Homa Hajjaran1*†
Reza Saberi2,3*†
Alireza Borjian1†
Mahdi Fakhar2†
Seyed Abdollah Hosseini2†
Sajjad Ghodrati1
Mehdi Mohebali1†- 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
- 2Toxoplasmosis Research Center, Communicable Diseases Institute, Iranian National Registry Center for Lophomoniasis and Toxoplasmosis, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
- 3Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran
Leishmaniasis is one of the most common vector-borne parasitic diseases in Iran. Leishmania species identification is necessary for epidemiological aspects, precise prognosis, control and treatment of the disease. We systematically searched all the studies, reports, and documentation related to species identification and geographical distribution of causative agents of cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL) using DNA-based molecular diagnostic techniques in Iran. International databases including PubMed, ScienceDirect, Embase, Google Scholar, Scopus, and Web of Science were systemically searched for English articles and Iran's databases including SID, IranMedex and Magiran were searched for Persian reports and articles. Searches were performed from 1999 to 2019 (20 years). The current review was conducted using the keywords: cutaneous leishmaniasis, visceral leishmaniasis, Leishmania species, Human, Molecular, PCR, and Iran. The study quality was evaluated using the NOS checklist. This meta-analysis procedure was accomplished using STATA, version 2.7.9. Of the 3,426 records identified in the initial search, 154 articles met inclusion criteria and qualified for the systematic review and meta-analysis. In subgroup analysis, the pooled frequency of causative agents of CL isolates was 67.3% (95% CI: 59.51–74.67%) for L. major and 32.1% (95% CI: 24.72–39.87%) for L. tropica. In addition, the pooled frequency of causative agents of VL isolates was 97.1% (95% CI: 94.6–98.8%) for L. infantum and 2.9% (95% CI: 1.12–5.37%) for L. tropica. The findings of this study showed that the main causative agents of CL and VL in Iran are L. major and L. infantum, respectively. Moreover, kinetoplast DNA (kDNA) and internal transcriber spacer (ITS) were the most used markers for identifying Leishmania species. The current study provides valuable data to encourage and direct researchers as well as public health managers in the comprehensive leishmaniasis control and prevention planning in Iran.
Introduction
Leishmaniasis is a neglected tropical disease (NTD) caused by the Leishmania parasites, which are transmitted by the bite of sand flies (1). There are four clinical forms of the disease: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL) and mucosal leishmaniasis (ML) (2). Despite universal scientific community efforts to reduce cases of human leishmaniasis, numerous cases of such devastating disease are still reported worldwide (3). The disease currently affects 12 million people with 350 million people are living in regions with a high risk of infection. World Health Organization (WHO) estimates the annual global incidence of 0.7–1.2 million cases of CL and 0.1–0.4 million cases of VL (4). At present, the majority (about 90%) of CL cases occur in eight countries mainly including Asian and South American countries (4). Moreover, more than 90% of global cases of VL had been reported from seven countries mainly including African and South American countries (4, 5). In Iran, CL is the most common form of the disease and recent reports estimates >20,000 annual cases (6), but VL has been reported sporadically, with about 100–300 new serologically positive cases of VL reported annually (7).
Species discrimination is important, because of differences among the Leishmania species in levels of virulence and responses to the various chemical drugs (8, 9). As a result, distinguishing Leishmania spp. is critical for accurate diagnosis and appropriate treatment (9). Morphological identification of Leishmania species is not possible, but a variety of DNA-based molecular diagnostic techniques, including restriction fragment length polymorphism (RFLP), nested-PCR methods as well as high-resolution melting analysis PCR (HRM-PCR) have been reported for identification of Leishmania on different taxonomical levels (genus and species) (10). According to our literature review, several target markers were used to identify Leishmania species, including minicircle kinetoplastic DNA, heat shock protein 70 gene, N-acetylglucosamine-1-phosphate transferase (nagt) gene, and internal transcription spacer (ITS1 & 2).
