Prevalence of Mycobacterium leprae in armadillos in Brazil: A systematic review and meta-analysis.

Understanding the prevalence of M. leprae infection in armadillos is important because of evidence from Brazil and other countries of an association between contact with armadillos and the development of Hansen's Disease (leprosy). Our aim was to characterize studies which have investigated natural M. leprae infection in wild armadillos in Brazil, and to quantify and explore variability in the reported prevalence of infection. We conducted a systematic review (PROSPERO CRD42019155277) of publications in MEDLINE, EMBASE, Global Health, Scopus, LILACS, Biblioteca Digital Brasileira de Teses e Dissertações, Catálogo de Teses e Dissertações de CAPES, and Biblioteca Virtual em Saúde up to 10/2019 using Mesh and text search terms (in English, Portuguese, Spanish, and French). The 10 included studies represented a total sample of 302 armadillos comprising 207 (69%) Dasypus novemcinctus, 67 (22%) Euphractus sexcinctus, 16 (5%) Priodontes maximus, 10 (3%) Cabassous unicinctus, and 2 (1%) Cabassous tatouay from 7 different states. Methods used included histopathology (4 studies), PGL-1 and LID-1 antigen detection (4 studies) and examination for clinical signs of disease (4 studies). Eight studies used PCR of which 7 targeted the RLEP repetitive element and 3 tested for inhibitory substances. M. leprae prevalence by PCR ranged from 0% (in 3 studies) to 100% in one study, with a summary estimate of 9.4% (95% CI 0.4% to 73.1%) and a predictive interval of 0-100%. The average prevalence is equivalent to 1 in 10 armadillos in Brazil being infected with M. leprae, but wide variation in sample estimates means that the prevalence in any similar study would be entirely unpredictable. We propose instead that future studies aim to investigate transmission and persistence of M. leprae within and between armadillo populations, meanwhile adopting the precautionary principle to protect human health and an endangered species in Brazil.

Introduction

Understanding the prevalence of M. leprae infection in armadillos is of public health importance because of epidemiological evidence from Brazil and other countries of an association between contact with armadillos and the development of Hansen’s Disease (leprosy) in people [1-5]. The first report of natural infection of M. leprae in wild armadillos in Brazil was a preliminary finding in 2002 based on PCR analysis of blood samples from 14 nine-banded armadillos (Dasypus novemcinctus) from the south-eastern state of Espírito Santo [6]. A later study confirmed these findings in Espírito Santo [7], and M. leprae was subsequently reported in wild armadillos from the northern states of Ceará [8] and Pará [3]. Conversely, studies in São Paulo and Mato Grosso do Sul [9] and Amazonas [10] found no M. leprae in wild armadillos.

Brazil is a high-burden country for Hansen’s Disease [11], with incidence varying according to geographic and socioeconomic determinants [12]. Although the disabling and disfiguring sequelae of Hansen’s Disease are entirely avoidable if diagnosed and treated early [13], the social stigma of ‘leprosy’ has not been entirely dispelled, and still has a profoundly negative impact on people diagnosed with this disease [14, 15]. The proportion of new cases in Brazil attributable to zoonotic transmission is unknown, but armadillo capture and consumption persists as a cultural habit in some parts of Brazil and wild armadillos in these areas may serve as a reservoir of M. leprae infection in humans [16].

The aim of this systematic review and meta-analysis was to characterize studies which have investigated natural M. leprae infection in wild armadillos in Brazil, and to quantify and explore variability in the prevalence of infection.

Methods

Review protocol

The protocol for this systematic review was defined in advance and registered with PROSPERO (CRD42019155277). A PRISMA checklist is provided as S1 PRISMA checklist.

Searches

We searched the following databases and libraries between October 26th-27th 2019: MEDLINE (Epub Ahead of Print, In-Process & Other Non-Indexed Citations and Daily 1946 to October 25, 2019), EMBASE (1974 to 2019 October 25), Global Health, Scopus, LILACS (Latin American and Caribbean Center on Health Sciences Information), Biblioteca Digital Brasileira de Teses e Dissertações (BDTD), Catálogo de Teses e Dissertações de CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Biblioteca Virtual em Saúde (BVS). Full search terms are provided in the supplementary appendix. In brief, we used Mesh and text search terms for: ("Mycobacterium leprae" OR "Leprosy") AND (“Armadillos” OR “Dasypus novemcinctus” OR “Dasypus septecinctus” OR “Euphractus sexcinctus”) AND “Brazil” in MEDLINE and EMBASE supplemented by Portuguese, Spanish and French equivalents in other databases (leprosy = lepra OR Hanseníase OR lepre; armadillo = tatu OR tatou; Brazil = Brasil OR Brésil). We imposed no date, language or publication type restrictions. Citations identified by the search were imported into EndNote (EndNote X9; Clarivate Analytics, Boston, MA 02210, USA) for de-duplication. Bibliographies of all included studies were searched manually.

