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Giardiasis in children and dogs, and the first report of assemblage E in dogs from northeastern Brazil

Giardíase em crianças e cães, e o primeiro relato de infecção por assemblage E em cães do nordeste brasileiro

Abstract

Diagnosis is crucial for controlling giardiasis. We determined the prevalence and genetically characterize isolates of Giardia duodenalis of children and dogs from rural communities in northeastern Brazil. G. duodenalis cysts were concentrated by centrifugal flotation/sedimentation. Molecular characterization was carried out using the loci ssu-rRNA, bg, tpi, and gdh. By parasitological techniques, Giardia spp. infection was detected in 72/192 children (37.5%; 95% CI: 30.6%-44.7%) and 24/139 dogs (17.3%; 95% CI: 11.4%-24.6%). By molecular analysis, infection was detected in 60/141 children (42.5%; 95% CI: 34.3%-51.2%) and 26/92 dogs (28.3%; 95% CI: 19.4%-38.6%). The total prevalence of giardiasis was 54.9% in children (106/193; 95% CI: 47.1%-61.6%) and 32.9% in dogs (47/143; 95% CI: 25.2%-41.2%). Zoonotic assemblages A and B of G. duodenalis were detected in children, and assemblage E of G. duodenalis was detected in one child and two dogs. Parallel use of parasitological and molecular techniques proved to be a more effective strategy for detecting giardiasis in children and dogs from endemic areas.

Keywords:
Diarrhea; dog population; parasitological diagnosis; protozoa; vulnerable communities; zoonoses

Resumo

O diagnóstico é crucial para o controle da giardíase. Foram determinadas as prevalências e as características genéticas isoladas de Giardia duodenalis, em crianças e cães de comunidades rurais do nordeste brasileiro. Os cistos de G. duodenalis foram concentrados por centrífugo-flutuação/sedimentação. A caracterização molecular foi realizada utilizando-se os loci ssu-rRNA, bg, tpi e gdh. Pelas técnicas parasitológicas, a infecção por Giardia spp. foi detectada em 72/192 crianças (37,5%; IC 95%: 30,6%-44,7%) e 24/139 cães (17,3%; IC 95%: 11,4%-24,6%). Molecularmente, a infecção foi detectada em 60/141 crianças (42,5%; IC 95%: 34,3%-51,2%) e 26/92 cães (28,3%; IC 95%: 19,4%-38,6%). A prevalência total de giardíase foi de 54,9% em crianças (106/193; IC 95%: 47,1%-61,6%) e 32,9% em cães (47/143; IC 95%: 25,2%-41,2%). Os assemblages zoonóticos A e B de G. duodenalis foram detectados em crianças, e o assemblage E de G. duodenalis foi detectado nas duas populações. O uso paralelo de técnicas parasitológicas e moleculares mostra-se uma estratégia mais eficaz para detecção de giardíase em crianças e cães de áreas endêmicas.

Palavras-chave:
Diarreia; população canina; diagnóstico parasitológico; protozoários; comunidades vulneráveis; zoonoses

Introduction

Giardia duodenalis is a zoonotic protozoa that causes diarrheal diseases in humans and animals (Buret et al., 2020Buret AG, Cacciò SM, Favennec L, Svärd S. Update on Giardia: highlights from the seventh International Giardia and Cryptosporidium Conference. Parasite 2020; 27: 49. http://dx.doi.org/10.1051/parasite/2020047. PMid:32788035.
http://dx.doi.org/10.1051/parasite/20200...
). Globally, more than one billion cases of diarrhea in children aged up to 5 years have been reported, among which more than five hundred thousand deaths have occurred annually (WHO, 2017World Health Organization - WHO. Diarrhoeal disease [online]. 2017 [cited 2022 Aug 20]. Available from: https://www.who.int/news-room/fact-sheets/detail/diarrhoeal-disease
https://www.who.int/news-room/fact-sheet...
). Acute diarrheal diseases are important causes of morbidity and mortality among Brazilians and are mainly associated with economically vulnerable populations (Brasil, 2018Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância de Doenças e Agravos não Transmissíveis e Promoção da Saúde. Saúde Brasil 2017: uma análise da situação de saúde e os desafios para o alcance dos objetivos de desenvolvimento sustentável [online]. 2018 [cited 2022 Aug 20]. Available from: http://bvsms.saude.gov.br/bvs/publicacoes/saude_brasil_2017_analise_situacao_saude_desafios_objetivos_desenvolvimento_sustetantavel.pdf
http://bvsms.saude.gov.br/bvs/publicacoe...
; Bahia, 2019Bahia. Secretaria da Saúde. Boletim epidemiológico perfil das doenças diarreicas agudas Bahia, 2019 [online]. 2019 [cited 2022 Aug 20]. Available from: http://www.saude.ba.gov.br/wp-content/uploads/2017/11/boletimDDA_n%C2%BA-01_2019.pdf
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), with more than four million human cases registered annually (Bahia, 2019Bahia. Secretaria da Saúde. Boletim epidemiológico perfil das doenças diarreicas agudas Bahia, 2019 [online]. 2019 [cited 2022 Aug 20]. Available from: http://www.saude.ba.gov.br/wp-content/uploads/2017/11/boletimDDA_n%C2%BA-01_2019.pdf
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). In dogs, these infections are more severe in puppies and can lead to death (Thompson et al., 2008Thompson RCA, Palmer CS, O’Handley R. The public health and clinical significance of Giardia and Cryptosporidium in domestic animals. Vet J 2008; 177(1): 18-25. http://dx.doi.org/10.1016/j.tvjl.2007.09.022. PMid:18032076.
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; Feng & Xiao, 2011Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011; 24(1): 110-140. http://dx.doi.org/10.1128/CMR.00033-10. PMid:21233509.
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).

Selection of diagnostic methods is essential for determining the correct prevalence that guides the planning of more effective surveillance and control actions for diarrheal diseases such as these infections (Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
; Bahia, 2019Bahia. Secretaria da Saúde. Boletim epidemiológico perfil das doenças diarreicas agudas Bahia, 2019 [online]. 2019 [cited 2022 Aug 20]. Available from: http://www.saude.ba.gov.br/wp-content/uploads/2017/11/boletimDDA_n%C2%BA-01_2019.pdf
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). Among the diagnostic factors that can lead to underestimation of prevalence, as well as weaken the generation of reliable epidemiological data on these infections are the lack of diagnostic standardization in research of protozoa in human and animal populations, type and quality of the samples, the profile of the population studied, technical capacity of the microscopist, and the use of an isolated technique (Dantas-Torres & Otranto, 2014Dantas-Torres F, Otranto D. Dogs, cats, parasites, and humans in Brazil: opening the black box. Parasit Vectors 2014; 7(1): 22. http://dx.doi.org/10.1186/1756-3305-7-22. PMid:24423244.
http://dx.doi.org/10.1186/1756-3305-7-22...
; Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
).

In Brazil, giardiasis is diagnosed through parasitological tests, which can underestimate prevalence due to variation in the sensitivity of techniques, as well as variation in the protocols. Molecular diagnosis is still scarce in the country and is restricted to academic research due to its high cost (Katagiri & Oliveira-Sequeira, 2007Katagiri S, Oliveira-Sequeira TCG. Zoonoses causadas por parasitas intestinais de cães e o problema do diagnóstico. Arq Inst Biol (Sao Paulo) 2007; 74(2): 175-184. http://dx.doi.org/10.1590/1808-1657v74p1752007.
http://dx.doi.org/10.1590/1808-1657v74p1...
; Dantas-Torres & Otranto, 2014Dantas-Torres F, Otranto D. Dogs, cats, parasites, and humans in Brazil: opening the black box. Parasit Vectors 2014; 7(1): 22. http://dx.doi.org/10.1186/1756-3305-7-22. PMid:24423244.
http://dx.doi.org/10.1186/1756-3305-7-22...
; Bahia, 2019Bahia. Secretaria da Saúde. Boletim epidemiológico perfil das doenças diarreicas agudas Bahia, 2019 [online]. 2019 [cited 2022 Aug 20]. Available from: http://www.saude.ba.gov.br/wp-content/uploads/2017/11/boletimDDA_n%C2%BA-01_2019.pdf
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; Hooshyar et al., 2019Hooshyar H, Rostamkhani P, Arbabi M, Delavari M. Giardia lamblia infection: review of current diagnostic strategies. Gastroenterol Hepatol Bed Bench 2019; 12(1): 3-12. PMid:30949313.).

The prevalence of giardiasis in Brazilian children ranges from 0.9% (Santos et al., 2014Santos CB, Araújo KCGM, Jardim-Botelho A, Santos MB, Rodrigues A, Dolabella SS, et al. Diarrhea incidence and intestinal infections among rotavirus vaccinated infants from a poor area in Brazil: a spatial analysis. BMC Public Health 2014; 14(1): 399. http://dx.doi.org/10.1186/1471-2458-14-399. PMid:24761937.
http://dx.doi.org/10.1186/1471-2458-14-3...
) to 31.2% (Mariano et al., 2015Mariano APM, Santos EN, Santos TN, Mota TN, Silva JA, Carvalho SMS, et al. Parasites in South Bahia: focus on giardiasis and ascariasis among preschoolers of Itabuna. Int J Health Sci (Qassim) 2015; 3(1): 61-75. http://dx.doi.org/10.15640/ijhs.v3n1a4.
http://dx.doi.org/10.15640/ijhs.v3n1a4...
); in dogs, it ranges from 1% to 41% (Mundim et al., 2003Mundim MJS, Souza SZ, Hortêncio SM, Cury MC. Freqüência de Giardia spp. por duas técnicas de diagnóstico em fezes de cães. Arq Bras Med Vet Zootec 2003; 55(6): 770-773. http://dx.doi.org/10.1590/S0102-09352003000600016.
http://dx.doi.org/10.1590/S0102-09352003...
; Campos et al., 2008Campos PC Fo, Barros LM, Campos JO, Braga VB, Carzorla IM, Albuquerque GR, et al. Parasitas zoonóticos em fezes de cães em praças públicas do município de Itabuna, Bahia, Brasil. Rev Bras Parasitol Vet 2008; 17(4): 206-209. http://dx.doi.org/10.1590/S1984-29612008000400007.
http://dx.doi.org/10.1590/S1984-29612008...
). G. duodenalis is composed of a complex of multiple species, which includes zoonotic assemblages (A, B), and species-specific assemblages (C, D, E, F, G, and H), being C and D specific to domestic and wild canines (Feng & Xiao, 2011Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011; 24(1): 110-140. http://dx.doi.org/10.1128/CMR.00033-10. PMid:21233509.
http://dx.doi.org/10.1128/CMR.00033-10...
). Assemblages A, B, and E circulate among Brazilian children, with a higher occurrence of assemblage A. The majority of infected children are reported from daycare centers in urban and peripheral areas (Kohli et al., 2008Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 2008; 102(7): 718-725. http://dx.doi.org/10.1016/j.trstmh.2008.03.002. PMid:18485429.
http://dx.doi.org/10.1016/j.trstmh.2008....
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Quadros et al., 2016Quadros RM, Weiss PHE, Marques SMT, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo 2016; 58(0): 66. http://dx.doi.org/10.1590/S1678-9946201658066. PMid:27680171.
http://dx.doi.org/10.1590/S1678-99462016...
; Scalia et al., 2016Scalia LAM, Fava NMN, Soares RM, Limongi JE, Cunha MJR, Pena IF, et al. Multilocus genotyping of Giardia duodenalis in Brazilian children. Trans R Soc Trop Med Hyg 2016; 110(6): 343-349. http://dx.doi.org/10.1093/trstmh/trw036. PMid:27317756.
http://dx.doi.org/10.1093/trstmh/trw036...
; Figueiredo Pacheco et al., 2020Figueiredo Pacheco FT, Novaes Rodrigues Silva RK, Souza de Carvalho S, Carvalho Rocha F, Trindade das Chagas GM, Chagas Gomes D, et al. Predominance of Giardia duodenalis AII sub-assemblage in young children from Salvador, Bahia, Brazil. Biomedica 2020; 40(3): 557-568. http://dx.doi.org/10.7705/biomedica.5161. PMid:33030834.
http://dx.doi.org/10.7705/biomedica.5161...
). Brazilian dogs are infected with assemblages A, B, C, D, and E, among which assemblage A predominates (Fantinatti et al., 2020Fantinatti M, Gonçalves-Pinto M, Lopes-Oliveira LAP, Da-Cruz AM. Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go. Mem Inst Oswaldo Cruz 2020; 115: e200431. http://dx.doi.org/10.1590/0074-02760200431. PMid:33503146.
http://dx.doi.org/10.1590/0074-027602004...
). This assemblage is also more prevalent in children and dogs from poor communities (Volotão et al., 2007Volotão AC, Costa-Macedo LM, Haddad FSM, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using β-giardin gene: a phylogenetic analysis. Acta Trop 2007; 102(1): 10-19. http://dx.doi.org/10.1016/j.actatropica.2007.02.010. PMid:17428432.
http://dx.doi.org/10.1016/j.actatropica....
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Quadros et al., 2016Quadros RM, Weiss PHE, Marques SMT, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo 2016; 58(0): 66. http://dx.doi.org/10.1590/S1678-9946201658066. PMid:27680171.
http://dx.doi.org/10.1590/S1678-99462016...
).

The aim of this study was to determine the prevalence and genetically characterize isolates of G. duodenalis from children and dogs from rural and semi-rural northeastern communities to identify priority areas to prevent and control diarrheal diseases in the rural area of the municipality of Ilhéus, Bahia, Brazil.

Material and Methods

Study area

The study was conducted in rural and semi-rural communities in the municipality of Ilhéus, State of Bahia, Brazil. Ilhéus has 184,236 inhabitants, of which 28,955 live in rural areas. The city has an average HDI (Human Development Index) of 0.69 and G (Gini Index) of 0.5; the infant mortality rate is 18.4/1000 live births, and the number of hospitalizations for diarrhea is 1.3/1000 inhabitants. These communities (Figure 1) are characterized by a high degree of social vulnerability, including the absence or low quality of health infrastructure, a high number of semi-restricted dogs, presence of farm animals on streets, frequent contact with wildlife, a high number of illiterates and people with incomplete primary education and low purchasing power. Only 65.9% of households are connected to the municipal sewage system (IBGE, 2010Instituto Brasileiro de Geografia e Estatatistica - IBGE. Censo Demográfico 2010 [online]. 2010 [cited 2022 Aug 20]. Available from: https://censo2010.ibge.gov.br/resultados.html
https://censo2010.ibge.gov.br/resultados...
; Harvey et al., 2020Harvey TV, Tang AM, Sevá AP, Santos CA, Carvalho SMS, Rocha CMBM, et al. Enteric parasitic infections in children and dogs in resource-poor communities in northeastern Brazil: identifying priority prevention and control areas. PLoS Negl Trop Dis 2020; 14(6): e0008378. http://dx.doi.org/10.1371/journal.pntd.0008378. PMid:32516309.
http://dx.doi.org/10.1371/journal.pntd.0...
).

Figure 1
A) Domestic activity on the Japu River; B) Presence of horses on a football field in the village of Castelo Novo. See the garbage dump near the camp; C) Example of a clay house present in several communities in Ilhéus; D) Black arrow indicating untreated water consumed in the community of Maria Jape; E) Cattle raised free in the community of Aritaguá. See the animal's interaction with open garbage. Source: Tatiani Harvey.

Ilhéus is located in the Atlantic Rainforest Biome, with a tropical and humid climate. Annual rainfall ranges from 1000 mm to 2700 mm, and the mean annual compensated temperature is approximately 25°C. In rural communities, rivers are widely used for household chores, personal and animal hygiene, and leisure (Harvey et al., 2020Harvey TV, Tang AM, Sevá AP, Santos CA, Carvalho SMS, Rocha CMBM, et al. Enteric parasitic infections in children and dogs in resource-poor communities in northeastern Brazil: identifying priority prevention and control areas. PLoS Negl Trop Dis 2020; 14(6): e0008378. http://dx.doi.org/10.1371/journal.pntd.0008378. PMid:32516309.
http://dx.doi.org/10.1371/journal.pntd.0...
). Most communities do not receive treated water (IBGE, 2010Instituto Brasileiro de Geografia e Estatatistica - IBGE. Censo Demográfico 2010 [online]. 2010 [cited 2022 Aug 20]. Available from: https://censo2010.ibge.gov.br/resultados.html
https://censo2010.ibge.gov.br/resultados...
).