There are several studies regarding the identification of Leishmania species causing CL and VL in Iran. The aim of this systematic review and meta-analysis was therefore to define the geographical distribution of Leishmania spp. among human populations as well as exploring molecular markers used for identifying Leishmania spp. in this population throughout two decades ago (1999–2019) in Iran.
Methods
Search Approach
This systematic study was achieved according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) (11). The present study was carried out to estimate the species identification and geographical distribution of causative agents of CL and VL cases in Iran. A search in literature was carried out via the nine English and Persian databases, including PubMed, Embase, Google Scholar, Science Direct, Scopus, Web of Science and SID, IranMedex and Magiran up to Sep 2019, respectively. The current review was conducted by the Medical Subject Headings (MeSH) terms including: “Cutaneous leishmaniasis”, “Visceral leishmaniasis,” “Leishmania”, “Species”, “Human,” “Molecular”, “PCR”, and “Iran”, alone or combined together with “OR” or/and “AND” operators.
Paper Screening
Initially, the titles and abstracts of searched articles were screened for eligibility by two authors independently, and those that did not describe identification of Leishmania species were removed. Data on the identification of Leishmania spp., were extracted from studies according to the following including criteria: (a) peer-reviewed original research, (b) papers studies that surveyed identification of Leishmania species using various polymerase chain reaction techniques, (c) studies published in English or Persian during 1999–2019 and (d) full-text articles were available. Additionally, the exclusion criteria were as follows; (a) duplicated data, (b) review studies, and (c) studies on animal reservoirs.
Data Extraction
Out of the retrieved papers, 154 papers were eligible for inclusion in this study. Required data were collected based on the first author, publication year, province, total sample, positive number, Leishmania spp., types of clinical manifestation, diagnostic methods, marker genetic used and quality assessment. Three independent authors extracted the above details carefully.
Quality Assessment
In the current study, the Newcastle-Ottawa Scale was used to evaluate the quality of studies. NOS score ranged from 0 and 7 [low quality, (1 and 2), moderate quality, (3–5), and high quality (6 and 7)] (12).
Statistical Analysis
This meta-analysis was completed using STATA software, as comprehensive meta-analysis software (http://statsdirect.com). The heterogeneity index was assessed using standard Cochran's Q- and I-squared statistics, with the random effects estimate they imply. Egger's test was used to assess potential publication bias. A p < 0.05 (≤0.05) is statistically significant.
Results
Characteristics and Quality of the Included Studies
Records retrieved in the mentioned electronic databases based on preparatory search strategies of nine databases yielded 3,426 papers; after removal of duplication papers, 2,244 papers were extracted. In the next step, using the abstract screening based on the inclusion/exclusion criteria, 1,683 other articles were excluded. Following that, 561 full-text articles were screened, of which 154 were found to be eligible for systematic review and meta-analysis. Figure 1 summarizes the flow chart presenting the study design process. The baseline characteristics of all included studies are tabulated in Tables 1, 2.
Figure 1. PRISMA flow diagram describing summaries of included/excluded studies.
Table 1. Baseline characteristics of the Leishmania species identification from CL cases in the systematic review and meta-analysis from 1999 to 2019.
Table 2. Baseline characteristics of the Leishmania species identification from the VL cases in the systematic review and meta-analysis from 2005 to 2019.
Results of the Meta-Analysis
In total, 10,586 CL isolates were identified, with two causatives of ZCL (L. major, n = 6,714) and ACL (L. tropica, n = 3,872) being reported in 19 provinces across Iran (Fars, Khuzestan, Isfahan, Golestan, Ilam, Razavi Khorasan, Kerman, Sistan & Balochistan, Tehran, Yazd, Hormozgan, Semnan. Most of the L. major isolates belonged to Fars (n = 992), Khuzestan (n = 844), and Isfahan (n = 687) provinces in the southern and central regions of Iran. In addition, the majority of L.tropica isolates belonged to Kerman (n = 1,142), Razavi Khorasan (n = 949) in the east, and Lorestan (n = 260) in the west (Figure 2). In contrast, 542 isolates for VL cases were determined in 11 provinces (Fars, Ardabil, Tehran, Kohgiluyeh and Boyer-Ahmad, Golestan, Ilam, Lorestan, East-Azerbaijan, Boushehr, Kerman, and Mazandaran), with the majority of VL isolates belonging to Fars (n = 230) in southwestern Iran and Ardabil (n = 107) in northwestern Iran (Figure 3).
Figure 2. Map of distribution of Leishmania spp. causing ZCL and ACL using molecular methods in different geographical areas of Iran.
Figure 3. Map of distribution of Leishmania spp. causing of visceral leishmaniasis using molecular methods in different geographical areas of Iran.
According to the literature review, nine genetic markers (kinetoplast DNA, internal transcribed spacer region-1 and 2, cytochrome b, heat shock protein 70, N -acetylglucosamine-1-phosphate transferase, pteridine reductase 1, tryparedoxin peroxidase, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and 7SL RNA) were used for identification Leishmania species that the most species were identified with kinetoplast DNA (n = 5,592) and ITS markers (n = 4,544). It should be noted that some species of Leishmania were identified by Random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) method using random primers. The used molecular methods of Leishmania species identification in most studies were nested PCR and PCR-RFLP.
In subgroup analysis, the pooled frequency of causative agents of CL isolates was 67.3% (95% CI: 59.51–74.67%) for L. major and 32.1% (95% CI: 24.72–39.87%) for L. tropica (Table 3 and Supplementary Figures 1, 2). Also of note, the 14 and four isolates were identified as L. infantum and L. turanica as causative agents of CL and ZCL cases, respectively (14, 22, 36, 47, 74, 89, 120, 122).
Table 3. The pooled frequency, heterogeneity, and publication bias of Leishmania species that causative VL and CL leishmaniasis.
In addition, the pooled frequency of causative agents of VL isolates was 97.1% (95% CI: 94.6–98.8%) for L. infantum and 2.9% (95% CI: 1.12–5.37%) for L. tropica (Table 3 and Supplementary Figures 3, 4). Also, other clinical forms of leishmaniasis were reported as follow: ML (n = 12), DCL (n = 5), MCL (n = 3), and PKDL (n = 2). ML cases caused by L. major (n = 7), L. tropica (n = 2), L. infantum (n = 2), and a mix of L. major/L. tropica (n = 1) whereas, all DCL and MCL cases caused by L. major, but two causative agents of PKDL were identified as L. infantum.
Discussion
Leishmaniasis remains a major community health-based challenge with worldwide distribution, particularly in Iran (165). Identification of species is essential in diagnosis, treatment and epidemiological studies (165). We attempted to determine the etiological agents of human cutaneous and visceral leishmaniasis and their geographical distribution in Iran over two decades ago in the current systematic review and meta-analysis study.
According to the finding of the meta-analysis, most Leishmania isolates identified in Iran belonged to CL than VL cases. Every year, a large number of CL cases with a wide distribution are reported in 19 of 31 Iranian provinces, primarily in the central, southwest, east, and northeast regions. Some evidence suggests that the CL incidence rate overall has been decreasing in recent years, from 37/100,000 in 2007 to 22/100,000 in 2013. This decrease in incidence could be because appropriate public health measures such as education to residents, case finding and management, treatment, control of reservoir hosts, and distribution of repellents and nets treated with permethrin in the endemic focus of the disease have been accomplished (166).
However, at the same time, it seems like the distribution of CL has been extended to a new area (166, 167). In contrast, VL is mainly endemic in restricted regions of Iran, notably the northwest (Ardabil province) and southwest (Fars province) (7).
As illustrated by the finding of the subgroup analysis, the pool frequency of L. major and L. tropica, as causative agents of CL was 67.32% (95% CI: 59.51–74.67%) and 32.1% (95% CI: 24.72–39.87%), respectively. It can be concluded that the distribution of ZCL is higher than ACL form. According to a systematic review study conducted by Foroutan et al. rodents are the most important reservoirs of Leishmania species in many foci of ZCL throughout Iran (168). The most important of these rodent reservoirs are Rhombomys opimus, Meriones libycus, and Nesokia indica. The finding of this study showed that L. major has been reported as the predominant species of these rodents. The role of rodents in the spread of ZCL is evident (168).
