatively sparse number of studies.

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? YES

-Is the study design appropriate to address the stated objectives? NO; BECAUSE THE TOPIC 2.1. Search strategy and eligibility criteria is not clear yet maybe a table for combination word estrategy will be necessary , modified in order to make it clearer

-Is the population clearly described and appropriate for the hypothesis being tested? YES

Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? NO

-Were correct statistical analysis used to support conclusions? YES

-Are there concerns about ethical or regulatory requirements being met? NO

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results clearly presented. Good tables and figures.

Reviewer #2: -Does the analysis presented match the analysis plan? YES

-Are the results clearly and completely presented? YES

-Are the figures (Tables, Images) of sufficient quality for clarity? YES

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions and limitations all supported.

Reviewer #2: -Are the conclusions supported by the data presented? YES

-Are the limitations of analysis clearly described? YES

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? YES

-Is public health relevance addressed? YES

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Much Improved manuscript which reads very clearly.

Reviewer #2: "Minor Revision"

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet maybe a table for combination word estrategy will be necessary , modified in order to make it clearer

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: A well presented paper

Reviewer #2: the study was made with heterogeneus and low quantity of data but in the other hand the authors get all avialible information to do the work

--------------------

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Reviewer #1: Yes: Dr Richard Weller

Reviewer #2: Yes: Eduardo Milton Ramos Sanchez

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

9 Apr 2022

Submitted filename: point-by point response.docx

Click here for additional data file.

18 Apr 2022

Dear Mr Shita,

We are pleased to inform you that your manuscript 'Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: a systematic review and meta-analysis' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

José Angelo Lauletta Lindoso

Guest Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The authors reviewed the manuscripts and accepted the suggestions of reviewer 2. The authors included the word search strategy to carry out this systematic review and meta-analysis. Therefore, I consider that the article should be accepted for publication.

25 May 2022

Dear Mr Shita,

We are delighted to inform you that your manuscript, "Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: a systematic review and meta-analysis," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases