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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 51-59

Molecular detection of mycobacterium tuberculosis complex species in triatomines found in urban households and Peridomiciles in a city with high prevalence of tuberculosis in Northeastern Brazil


1 Department of Pathology and Legal Medicine, Federal University of Ceará, Fortaleza, Brazil
2 Bacteriology and Mycology Section, Evandro Chagas Institute, Ananindeua; Postgraduate Program in Parasitic Biology in the Amazon, State University of Pará, Belém, Brazil
3 Postgraduate Program in Public Health, Fortaleza University, Fortaleza, Brazil
4 Bacteriology and Mycology Section, Evandro Chagas Institute, Ananindeua, Brazil
5 Medicine School, Federal University of Ceará Sobral, Brazil
6 Sobral Zoonoses Surveillance Unit, Sobral Health Secretary, Sobral, Brazil

Date of Submission14-Nov-2021
Date of Decision25-Nov-2021
Date of Acceptance19-Jan-2022
Date of Web Publication12-Mar-2022

Correspondence Address:
Cristiane Cunha Frota
Federal University of Ceará, Medicine School, Department of Pathology and Legal Medicine, Rua Monsenhor Furtado S/N, 60441-750 Fortaleza, CE
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_230_21

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  Abstract 


Background: Triatomine insects, native to northeastern Brazil, have been found in the urban peridomicile. The city of Sobral has a high number of tuberculosis cases and several triatomine species. This study investigates the presence of mycobacteria, particularly Mycobacterium tuberculosis complex (MTBC) species, in triatomines captured in the urban perimeter of Sobral. Methods: We analyzed 167 triatomines captured in urban households and peridomiciles of Sobral. Mycobacteria were identified by the PRA-hsp65 method followed by partial sequencing of the hsp65 and rpoB genes. The sequences confirmed as MTBC were also typed by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and spoligotyping. Results: Triatoma brasiliensis (38.6%), Triatoma pseudomaculata (32.9%), Panstrongylus lutzi (24.3%) were the most frequently identified. In 51.1% (70/167) of them, species of the Mycobacteriaceae family were detected by PRA-hsp65; of these, 31.4% (22/70) were identified as belonging to MTBC species. Nine (12.9%) of the triatomine samples were confirmed by sequencing as belonging to MTBC species. MIRU-VNTR genotyping suggests that the presence of different MTBC sublines in the triatomines should be investigated. Conclusion: This is the first report of MTBC lineages in triatomine insects. These results indicate the migration and adaptation of these insects in an urban setting.

Keywords: Hemiptera, mycobacteria, Mycobacterium tuberculosis, sequencing analysis, Triatominae, tuberculosis


How to cite this article:
Silva CS, Gondim Costa Lima LN, Batista Lima KV, Freitas de Almeida RL, da Conceição ML, Parente CC, Rocha AA, da Silva TC, Sancho Sd, Frota CC. Molecular detection of mycobacterium tuberculosis complex species in triatomines found in urban households and Peridomiciles in a city with high prevalence of tuberculosis in Northeastern Brazil. Int J Mycobacteriol 2022;11:51-9

How to cite this URL:
Silva CS, Gondim Costa Lima LN, Batista Lima KV, Freitas de Almeida RL, da Conceição ML, Parente CC, Rocha AA, da Silva TC, Sancho Sd, Frota CC. Molecular detection of mycobacterium tuberculosis complex species in triatomines found in urban households and Peridomiciles in a city with high prevalence of tuberculosis in Northeastern Brazil. Int J Mycobacteriol [serial online] 2022 [cited 2022 May 25];11:51-9. Available from: https://www.ijmyco.org/text.asp?2022/11/1/51/339508




  Introduction Top


The Mycobacteriaceae family has several bacteria of clinical interest, the most endemic species being Mycobacterium tuberculosis (TB), the agent of TB.[1] In 2019, the World Health Organization estimated an incidence of 10 million new cases worldwide.

Brazil is among the 30 countries responsible for 90% of the TB cases diagnosed worldwide. In 2019, 73,864 new cases were diagnosed, with an incidence of 35.0 cases/100,000 inhabitants. In the same year, the state of Ceará located in northeastern Brazil showed incidence higher than the national average, with 42.9 cases/100,000 inhabitants.[2] In 2017, the Ministry of Health of Brazil established the goal of reducing incidence to <10 cases/100,000 inhabitants.[3] In addition to TB, diseases caused by nontuberculous mycobacteria (NTM) are considered emerging in Brazil; however, notification is not mandatory, which hinders data estimation.[4],[5],[6]

The presence and growth of mycobacterial species, including pathogenic ones, in invertebrates has been demonstrated by several authors, including Mycobacterium marinum in Drosophila melanogaster,[7] Mycobacterium ulcerans in mosquitoes of the genus Aedes and in aquatic insects of the Naucoridae family,[8] M. tuberculosis in Galleria mellonella,[9] and several species of NTM in cockroaches,[10],[11] flies, and ticks.[12],[13] Studies have demonstrated the presence of mycobacteria in the microbiome of triatomine species.[14],[15] In addition, the leprosy bacillus has been shown to multiply and be excreted in its viable form in the feces of these insects.[16]

In Ceará, the city of Sobral has reported a high number of TB cases,[17] with the incidence of cases within the municipality varying from a few cases to 949 cases/100,000 inhabitants.[2] In this scenario with numerous TB cases, Sobral also has a high number of leprosy cases,[18] and Mycobacterium leprae bacillus mRNA has even been detected in water collections near the residences of people with leprosy.[19] The municipality is also characterized by the presence of several species of triatomines throughout the urban perimeter.[20]

Triatomines have the ability to transmit diseases among humans through their bites via saliva or contaminated feces, being considered vectors of human bacterial pathogens.[21] Despite several reports of NTM isolation in environmental samples, few studies have investigated the presence of pathogenic mycobacteria in potential vectors cohabiting the peridomicile in TB endemic regions. In this sense, this study investigated the presence of mycobacteria, particularly M. tuberculosis species, in triatomines collected within the urban perimeter of Sobral. The results are important in drawing the attention of health professionals to the fact that agents in the environment, particularly hemipteran insects, can act as carriers of TB transmission in endemic regions.


  Methods Top


The study area

Sobral is located 410 km from the equator (3°40'58” S; 40°21'4” W) and is one of the largest cities in the state of Ceará. It has a tropical hot semi-arid (dry) climate and is considered an important commercial, industrial, and educational center of northern Ceará. The municipality spans 2068 km2 with an estimated population of 210,711 habitants in 2020.[22] The human development index is 0.714, with about 10% of the population living in extreme poverty.[23] The city is a center of migration for people from rural localities living in poverty within the urban district of the city. In its geographical limits, two mountains with dense vegetation, Meruoca and Massapê, are located 18 km away.

Collection and identification of triatomines

Between January 2019 and April 2020, a total of 167 triatomines were collected, including adult insects and nymphs captured in urban households and peridomiciles of Sobral. The insects were captured in two ways: specimens were captured and sent by the population of Sobral to the Triatomine Information Posts (acronym in Portuguese) installed in the health posts of the municipality or were collected by zoonosis researchers during visits to the residences in the districts of Sobral [Figure 1]. The geographical location of each residence where the insects were found was recorded.
Figure 1: Landscapes of Sobral, Northeast Brazil. (a) Clay house with cracks vulnerable to triatomines. (b) Yard surrounded by rocks and plants. (c) Food and waste accumulated inside the house. (d) Chickens and rocks in the peridomicile, areas potentially harbouring triatomines

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Insects collected alive were stored individually in sterile flasks and then sent to the Zoonosis Control Centre of Sobral. Before dissection, the insects were washed with 0.5% sodium hypochlorite solution and rinsed with sterile distilled water. Then, the insects were taxonomically characterized by an entomologist by dissection with the aid of a stereoscopic microscope (magnification × 12).[24] Then, they were kept at −20°C and sent to the laboratory in the capital of Ceará.

DNA extraction

The insects were thawed to room temperature and then placed separately in sterile  Petri dish More Detailses. With the aid of a sterile scalpel, a sample was collected from the intestine of each insect (from the pro ventricle to the rectum). DNA was extracted using the phenol-chloroform technique as previously published.[25]

Identification of mycobacteria by polymerase chain reaction

Mycobacteria amplification was performed using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the hsp65 gene (PRA-hsp65) as described by Telenti et al.[26] The algorithm used in the identification of mycobacteria is available on the PRASITE website.[27]

Sequencing

All samples identified as mycobacteria were submitted to partial sequencing of the hsp65 gene; those that did not amplify were sequenced with the rpoB gene. For the amplification of the rpoB target, the primers Myco-F (5'-GGCAAGGTCACCCCGAAGGG-3') and Myco-R (5'-AGCGGCTGCTGGGTGATCATCATC-3') were used,[28] and for the hsp65 region, the primers Tb11 (5'-ACCAACGATGGTGTGTCCAT-3') and Tb12 (5'-CTTGTCGAACCGCATACCCT-3').[26]

PCR products were purified using the EasyPure® PCR Purification Kit (TransGen Biotech Co, Beijing, China) and then sequenced in the Applied Biosystems DNA Sequencer equipment (Applied Biosystems™, Carlsbad, CA, USA) using the BigDyeTM Terminator Cycle Sequencing Kit. Sequences were analyzed using SecScape software v. 2.7 (Applied Biosystems™, Carlsbad, CA, USA) and BioEdit Sequence Alignment Editor version 7.2.5 (BioEdit, Carlsbad, CA, USA). The sequence of the M. tuberculosis H37Rv strain was used for sequence alignment and analysis. The results were noted in Microsoft Excel 2019, and then descriptive analysis was performed.

Mycobacterium tuberculosis complex genotyping

Genotyping by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) included the 24 loci proposed by Supply et al.[29] and used the duplex PCR protocol proposed by Yasmin et al.,[30] with 0.66 or 1 μM of each primer, and other conditions were adapted to those recommended by the manufacturer of the Platinum™ Multiplex PCR Master Mix enzyme (Applied Biosystems™, Carlsbad, CA, USA). Electrophoresis of the PCR product was performed on a 2% agarose gel.

The MIRU-VNTR profiles were compared with the profiles of Brazilian isolates analysed by Conceição et al.[31] Brazilian clinical isolates with the same VNTR values at all loci as those obtained from triatomines were selected. This set was compared with the online database MIRU-VNTRplus[32] to evaluate the repeat units present and to obtain the phylogenetic tree.

Spoligotyping was performed by reverse hybridization as previously described[33] using nylon membrane.[34] The patterns obtained were interpreted as binary codes, analyzed in the online database,[35] and classified according to spoligotype international types (SIT).

Ethics

In this study only invertebrate animals were included and ethical approval was not required.


  Results Top


Of all triatomines collected, species of the Mycobacteriaceae family were detected by PRA-hsp65 in 51.1% (70/167) of them [Table 1]. The most frequent mycobacterial species were: Mycobacterium tuberculosis complex (MTBC) in 22 (31.4%) triatomines, followed by Mycolicibacterium fortuitum/Mycolicibacterium senegalense type 1 in 12 (17.1%), species with the digestion pattern of Mycolicibacterium smegmatis/Mycolicibacterium wolinski type 1/Mycolicibacterium mageritense in 19 (12.9%), M. senegalense in 8 (11.4%), and Mycobacterium gordonae in 5 (7.1%).
Table 1: Species of the Mycobacteriaceae family identified by the polymorphism analysis-hsp65 method in triatomines collected in Sobral, Brazil

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Among the PRA-hsp65-positive triatomines, Triatoma brasiliensis, Triatoma pseudomaculata, and Panstrongylus lutzi were the most common species at 38.6%, 32.9%, and 24.3%, respectively [Table 2]. The other species with lower frequencies were Rhodnius nasutus (2.8%; 2/70) and Panstrongylus megistus (1.4%; 1/70). Adult insects represented 77.1% of the captured insects, indicating colonization of the collection sites and neighbourhood. Triatomines were found in adult stages (males and females) or only in nymphs among the species T. brasiliensis (88.9% and 11.1%, respectively) and T. pseudomaculata (43.5% and 56.5%, respectively).
Table 2: Description of triatomine species that were positive for mycobacteria

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Sequences were obtained in 49 of the 70 PRA-hsp65-positive triatomine samples (70%), 19 samples being for the hsp65 gene and 30 for the rpoB gene. There were no samples sequenced for both genes.