The findings of this study demonstrated that the main causative agent of ZCL cases in the 14 provinces is L. major and the main causative agent of ACL cases in the five provinces is L. tropica (Figure 2). Although L. tropica was formerly common in many large urban areas, it has also been observed in rural areas and small cities in Iran (169). According to the findings of the two studies, four isolates of L. turanica were found in CL patients in the Gonbad-Kavous and Turkmen Sahara districts of Golestan province, which are the known oldest ZCL foci (90, 121). Nevertheless, it should be noted that L. major is the principal agent of ZCL in Iran. Besides, 14 isolates of L. infantum have been reported to cause CL cases (14, 22, 36, 47, 74, 122). A review of the literature showed that cases of L. infantum as the causative agent of CL have previously been identified in the Mediterranean (170), Southeast European countries, such as Portugal, Spain, Italy, and France (171) and the Americas (172), which is consistent with the findings.
On the other hand, the pool frequency of causative agents of VL isolates was 97.1% (95% CI: 94.6–98.8%) for L. infantum and 2.9% (95% CI: 1.12–5.37%) for L. tropica. The result of this study revealed that the main causative agent of VL in Iran is L. infantum. According to the results of a recent systematic review, the prevalence of HVL infection has been decreased in Iran throughout the last two decades. The maximum (3%, 95% CI: 1–5%) and minimum (0.5%, 95% CI, 0.2–0.7%) pooled prevalence of HVL was estimated in the northern and western Iranian provinces, respectively (173). It should be noted that the reason for reporting the relatively high number of cases of VL in Tehran, central Iran, as shown in Figure 3, is that these patients were referred to Tehran University Hospitals from other regions for diagnosis and treatment follow-up. Therefore, these reported cases did not belong to Tehran.
Nonetheless, despite the Iranian Center for Disease Control's (CDC) efforts to monitor and prevent HVL, new human cases of VL continue to emerge in old endemic foci. On the other hand, the disease has also emerged in new non-endemic areas of the country, such as Golestan province in north-eastern Iran (174). However, geo-climatic and environmental factors play the most important role in the emergence/reemergence of HVL in an area (174). Reasonable steps to monitor VL and prevent its spread to other areas should be taken in this respect.
Currently, molecular approaches are used for species identification, genotyping, and determine polymorphisms in Leishmania parasites (175). In most cases, these methods have replaced the isoenzyme method, which is the standard method for determining the species and strain of the Leishmania parasite (176). Molecular techniques have the potential to be more sensitive and rapid. In addition to high sensitivity and specificity, molecular methods can differentiate relapse from reinfection of disease (177).
Several DNA markers were used for DNA amplification of Leishmania spp. in the included articles, in which most kDNA and ITS1markers were used for the diagnosis of identification of species. Our finding showed that the kDNA-based PCR was the most sensitive diagnostic method for leishmaniasis and the ITS1-based PCR could be used as a sensitive/specific method to identify the Leishmania species. It is interesting to know that ITS1 is less sensitive compared to kDNA minicircles, because the copy number of rDNA (<200) is lower than the copy number of kDNA minicircles (tens of thousands). Therefore, it is more desirable to use specific primers for ITS regions and kDNA genes to diagnose the disease (103).
Phylogenetic analyses targeting the ITS1 gene are valuable and reliable tools in genetic analytical characterization of Leishmania parasite. This region is highly conserved among species (178). The ITS region as a target for differentiation of Leishmania at species and strain level has been used in different studies (102, 105, 132). As a whole, it should be noted that apply of two genetic markers simultaneously could provide more data regarding genetic map of the Leishmania parasite particularly in an endemic focus.
Notwithstanding that the DNA-based methods have proven to be very efficient in the identification and distinguish of Leishmania species, these methods also have limitations. One of these limitations is the exquisite sensitivity of these methods, and consequently false-positive PCR (179). For resolving this problem, it is necessary to use positive and negative control in each experiment simultaneously. Furthermore, preventing PCR contamination requires that this method be performed in reference laboratories. The specificity of PCR is generally controlled by several variables, including primer design, target genes, amount and purity of DNA, and type of enzyme (180).