Screening, inclusion/exclusion, quality assessment and data extraction

Screening and quality assessment were conducted independently and in parallel by three reviewers: title and abstract SC and PD; full text SC and JMA; quality assessment SC, JMA and PD. References were included if they described Mycobacterium leprae carriage or natural infection in wild armadillos in Brazil, regardless of armadillo species or microbiological method. Studies involving experimental infection and/or involving animals that were already captive were excluded. The methodological quality of each included study was rated using a 10-item quality assessment tool adapted from the NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies () [17]. The adaptation allowed for the assessment of data quality pertaining specifically to animal pathogen carriage/infection studies. Each study was rated as being of 'good', 'fair' or 'poor' quality based on the average score of the two reviewers. Data extraction was done independently and in parallel by two reviewers (SC and JMA) into a spreadsheet (). The primary outcome for data extraction was the proportion of the captured armadillos which tested positive for M. leprae. Other extracted variables included: geographical region of Brazil; armadillo species; diagnostic method (e.g. PCR, ELISA); specimen type (e.g. tissue, blood); and tissue type (e.g. liver, brain, skin, etc.).

Analysis

Key characteristics of each included study were summarized qualitatively. Binomial-normal random effects meta-analysis of the proportion of captured armadillos in which M. leprae was detected was performed in Stata (StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX, USA) using metaprop_one [18]. In this approach, the binomial distribution is used to model within-study variability, and the normal distribution is used to model the random effects. Between-study heterogeneity was estimated as τ2, and evidence of heterogeneity was tested by Likelihood Ratio (LR) test comparing random and fixed effects models. The proportion of overall heterogeneity attributable to between-study variance was quantified using a formulation of the I2 statistic for binary variables [19]. Prediction intervals were estimated to show the expected prevalence of M. leprae (% positive armadillos) accounting for between-study variability [20, 21]. Meta-analysis defaulted to fixed effects if 3 or fewer studies were included. We used Egger’s test to detect small-study bias. Subgroup analyses specified a priori (subject to sufficient data) were by geographic region, armadillo species, diagnostic method, specimen type, and tissue type.

Results

Database searches identified 122 references (). A study known to be under review at the time of database searching was also included [22]. After de-duplication and screening by title and abstract, 13 references were retained for full text review, of which 10 were included for data extraction. Quality assessment rated 8 as ‘good’ and 2 as ‘fair’ quality (reviewer agreement 9/10) (). Five of the full text articles assessed for eligibility were theses or dissertations [7, 23–25, 28]. The full PCR results from two of these had been published in peer-reviewed papers that we included for data extraction: Pedrini 2006 thesis [24] in Pedrini et al 2010 [9]; Portela 2015 dissertation [25] in da Silva et al 2018 [3]. PCR results from the Deps 2003 thesis [23] were published as preliminary findings in Deps et al 2002 [6]; PCR results from the Antunes 2007 dissertation [7] and de Souza dissertation [28] had not yet been published. Key features and findings of the 10 included studies are summarized in , with further details of each study provided in .

Study sites and capture of armadillos

The geographical locations of the 10 included studies are shown in . Four studies were based in Espírito Santo state in the south east region of Brazil, 3 of which (Deps and Deps et al.) used all or part of a total sample of 66 armadillos (all Dasypus novemcinctus) collected between June 2000 and July 2001 [23, 26, 27], one (Antunes) a later sample of 69 Dasypus novemcinctus caught mainly in a different part of the state between July 2004 and July 2005 [7].

Locations of studies investigating M. leprae infection in wild armadillos in Brazil.

1—Deps, 2003, Espírito Santo; 2—Deps et al., 2007, Espírito Santo; 3—Deps et al., 2008, Espírito Santo; 4—Antunes, 2007, Espírito Santo; 5—Pedrini et al., 2010, São Paulo and Mato Grosso do Sul; 6—Frota et al., 2012, Ceará; 7—de Souza, 2016, Mato Grosso do Sul; 8—da Silva et al., 2018, Pará; 9—Stefani et al., 2019, Amazonas; 10a-10e - da Silva Ferreira et al., 2020, Rio Grande do Norte. Map produced using QGIS, Open Source Geospatial Foundation Project http://qgis.osgeo.org.

Two studies were based in the adjacent São Paulo (south east Region) and/or Mato Grosso do Sul (central west region) states, the 2010 study collecting a sample of 44 armadillos (mainly Dasypus novemcinctus and Euphractus sexcinctus) from both states at unspecified dates [9], the 2016 study a sample of 43 live (mostly Euphractus sexcinctus and Priodontes maximus) and 7 roadkill armadillos collected between June 2011 and January 2015 from the same ecoregion (Pantanal da Nhecolândia) in Mato Grosso do Sul as the earlier study [28].

The four remaining studies were located in the north or north east of Brazil: a 2012 study caught 29 armadillos (27 Dasypus novemcinctus, 2 Euphractus sexcinctus) between July and August 2007 in the north east region state of Ceará [8]; two more recent studies caught 16 and 12 Dasypus novemcinctus from the states of Pará (unspecified dates) and Amazonas (expedition in August 2015) [10], respectively; the most recent study (2019) caught 20 Euphractus sexcinctus between May and June 2016 in the north east region state of Rio Grande do Norte [22].

The 10 included studies yielded a total sample of 302 armadillos (295 live, 7 roadkill), comprising 207 (69%) Dasypus novemcinctus (‘Nine-banded’), 67 (22%) Euphractus sexcinctus (‘Six-banded’), 16 (5%) Priodontes maximus (‘Giant’), 10 (3%) Cabassous unicinctus (‘Southern Naked-tailed’), and 2 (1%) Cabassous tatouay (‘Greater Naked-tailed’) (). Armadillos were captured by local hunters in 7 studies [3, 8, 10, 22, 23, 26, 27], by veterinarians or wildlife specialists in 2 studies [7, 28] and the method was not reported in one study [9].

Armadillo species investigated for natural M. leprae infection in Brazil with geographic distributions.

a) Dasypus novemcinctus (Nine-banded armadillo, common long nosed armadillo); b) Euphractus sexcinctus (Six-banded armadillo, yellow armadillo); c) Cabassous tatouay (Greater naked-tailed armadillo); d) Priodontes maximus (Giant armadillo); e) Cabassous unicinctus (Southern naked-tailed armadillo, leathered tail armadillo). Credits: a) the authors (JMA); b) Laboratory of Studies in Immunology and Wildlife at UFERSA, Mossoró-RN, Brazil; c) & e) Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Brazil; d) Carly Vynne; all species distribution maps from Wikimedia commons, attribution www.iucnredlist.org, accessed 11/12/2019.

Biological samples

Armadillos were anaesthetized and euthanised in 5 studies [7–10, 22], captured and released in 4 studies [23, 26–28] or specimens were obtained from animals recently killed by local hunters [3]. Blood specimens were taken in 7 studies [7, 9, 22, 23, 26–28] but only three authors (Deps [23, 26, 27], Pedrini et al [9], da Silva Ferreira et al [22]) used these to test for M. leprae. A wide range of tissue types were sampled (), with all except 2 studies (Pedrini et al [9], da Silva Ferreira et al [22]) using spleen specimens and all except 2 (Stefani et al [10], da Silva et al [3]) using liver and/or ear tissue specimens. One study (Pedrini et al [9]) also tested for M. leprae in one faeces specimen and a small number (5) of nostril swabs.

DNA

Test results using PCR to detect M. leprae DNA were reported in all studies except Deps et al 2007 [26] and Deps et al 2008 [27] but this author had reported preliminary PCR results in 2002 [6] and full PCR results were included in our meta-analysis [23]. All but one of the 8 PCR studies targeted the RLEP M. leprae-specific repetitive element [3, 7–10, 28]; Deps targeted a 372bp groE-L gene sequence encoding the 65kDa protein [23] and Antunes (in addition to RLEP) targeted a 360bp sequence encoding the 18kDa protein [7]. The 7 RLEP studies differed with respect to PCR methods (primers, testing for inhibitory substances, nested PCR) and whether positive PCR results were confirmed as M. leprae by other genomic methods (sequencing, VNTR, RFLP) ().

Three of the 5 studies which used a single round of PCR for RLEP reported 0% positivity [9, 10, 28], with the other two reporting 6% (4/65) and 63% (10/16) positivity [3, 7]; the two studies which used nested PCR for RLEP reported 21% (6/29) and 100% (20/20) positivity [8, 22]. Inhibitory substances were tested for in 3 studies [8, 10, 22], being detected in 0/12, 1/29, 1/20 of samples where the corresponding PCR results were 0% (0/12), 21% (6/29) and 100% (20/20), respectively. The two non-RLEP PCR studies reported 53% (19/36) and 3% (2/65) positivity based on 65kDa conventional and 18kDa real-time PCR, respectively [7, 23]. The two 18kDA-positive samples were among 4 samples which were RLEP-positive [7].

Other genomic methods to confirm PCR results were described in 6 studies but were not used in two of these studies because PCR results were negative [9, 28]. In the remaining 4 studies, PCR results were confirmed to be M. leprae in 2/4 positive samples by RLEP copy sequence [7], in 6/6 by gyrA gene sequence [8], in 10/10 by RLEP sequence [3], and in 20/20 by RFLP [22].

Overall test results (M. leprae PCR-positive) from the 8 PCR studies are summarized in and . M. leprae prevalence by PCR ranged from 0% in 3 studies (12 Dasypus novemcinctus in Amazonas state [10], 50 armadillos of various species in Mato Grosso do Sul [28], 44 of various species in Mato Grosso do Sul and São Paulo [9]) to 100% in 20 Euphractus sexcinctus in Rio Grande do Norte state [22]. The other four PCR studies reported prevalences of 6% in 65 Dasypus novemcinctus in Espírito Santo [7], 21% in 29 mostly Dasypus novemcinctus in Ceará [8], 53% in 36 Dasypus novemcinctus in Espírito Santo [23], and 63% in 16 Dasypus novemcinctus in Pará state [3].

Prevalence of natural M. leprae infection in wild armadillos in Brazil detected using PCR methods.

PCR = Polymerase Chain Reaction; RLEP = M. leprae-specific repetitive element; (inh) = inhibitory substances tested for in negative DNA samples.

The summary estimate for M. leprae prevalence was 9.4% (95% CI 0.4% to 73.1%) (), with between-study heterogeneity (τ2 = 17.7) representing a negligible proportion of overall variance in this estimate (I2 = 1%, p<0.01). The predictive interval shows that the prevalence in a hypothetical future study with characteristics similar to the included studies would be entirely unpredictable, i.e. prevalence could be between 0% and 100%.

Egger’s regression test for funnel plot asymmetry did not indicate small-study bias (p = 0.16) although the 100% prevalence study was outside the pseudo 95% confidence limits ().

Differences in prevalence by tissue type in studies which found non-zero prevalence and tested multiple tissues types is shown in . Moderate heterogeneity between tissue types within the Deps study was weakly supported by statistical evidence (I2 = 35%, p = 0.1); there was no heterogeneity between tissue types for Frota et al (I2 = 0%, p = 1.0).

M. leprae prevalence (detected by PCR) in the two most commonly sampled armadillo species (Dasypus novemcinctus and Euphractus sexcinctus) is shown in . All other sampled armadillo species had zero prevalence, Cabassous unicinctus (0/2 and 0/8 animals) [9, 28], Cabassous tatouay (0/2) [9] and Priodontes maximus (0/16) [28], but these results were reported by studies which also found no M. leprae in Dasypus novemcinctus and Euphractus sexcinctus. The included studies did not provide sufficient data to support meaningful subgroup analyses by geographic region, diagnostic method or specimen type.

Histopathology

Four studies used staining techniques including Ziehl-Neelsen (ZN), haematoxylin and eosin (HE) and Fite Faraco (FF) to identify M. leprae in tissue samples: Deps 2003 (ZN, HE) [23]; Pedrini et al (ZN) [9]; da Silva et al (HE, FF, SYBR Gold, and auramine/rhodamine) [3]; and Stefani et al (HE, FF) [10]. Deps 2003 also used Bacillus Calmette-Guérin (BCG) antigen immunohistochemistry [23] and Pedrini et al inoculated liver, spleen and mesenteric lymph node specimens in LJ culture medium [9].

Deps reported 0/50 and 0/47 positive results in ear tissues samples by ZN and BCG techniques, respectively [23]. By contrast, HE results showed scarce infiltration in 15/48 (31%) ear tissue samples, moderate in 21/48 (44%) and intense in 10/48 (21%).

Pedrini et al reported entirely negative ZN results, consistent with their PCR results [9]. Stefani et al reported negative FF acid-fast bacilli results in 48 specimens from 12 armadillos, whilst HE stained tissue sections did not show histopathological features of M. leprae infection except for one skin fragment that presented unspecific inflammatory infiltrate suggestive of indeterminate leprosy [10]. Da Silva et al reported positive histopathological findings in tissue specimens from animals that had tested positive for M. leprae by PCR, but did not report overall positivity by each technique in all of the armadillos in their sample (10 PCR-positive, 6 PCR-negative) [3].

PGL-1 and LID-1

Three studies tested for M. leprae phenolic glycolipid 1 (PGL-1) antigen [3, 26, 27], and one study tested for both PGL-1 and LID-1 reactivity [22]. Deps et al 2007 used ML Flow rapid immunochromatographic serology [26] and ELISA [27], reporting 11/37 (47%) positive by ML Flow compared with 5/47 (11%) positive by ELISA. The tests were conducted at different times and concordance was not reported for paired blood samples. Da Silva Ferreira et al. reported 4/20 ML Flow negative and 3/20 NDO-LID negative (20/20 were PGL-1 ELISA and RLEP PCR positive), and suggested that these false negative lateral flow test results could be related to stage of infection because the false-negative animals showed lower anti-PGL-1 reactivity [22]. Da Silva et al used polyclonal rabbit antibody to localise PGL-1 antigen in spleen sections but did not report overall positivity in all the armadillos in their sample [3].

Clinical signs

Four studies investigated clinical signs of leprosy, three with positive findings (Deps [23], Antunes [7], da Silva Ferreira et al [22]), one negative (de Souza [28]). Deps found head or body ulcers in 12% (6/52) and ulcerated lesions on the paws and/or internal carapace of 96% (50/52) of armadillos in a study with 53% (19/36) positivity by PCR [23]; none of the animals had a typical clinical picture of disseminated disease similar to human Virchowian leprosy [31]. In 65 armadillos in a study with 6% (4/65) PCR-positivity, Antunes identified clinical alterations in 95% (62), including external lesions (20%), lymphadenomegaly (49%), liver (30%) and splenic (35%) lesions, splenomegaly (27%) and hepatomegaly (24%) [7]. In a study which found 100% positivity by PCR in 20 armadillos, skin lesions were identified in 6 animals (30%), splenomegaly in 4 (20%) and lymphadenopathy in 7 (35%) [22]. Complete absence of clinical signs in 50 armadillos as reported by de Souza were consistent with the entirely negative PCR results in this study [28].

Discussion

This is the first systematic review of natural Mycobacterium leprae infection in wild armadillos in Brazil, 17 years after the first report of M. leprae in nine-banded armadillos (Dasypus novemcinctus) caught in the south-eastern state of Espírito Santo [6]. Our review shows that the prevalence of M. leprae in samples from armadillo populations in Brazil varies from 0% to 100% (pooled average 9.4%), that M. leprae infects the two main species (Dasypus novemcinctus and Euphractus sexcinctus), and that natural infection has been reported from the north and north eastern states of Pará [3], Ceará [8] and Rio Grande do Norte [22] and as far south as Espírito Santo state [7, 23, 26, 27]. Whether the negative findings of two studies conducted further to the south and east of Espírito Santo indicate a limit to the spread of M. leprae infection in armadillos is uncertain, although both studies were relatively large [9, 28]. Similarly, absence of M. leprae infection (except for a possible paucibacillary case determined by histopathology) in armadillos captured in Amazonas state, a Hansen’s Disease endemic region, does not provide conclusive evidence of absence given the small sample size [10]. Indeed, relatively small sample sizes in all the included studies meant that the observed overall variation in M. leprae prevalence could be entirely attributable to sampling error [32], rather than to real variation i.e. some armadillo populations being heavily infected with M. leprae whilst others are disease-free or to artefactual variation, i.e. arising from differences in methods.

The most important methodological differences that could explain some of the observed variation in M. leprae prevalence as detected by RLEP PCR relate to biological samples (methods of specimen collection, processing and storage) and the presence of PCR inhibitors. Regarding the latter, the authors’ own experience of using PCR to detect M. leprae DNA is that the amount of inhibitors varies considerably depending on sample methods, leading to false negative results. The use of positive controls (purified M. leprae DNA) does not solve the problem because it only gives certainty that the PCR reaction worked but does not detect inhibitors present in the sample. Instead it is necessary to make a control of inhibitors directly in the samples by reconstituting negative ones with M. leprae DNA and repeating the PCR. Inhibitory substances were tested for in only 3 of the 8 PCR studies [8, 10, 22], and although they were detected in only a small proportion of samples we cannot discount false negative results possible affecting prevalence estimates in the other 5 studies, two of which reported 0% M. leprae prevalence [9, 28].

With regard to single vs. nested PCR, the former method was used by the three studies which reported 0% M. leprae positivity [9, 10, 28] and by the study with the lowest non-zero prevalence [7]. However, all four studies used positive controls and da Silva Ferreira et al, in their study which found 20/20 positive, reported that nested PCR had detected only one additional positive animal after the first round of PCR [22]. All but one of the 8 PCR studies targeted RLEP, albeit with some differences in the primers used, therefore differences in M. leprae DNA targets are unlikely to account for overall variation. Also, the one study with a different target (65kDa) reported 53% positive samples [23]. Whilst these methodological differences might contribute to uncertainty in quantifying accurately M. leprae prevalence in a given armadillo population, they are unlikely to account for the very wide variation that we found in our review. However, we concur with da Silva Ferreira et al who argued that it would be of benefit to future studies in this area to standardize methods [22], with a protocol based on best practice in specimen collection, handling and processing and standardized PCR methods in terms of primers and testing for inhibitory substances.

Another methodological aspect in which we find ourselves in agreement with da Silva Ferreira et al is that simpler and more rapid methods for M. leprae testing in wild armadillos, such as the ML Flow and NDO-LID tests, should be considered for future studies. Indeed, the ML Flow test was first evaluated as being potentially suitable by one of our authors in 2007 [26]. We would argue that, depending on the scientific question being asked, the lower sensitivities of such tests might be outweighed by their ease of use and non-lethality. As with PCR, these methods would need to be standardized to ensure comparability between studies. Conversely, a qualitative appraisal of results from the studies in our review suggests that histopathological methods and clinical examination for signs of leprosy are less useful. The former have the disadvantages of requiring tissue samples, being difficult to perform in the field, and being less sensitive than PCR as shown by discordant results in studies where both were performed. The discriminatory utility of clinical signs, although characterized to some extent in laboratory animals [33], is unknown in wild armadillos and is probably susceptible to observer bias (dependent on expertise and experience) and to selection bias (if animals with advanced disease are more or less likely to be caught).

Variation in the prevalence of M. leprae infection in armadillo populations in Brazil as a real natural phenomenon merits further investigation and requires studies very different in design from those reviewed here. Indeed, we would argue that further studies based on small (N<100) samples from selected locations are not going to further our understanding. Instead, much larger and longer-term studies conducted in partnership with national or regional animal conservation and ecology groups are needed to map M. leprae infection in armadillos across Brazil. At the same time, data must be gathered in studies focused on subpopulations of armadillos in endemic areas of Brazil to characterize M. leprae transmission and persistence within groups of animals, for example, using trackers and repeated sampling over the armadillo lifespan, as has been done in the USA to gather data which were then used to model M. leprae spread within the armadillo population [34]. Such studies could also test for other non-tuberculosis mycobacteria, including M. lepromatosis, the other causal agent of Hansen’s Disease [35].

Variability of the prevalence among armadillos could be related to different habitats. The observed zero prevalence among armadillos in the Central-West Region could be a consequence of seasonal floods in the Pantanal that. This environment favours larger populations of wild animals, including armadillos, and representative samples would require many more animals to estimate the true prevalence of M. leprae infection in such areas.

Of note is that the Nine-banded armadillo, Dasypus novemcinctus, has tended to be the focus of studies regarding M. leprae in armadillos [33], representing 69% of our pooled sample and providing 7 prevalence estimates compared with 4 for Euphractus sexcinctus. However, data from a case-control study of Hansen’s Disease risk in relation to armadillo contact [1] show that Euphractus sexcinctus was eaten almost as frequently (by 63% (94/149) of respondents) as Dasypus novemcinctus (74% (110/149)), Priodontes maximus by 12% (18/149) and Tolypeutes tricinctus by 11% (17/149)), and the recent study by da Silva Ferreira et al reported 100% M. leprae prevalence in 20 Euphractus sexcinctus [22]. A wide variety of contact with armadillos through hunting, cooking and consumption of armadillo meat was described among residents of the State of Ceará, in north-eastern Brazil [36]. The complexities of human-armadillo interaction in relation to M. leprae include the suggestion that transmission can occur in the opposite direction [29] and evidence that infection in armadillos is part of a wider environmental pool of M. leprae [37].

Strengths and limitations

The main strength of our review is that its scope was very focused, and we are confident that all relevant studies have been identified, including 5 theses or dissertations [7, 23–25, 28]. Three of these were included in our review because they provided data that had not been published [7, 23, 28]; two were rated as ‘good’ quality. We were also able to include a very recent study which had not been indexed when the databases were searched [22]. Another strength is that the methods used by the included studies to obtain the estimates of M. leprae prevalence that we used in our main meta-analysis were reasonably homogeneous, i.e. PCR targeting RLEP (M. leprae-specific repetitive element). The main limitation is that the relatively small sizes of the included studies (all but one of the eight studies contributing to the main PCR meta-analysis had ≤50 armadillos) combined with the range of prevalences (including several studies with zero positive animals) yielded a very wide predictive interval. This means that we cannot ascertain how much variability in M. leprae prevalence might be attributable to differences in methods or how much it represents real variation in M. leprae prevalence across armadillo populations in Brazil.

Conclusion

The true risk to human health of contact with M. leprae-infected armadillos has not been systematically reviewed, but evidence from Brazil and other countries indicates an association between contact with armadillos and increased risk of Hansen’s Disease [1-5]. Whilst Hansen’s Disease is officially recognized as zoonotic in the USA, with recommendations regarding contact with armadillos [38], no recommendations have been made by the Ministry of Health in Brazil or the National Programme for Control of Hansen’s Disease. The fraction of Hansen’s Disease in the human population that is attributable to contact with armadillos will depend on the magnitude of the risk, the type and frequency of contact and consumption and how common these practices are in the population, the role of other (human-to-human) transmission routes for M. leprae, and the immunological susceptibility of the individual. Our review has shown that one other possible factor, the prevalence of M. leprae in wild armadillos, cannot be predicted with any certainty based on data from studies conducted to date, although average prevalence is equivalent to 1 in 10 armadillos in Brazil being infected. The large-scale long-term studies that we suggest for future research could attempt to correlate M. leprae in wild armadillos with proximity to human habitats. In the meantime, the precautionary principle should prevail, with public health and educational efforts directed towards improving community knowledge and changing behaviour to protect human and armadillo populations.

PRISMA checklist.

(DOCX)

Click here for additional data file.

Funnel plot with pseudo 95% confidence limits of M. leprae prevalence in wild armadillos in Brazil detected using PCR methods (corresponding to Fig 4).

(TIF)

Click here for additional data file.

Detailed description of included studies investigating M. leprae infection in wild armadillos in Brazil.

(DOCX)

Click here for additional data file.

Supplementary Appendices (search terms, references after de-duplication, quality assessment (QA) tool, QA scores, extracted data).

(XLSX)

Click here for additional data file.

PRISMA flow diagram.

(TIF)

Click here for additional data file.

6 Feb 2020

Dear Professor Deps,

Thank you very much for submitting your manuscript "Prevalence of Mycobacterium leprae in armadillos in Brazil: 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.

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,

Carlos Franco-Paredes

Associate Editor

PLOS Neglected Tropical Diseases

Gerd Pluschke

Deputy Editor

PLOS Neglected Tropical Diseases

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

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: Yes, objective is clear and robust, as well as the method used. No ethical concerns to comment.

Reviewer #2: The methods are clearly articulated, the design is appropriate to address the objectives the authors proposed, the chosen criteria for the papers was clear, the sample size is a concern as acknowledged, the statistical analysis was appropriate and supports the conclusions, no concern related to ethical issues.

Reviewer #3: Prevalence of Mycobacterium leprae in armadillos in Brazil: a systematic review and meta-analysis

1 investigate natural M leprae infection in wild armadillos in endemic country to disease in human that have a control program but the new cases are maintenance and as armadillos are eaten in theses areas they can be infection to human and human to armadillos but quantify and explore the variability in prevalence of armadillos infection will be very difficult with this systemic review.

2. It is a systematic review protocol, but there are many limitations by the variability of methodology between the studies.

3. the armadillos live in endemic areas of hansen disease human and are animals susceptible to infection of M leprae, but as they no are domestic animals perhaps all sample had few size.

4 and 5. my knowledge is poor in meta-analysys, but the discussion and conclusions showed are supported by the revision.

6. the authors suggest new study only with blood and in few studies the armadillos were euthanised and probable when ethical or regulatory requirements were more light to experimental in animals.

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

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: This is a systematic review and meta-analysis proposal. Results are clearly stated. Tables are necessary and help readers to clearly understand the method and findings.

Reviewer #2: The analysis matches the plan, the results are clearly presented, tables have clarity but images are of low quality, suggest replacement.

Reviewer #3: The analysis presented match the analysis plan with one systematic review protocol and the bibliographies included non-indexed citations as theses were justified

line 113-4: M leprae was detected is important defined what is this? Same line 127: prevalence of M leprae.

Table 1 is better figure 1:

Deps 69 or 66 cases included 52 need more explain about this. line 151-3

Antunes line 202 (2 or 4 /65)

Frota 27 + 2 = 2

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

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: To some extent, yes. The conclusion clearly state the main finding in what relates the proposed objective.

However, some digresion to the main objective seems to be unnecessary and somewhat tendentious to a subject leprosy as a zoonotic disease) not strictly relate to the main objective (see below).

Reviewer #2: The conclusion and limitations are well stated and discussed, as well as the meaning of their findings. The public health relevance of the study is also addressed.

Reviewer #3: Objectives of quantify and explore the variability in prevalence of armadillos infection will be very difficult with this systemic review, because of the limitations by the differences in methodology of the studies and because the definition of infection in leprosy is yet very difficult if the same methodology is used, excepted when the bacillos are found in tissue for stain specific but few studies used this methodology.

The suggestion of recognized the armadillos infection by Brazil govern is the most important conclusion of this research and the changing behaviour to protect human and armadillos.

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

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: L. 72 - …sequelae of Hansen’s Disease are entirely avoidable if diagnosed and treated early [13]…

I would suggest to remove “entirely” – sometimes this is not true.

L. 74-77 - Whilst the proportion of new cases in Brazil attributable to zoonotic transmission is unknown, the persistence of armadillo capture and consumption as a cultural habit in some parts of Brazil means that the role of wild armadillos as a reservoir of M. leprae is relevant to efforts to eliminate the disease [16].

This paragraph has an intrinsic disruption. There is no sense to link “the persistence of armadillo capture and consumption” to the possibility of armadillos being or not a reservoir of M. lerprae and, chiefly, to link this to efforts to eliminate leprosy! This paragraph is equivocal and should be deleted.

Reviewer #2: I recommend minor revisions.

Reviewer #3: line 113-4: M leprae was detected is important defined better and line 127: prevalence of M leprae defined better.

Table 1 suggest figure 1:

Deps 69 or 66 cases included 52 need more explain better this. line 151-3

Antunes line 202 (2 or 4 /65)

Frota 27 + 2 = 2

the bacillos are found in tissue for stain specific but few studies used this methodology explain more about this limitations, or the definition of infection.

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

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: The method used has enough strength to congratulate authors for their paper. The final paragraph in ”Strengths and Limitations” is relevant to the entire study.

Indeed, difficult to ascertain how much the variability in prevalence might be attributed to differences in methods or how much it represents a real variation in the prevalence of M. leprae in the armadillos.

This reported limitation recommends some extra caution in their conclusions: leprosy as a zoonosis in USA is a somewhat "political" declaration and to me it is a wise decision of the Brazilian health authorities not to take this seriously for Brazil. It seems that authors do not agree with this cautious and correct decision - however, the results of their review paper is in itself a strong reason for not doing so!

Authors should be congratulated for intensively recommend new and lengthy studies in the attempt to correlate M. leprae in wild armadillos with proximity to human habitats. Meanwhile, considering their results & conclusions, let us not blame armadillos for playing any key role in the epidemiology of leprosy in Brazil – there are no clear evidences for that so far. In this sense, we would recommend authors to review some parts of their text (namely in Introduction and Conclusions) where a tendency to speak in favor of this thesis is strongly biased and intentionally suggested. Their main objective is to discuss prevalence of M. leprae in wild armadillos, not to claim that armadillos are responsible for the endemy of leprosy in Brazil, as they tend to suggest.

Reviewer #2: The authors properly discuss the variability in the prevalence of leprosy among the armadillos of the articles chosen. They propose laboratory tests that are appropriate and might be chosen and standardized in future studies.

The authors could discuss in some more considerable depth both the implications of their small sample size, and the great variability of prevalence among the armadillos from the different studies.

The authors fail to cite a study that mentions the possibilities of transmission between armadillos and humans. They also fail to mention/cite that the transmission may occur in the opposite direction as well.

In general, the article is very well written.

Reviewer #3: Hansen disease is very important public health in endemic countries as Brazil, that is in the first tax of detection new cases of the world and about 40 years ago have a specific and free multi-drug-therapy. As armadillos could be important to the infection to human and human to armadillos this revision can contribution to call attention of the govern and the researchers for the importance of studies in this theme neglected disease if treated early no disability and no transmission..

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

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Submitted filename: Review Deps meta-analysis_2.docx

Click here for additional data file.

10 Feb 2020

Submitted filename: Responses to Reviewers Comments.docx

Click here for additional data file.

10 Feb 2020

Dear Professor Deps,

We are pleased to inform you that your manuscript 'Prevalence of Mycobacterium leprae in armadillos in Brazil: 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 within two working days 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,

Carlos Franco-Paredes

Associate Editor

PLOS Neglected Tropical Diseases

Gerd Pluschke

Deputy Editor

PLOS Neglected Tropical Diseases

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

11 Mar 2020

Dear Professor Deps,

We are delighted to inform you that your manuscript, "Prevalence of Mycobacterium leprae in armadillos in Brazil: 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,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases

Table 1

Main characteristics and findings of included studies investigating M. leprae infection in wild armadillos in Brazil.