Species belonging to MTBC were detected in nine (18.3%) of the triatomine samples: eight with M. tuberculosis and one with Mycobacterium africanum. Species of the genus Brevibacterium were detected in 24.5% (12/49), followed by Cutibacterium acnes in 12.2% (6/49). The other genera detected were Amycolatopsis, Gordonia, Microbacterium, Corynebacterium, Streptomyces, Dietzia, Brachybacterium, Tsukamurella, Nocardiopsis, and Kocuria [Table 3].
Table 3: Species identified by polymorphism analysis-hsp65 and after analysis of the genetic sequences of triatomine samples in the nucleotide database GenBank (National Center for Biotechnology Information)

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The MTBC-positive triatomine samples showed identity ranging from 93.9 to 99.4%. The nine samples identified as MTBC were detected in four P. lutzi (adults), two T. pseudomaculata (one adult and one nymph), two T. braziliensis (one adult and one nymph), and one R. nasutus (adults) sample. These sequences were identified by PRA-hsp65 as MTBC in seven samples of triatomines, while the other two were identified as Mycolicibacterium agri type 1 (sample number 130) and M. senegalense (sample number 143).

The spoligotyping profile was inconclusive for M. tuberculosis samples. Complete MIRU-VNTR profiles were obtained for five of the six samples genotyped at 24 loci. All profiles were different from each other and were unique compared with those in the MIRU-VNTRplus database and to clinical isolates investigated in Brazil. Only one sample presented a single copy number for all the VNTR investigated. Two samples showed copy number variation only at locus 2163b, one sample had variations at six loci, and another showed variation at nine of the 24 loci [Table 4]. All VNTR copy numbers of the triatomine samples were compared with those of the 1613 Brazilian clinical isolates obtained in a previous study,[31] resulting in one isolate from the state of Pará and one isolate from the state of Rio de Janeiro being closer to those of this study. There were no samples from Ceará in the database. In addition to the copy variability in the VNTRs in each of the samples and among them, the phylogenetic tree based on VNTRs generated through the MIRU-VNTRplus tool revealed clustering of one of the samples with the Delhi/CAS lineage, one clustering with the LAM lineage, and three close to the S lineage, together with a clinical isolate from Pará and another from Rio de Janeiro selected among the 1613 Brazilian isolates [Figure 2] and [Figure S1].
Table 4: Mycobacterial interspaced repetitive units-variable number of tandem repeats fingerprinting results for five Mycobacterium tuberculosis complex triatomine isolates

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Figure 2: Phylogenetic tree of triatomine samples against the MIRU-VNTRplus database. Each box per tree branch contains the strain followed by the sample identification. Yellow-highlighted boxes refer to the MIRU-VNTR profiles of triatomine samples (IEC_number) and Brazilian clinical isolates from Pará state and Rio de Janeiro state.[46] Columns indicate the family referring to the SIT and MIRU profiles 24 loci. (a) Clustering of one sample to the branch from the Delhi/CAS family and one to the LAM family. (b) Clustering of three samples and two Brazilian strains to the branch of family S. The complete tree obtained using the MIRU-VNTRplus tool is presented in the supplementary material. MIRU-VNTR: mycobacterial interspaced repetitive units-variable number of tandem repeats

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  Discussion Top


This is the first report on the detection of MTBC species in triatomine insects, which were collected in the urban peridomicile of Sobral, a densely inhabited region in northeastern Brazil. These results indicate the adaptation of these insects to the urban setting.[20],[21]

The city of Sobral displays favorable eco-epidemiological characteristics for the colonization of these insects. It presents favorable environmental conditions with shrub vegetation typical of the Caatinga. The households and peridomiciles in Sobral where these insects were captured usually house several domestic animals, such as chickens, ducks, cats, and dogs. Residents usually accumulate food grains and seeds (bean pods and corn kernels), tiles, bricks, sand, and straw. These environmental characteristics are favorable for the multiplication and colonization of these insects near humans.