In the end, the issue of the Leishmania RNA virus has become an interesting topic (181). Leishmania RNA virus (or LRV) is a genus of double-stranded RNA (dsRNA) virus in the family Totiviridae. LRVs exist within many species of the Leishmania isolates (181). Nowadays, Leishmania RNA virus is being extensively surveyed because it might be an important virulence factor of the infection (182). According to previous evidence, studies have been conducted to investigate the presence of Leishmania RNA virus in Iran. It is interesting to know that Leishmania RNA virus has been detected in many L. major species and one L. infantum isolated from a VL patient, and one L. tropica isolated from a CL patient in Iran (110, 183).
Limitations
One of the limitations of this study was that some authors did not report isolates to belong to which province and isolates were introduced to as Iranian isolates. In addition, the limitations of the present study include: (a) use of different diagnostic techniques in the two included studies without similar results, (b) available studies with no sufficient information on identification of Leishmania species, and (c) variability of the sample size of the included studies. Also, people commuting between urban and rural areas has made it difficult to determine the main source of infection.
Conclusion
Our study reconfirms that CL and VL remain important infectious diseases in Iran. In this regard, the main causative agent of ZCL and ACL in Iran is L. major and L. tropica, respectively. In addition, the findings of this study demonstrated that the main causative agent of VL in Iran is L. infantum. The current study provides the geographical distribution of causative species in CL and VL forms in Iran and is a source of data to help researchers and public health workers in comprehensive investigations and developing prevention programs. Based on current findings, two markers kDNA and ITS1 can be used to accurately diagnose and determine Leishmania species using molecular methods. Our findings highlight the need for the implementation of control measures among the patients of both CL and VL. Further attention and monitoring will be needed to improve the surveillance and effective control to reduce the incidence of leishmaniasis in Iran.
Data Availability Statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
Author Contributions
HH and RS conceived the presented idea and wrote the manuscript. MF and MM reviewed and commented on the findings of this work. HH, AB, and SG initially searched the literature studies and collected the data. SH analyzed and interpreted the data and methods. All authors provided critical feedback and agreed to the published version of the manuscript.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
We would like to express their thankfulness to all authors that their valuable publications were included in the current review.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh.2021.661674/full#supplementary-material
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Keywords: Leishmania major, Leishmania tropica, Leishmania infantum, DNA-based molecular method, human, Iran
Citation: Hajjaran H, Saberi R, Borjian A, Fakhar M, Hosseini SA, Ghodrati S and Mohebali M (2021) The Geographical Distribution of Human Cutaneous and Visceral Leishmania Species Identified by Molecular Methods in Iran: A Systematic Review With Meta-Analysis. Front. Public Health 9:661674. doi: 10.3389/fpubh.2021.661674
Received: 31 January 2021; Accepted: 31 May 2021;
Published: 25 June 2021.
Edited by:
Herbert Leonel de Matos Guedes, Federal University of Rio de Janeiro, BrazilReviewed by:
Muhammad Imran Khan, The University of Haripur, PakistanMehmet Karakus, University of Health Sciences, Turkey
Yusuf Ozbel, Ege University, Turkey
Copyright © 2021 Hajjaran, Saberi, Borjian, Fakhar, Hosseini, Ghodrati and Mohebali. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Homa Hajjaran, hajaranh@tums.ac.ir; Reza Saberi, r.saberi@mazums.ac.ir
†ORCID: Homa Hajjaran orcid.org/0000-0001-5877-2845
Reza Saberi orcid.org/0000-0002-7906-7034
Alireza Borjian orcid.org/0000-0003-1440-4922
Mahdi Fakhar orcid.org/0000-0002-4690-6938
Seyed Abdollah Hosseini orcid.org/0000-0002-2990-1123
Mehdi Mohebali orcid.org/0000-0002-4164-9514