Author	Year	Location	Ref	Armadillo species	Total number caught	Test method(s)a, b	Positive animals (tissue)	Positive animals (blood)	Test method detailsa
Deps	2003	Espírito Santo	[23]	Dasypus novemcinctus	52	PCRc	19/36 (53%)	5/42 (12%)	single-round 65 kDa (+ Southern Blot Hybridization)
				Dasypus novemcinctus	52	BCG, HE, ZN	0/47 (BCG) 0/48 (HE) 0/50 (ZN)		Ear tissue
Deps et al	2007	Espírito Santo	[26]	Dasypus novemcinctus	52	ILF	-	11/37 (30%)	PGL-1 rapid test
Deps et al	2008	Espírito Santo	[27]	Dasypus novemcinctus	66	ELISA	-	5/47 (11%)	PGL-1 IgM
Antunes	2007	Espírito Santo	[7]	Dasypus novemcinctus	65	PCR	4/65 (6%)	-	single-round 18kDa, RLEP (+ qPCR + sequencing)
Pedrini et al	2010	São Paulo + Mato Grosso do Sul	[9]	Dasypus novemcinctus	18	PCR	0/18	0/2	single-round RLEP (+ MegaBACE 1000 sequencing)
				Euphractus sexcinctus	22	PCR	0/22	0/19	single-round RLEP (+ MegaBACE 1000 sequencing)
				Cabassous tatouay	2	PCR	0/2	0/2	single-round RLEP (+ MegaBACE 1000 sequencing)
				Cabassous unicinctus	2	PCR	0/2	0/2	single-round RLEP (+ MegaBACE 1000 sequencing)
				All species as above	44	ZN	0/44	-
Frota et al	2012	Ceará	[8]	Dasypus novemcinctus	27	PCR	5/27 (19%)	-	nested RLEPinh (+ gyrA sequencingd)
				Euphractus sexcinctus	2	PCR	1/2 (50%)	-	nested RLEPinh (+ gyrA sequencingd)
de Souza	2016	Mato Grosso do Sul	[28]	Priodontes maximus	16	PCR	0/16	-	single-round RLEP (+ qPCR + mPCR + VNTR)
				Euphractus sexcinctus	17 + 6e	PCR	0/23	-	single-round RLEP (+ qPCR + mPCR + VNTR)
				Dasypus novemcinctus	2 + 1e	PCR	0/3	-	single-round RLEP (+ qPCR + mPCR + VNTR)
				Cabassous unicinctus	8	PCR	0/8	-	single-round RLEP (+ qPCR + mPCR + VNTR)
da Silva et al	2018	Pará	[3]	Dasypus novemcinctus	16	PCRf	10/16 (63%)	-	single-round RLEP (+ WGS)
Stefani et al	2019	Amazonas	[10]	Dasypus novemcinctus	12	PCR	0/12	-	single-round RLEPinh
				Dasypus novemcinctus	12	HE, FFg	0/12	-
da Silva Ferreira et al	2020	Rio Grande do Norte	[22]	Euphractus sexcinctus	20	PCR	20/20	-	nested RLEPinh (+ RFLP)
				Euphractus sexcinctus	20	ELISA, ILFh	-	20/20	PGL-1 IgM, LID-1 IgG

a BCG = Bacillus Calmette–Guérin (antigen immunohistochemistry); ELISA = enzyme-linked immunosorbent assay; FF = Fite Faraco; HE = haematoxylin and eosin stain; ILF = immunochromatographic lateral flow test; PCR = Polymerase Chain Reaction (mPCR = multiplex PCR; qPCR = Real Time PCR); PGL-1 = phenolic glycolipid 1; RFLP = restriction fragment length polymorphism analysis; RLEP = M. leprae-specific repetitive element; VNTR = variable number tandem repeat (genotyping); WGS = whole genome sequencing; ZN = Ziehl-Neelsen (bacilloscopy)

b Four studies investigated clinical signs of leprosy, two with positive findings (Deps [23] and Antunes [7]), one negative (de Souza [28]) and one inconclusive (Stefani et al [10])

c Deps reported ML Flow rapid immunochromatographic serology (PGL-1) results in Deps et al 2007 [26] and ELISA (PGL-1) results in Deps et al 2008 [27]

d the analysed samples belonged to the gyrAT (SNP type 3) population, which was also identified in wild armadillos in the USA [29] and in humans in Brazil [30]

e roadkill animals

f da Silva et al used SYBR Gold and auramine/rhodamine staining techniques (staining of mycobacteria in situ), detection of PGL-1 antigen using polyclonal rabbit antibody and acid-fast staining of bacilli using HE and FF techniques in spleen sections from PCR-positive wild armadillos, but total numbers of samples tested using these techniques and overall concordance with PCR results was not reported [3]

g Following complete dermato-neurological examination by a dermatologist, skin lesions suspect of leprosy were biopsied. Skin sections were further prepared for histopathological examination after HE and FF staining for bacilli identification. 48 skin sections on 96 slides were tested, all were negative, but one armadillo showed skin histopathology compatible with paucibacillary leprosy, another showed granulomas with epithelioid and Langerhans cells [10]

h ELISA IgM against PGL-1 and IgG against LID-1 antigens; NDO-LID rapid ILF test (Orange Life, Rio de Janeiro, Brazil); ML Flow ILF test (acquired from Dr. Samira Bührer-Sékula, Royal Tropical Institute, KIT Biomedical Research, Amsterdam, the Netherlands)

inh inhibitory substances tested for in negative DNA samples