Previous experimental and epidemiological studies have shown the presence and viability of M. tuberculosis in natural environments, such as soil, water, and in insects, where bacilli can be isolated from soil for up to 4 weeks after contamination.[15],[36],[37],[38] We cannot infer how triatomines acquired the bacilli, whether through soil contamination or after biting a person with TB. Due to the low number of M. tuberculosis-positive triatomines, we cannot relate our findings to the locations of patients diagnosed with TB. Sobral has high endemicity of this disease, with a treatment abandonment rate of about 11%,[17] much higher than that recommended by the WHO.[39] However, this study highlights the importance of investigating the role of these insects in TB transmission using molecular methods in regions where TB is highly prevalent.

Fifteen bacterial species were identified by sequencing in the samples analyzed, all belonging to the phylum Actinobacteria. The sequencing analyses identified predominant bacteria from the insect microbiota. The genus Brevibacterium was identified in 24.5% of the sequences. Other genera also appeared at a lower frequency, such as Gordonia, Dietzia, Corynebacterium, Nocardia, and Kocuria. These genera are recognized as participating in the microbiota of triatomines, where they are found at all stages of triatomine development and may play an important role in the survival and reproduction of these insects.[40],[41] During hematophagy, these insects acquire microorganisms that then colonise the gastrointestinal tract. Previous studies have shown that the species T. brasiliensis and T. pseudomaculata have a predominance of Proteobacteria and Actinobacteria among their microbiota.[14],[41],[42]

Our results showed that 51.1% of the samples tested were positive for the hsp65 gene, and of those, only seven samples (10%; 7/70) had concordant results between PRA-hsp65 and sequencing, which were identified as MTBC. These data show that PRA-hsp65 results should be interpreted with caution. Studies have indicated that the detection of this gene in non-cultured samples leads to the identification and differentiation of mycobacteria, and its association with other methods of molecular diagnosis is necessary to assist in the epidemiological surveillance of mycobacteria, especially NTM.[6] A study by Tortone et al.[44] found that only 30% of the PRA-hsp65 NTM samples showed results concordant with sequencing.

PRA-hsp65 is characterized as an effective support technique for the identification of mycobacteria. However, like sequencing, the sensitivity and specificity of the results are affected by the quality of the samples analyzed.[44] Studies agree that PRA is not adequate for the identification of new or rarely observed species, requiring other techniques for definitive identification. Nevertheless, it is still an accessible technique for the identification of NTM due to its cost effectiveness and ease of execution in laboratories.[43],[44],[45]

The genotyping of MTBC species by spoligotyping would allow the determination of families and lineages to better understand the origin of the strains circulating in triatomines. The inconclusive profiles may be related to the presence of more than one mycobacterial species in the sample, which could be resolved by culturing and later isolating both species; however, this was not possible. Since hybridization and detection of variable regions are involved, the results overlap in the presence of more than one strain. In this case, MIRU-VNTR was more enlightening.

MIRU-VNTR genotyping demonstrated great genetic diversity in the samples analyzed, with copy variation at various loci, including variations within the same sample. The fact that one of the samples presented variations in six loci and another presented variation in nine of the 24 loci suggests the presence of more than one strain, requiring cultivation and isolation for confirmation in future investigations.

When compared with Brazilian strains, great genotypic proximity of triatomine samples containing VNTR values similar to those of one strain from the Brazilian state of Pará and another from the state of Rio de Janeiro was observed, as reported in previous genotyping studies of human isolates.[31],[46] Phylogenetically, in database analysis, the MIRU-VNTR profile of 24 loci of an investigated triatomine sample clustered together the CAS-Delhi (Central Asian-Delhi) family (strain 3) predominantly found in Afghanistan, Pakistan, North India, Nepal, Middle East, Libya, Sudan, Ethiopia, Kenya, and Tanzania. The CAS strains are related to the Beijing family strains and are probable precursor strains of the latter, as they were observed geographically close to the region where the Beijing strain originated.[47] One sample was clustered in the branch of the LAM family (strain 4), which is one of the most prevalent families of M. tuberculosis worldwide and is most commonly found in South America,[48] and the remaining three samples formed clusters in the S family (strain 4), along with clinical isolates from Pará and Rio de Janeiro. Therefore, the identified profiles predominantly belong to strain 4, which is the most widely dispersed, affecting humans worldwide.[49] The presence of profiles similar to those found predominantly causing disease indicates the need for surveillance of strains present in the municipality to determine the possibility of transmission of MTBC strains from the environment or vectors to human or animal populations in Sobral.

In this study, the species T. brasiliensis (38.6%) and T. pseudomaculata (32.9%) predominated among the captured triatomine species. The high prevalence of these two species is explained by the fact that both are the most commonly found species in northeastern Brazil, a region with high triatomine diversity. Besides their natural microhabitat, these insects are also commonly found inside households and are associated with habitats of peridomestic animals such as dogs and chickens.[20],[50] The colonisation of these triatomines (adults and nymphs) near and/or living with humans can influence their microbiota, causing them to acquire a different microbiota from those in the wild habitat.[40] In Sobral, there is a predominance of hyperxerophilic Caatinga with shrubs and intense deforestation, and palm trees (Acrocomia aculeata and Copernicia prunifera) are also found in the vegetation. The intense deforestation and anthropogenic invasion of the natural habitat of triatomines has led to urbanization of these insects, which make new nests in peridomestic and urban households.[20],[51]

The control of these insects, being native to the northeastern region of Brazil, has been challenging. They are able to disperse by flying from their natural habitat towards artificial lights, which favors the invasion and colonization of houses. Other authors have reported the capture of T. pseudomaculata species inside houses and feeding on human blood, indicating the potential of these species in the intradomiciliary colonization and transmission of diseases.[50],[52] Furthermore, bacilli of M. tuberculosis or other microorganisms can be transmitted to colonized nymphs by vertical transmission or horizontal transmission through coprophagy, cannibalism, and environmental contact.[40]


  Conclusions Top


This study is the first to describe the identification of MTBC species in triatomines in the urban peridomicile. Understanding TB transmission and its relationship with the habitat can help establish complementary measures to control the disease, not just patient-centered ones. The detection of MTBC in environmental samples suggests that the transmission routes may be more extensive and varied, or that it is an adapted mycobacterial species. In this context, the possibility of insects acting as vectors of these pathogens would form new chains of transmission, where further investigations would be needed to understand transmission by these vectors.[21],[38] Moreover, we emphasize the need for further studies investigating the role of the environment and hematophagous insects as vectors of diseases as well as the relationship between the dispersion of these vectors and the process of uncontrolled urbanization in northeastern Brazil.

Limitation of study

As for the limitations of the present study, we highlight the impossibility of cultivation in culture media for mycobacteria from the collected triatomines. Cultivation of triatomines was conducted, but without success. The handling of insects done by the technicians from the Zoonoses Center for taxonomic characterization and for the detection of T. cruzi in insects may have interfered with the necessary viability for cultivation. Another limitation presented was the failure to obtain data sequences by sequencing all 70 samples tested. About 30% (21/70) of the samples were not sequenced, for one of the following reasons: non-amplification of rpoB or hsp65 products, poor-quality nucleotide sequence, and insufficient amount of DNA.

Acknowledgments

We would like to thank the Zoonosis Control Centre of the Sobral Health Secretary for providing access to the data used in this study, CE, Brazil.

Financial support and sponsorship

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP)/CAPES Grant Number 88887.165958/2018-00.

Conflicts of interest

There are no conflicts of interest.



 
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