Study population and sample collection

Sample size calculation was based on an estimated proportion of 50% ± 5% accuracy with 95% confidence and population size of 3,851 for children and 5,348 for dogs. The estimated sample size was 350 for children and 359 for dogs; the sampling was stratified by districts and systematized. Sample sizes were calculated using EpiTools (https://epitools.ausvet.com.au/oneproportion). The total number of collector flasks calculated for each population, for each type of analysis, was given to parents and dog owners. Between March and November 2016, fecal samples from all ten districts of Ilhéus, Bahia, Brazil, were delivered by only 193 children aged from 1 to 5 years, 192 for parasitological analysis and 141 for molecular analysis. Also, only 143 canine fecal samples were delivered by dog owners, 139 for parasitological analysis and 92 for molecular analysis. For parasitological analysis, three samples were collected per participant, on alternate days, using the TF-Test® Conventional commercial kit (Immunoassay Indústria e Comércio Ltda., Brazil). A fourth fecal sample was collected in a collector flask without preservatives, for molecular analysis. Health clinics, public schools, and volunteer residents received and stored the samples in cool boxes containing reusable ice packs on the day scheduled to collect samples.

Parasitological analysis

The samples collected were stored at 4 oC until processing in a maximum of 5 days. The analyses were made following the TF-TestTM kit protocol (Gomes et al., 2004Gomes JF, Hoshino-Shimizu S, Dias LCS, Araujo AJSA, Castilho VLP, Neves FAMA. Evaluation of a novel kit (TF-Test) for the diagnosis of intestinal parasitic infections. J Clin Lab Anal 2004; 18(2): 132-138. http://dx.doi.org/10.1002/jcla.20011. PMid:15065214.
http://dx.doi.org/10.1002/jcla.20011...
; Coelho et al., 2013Coelho WMD, Gomes JF, Amarante AFT, Bresciani KDS, Lumina G, Koshino-Shimizu S, et al. A new laboratorial method for the diagnosis of gastrointestinal parasites in dogs. Rev Bras Parasitol Vet 2013; 22(1): 1-5. http://dx.doi.org/10.1590/S1984-29612013000100002. PMid:24252948.
http://dx.doi.org/10.1590/S1984-29612013...
), which uses the sedimentation principle (TFC), and the Faust et al. (1938)Faust EC, D’Antoni JS, Odom V, Miller MJ, Peres C, Sawitz W, et al. A critical study of clinical laboratory technics for the diagnosis of protozoan cysts and helminth eggs in feces. Am J Trop Med Hyg 1938;18(2 Suppl 1): 169-183. http://dx.doi.org/10.4269/ajtmh.1938.s1-18.169.
http://dx.doi.org/10.4269/ajtmh.1938.s1-...
method modified by Sloss et al. (1999)Sloss MW, Zajac AM, Kemp RL. Parasitologia clínica veterinária. 1. ed. São Paulo: Manole; 1999. , which is based on centrifugal-float method (CF). Kit samples were divided into two equivalent aliquots for further processing with each technique. All samples were analyzed by the same microscopist on slides containing a drop of Lugol solution and subsequently observed at 40X under optical microscopy to detect cysts or trophozoites of G. duodenalis.

DNA extraction, molecular analysis, and sequencing

The samples collected for molecular analysis were immediately homogenized, and a 2g- aliquot of feces was separated into a microtube and stored at -20 °C for approximately 1 year. DNA was extracted from 200 mg of feces, using the Purelink™ Microbiome DNA Purification kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations, with the following adaptations: a) after resuspension in lysis buffer and lysis enhancer, the samples were incubated at 65 °C for 10 min; b) 5 cycles of freeze-thawing were applied by freezing samples in liquid nitrogen for 1 minute, followed by thawing at 65 °C; c) the recommended amount of the elution buffer was divided in half and samples processed according to the protocol. This step was repeated once more to optimize DNA recovery. Genetic characterization of G. duodenalis was made by nested PCR (nPCR) targeting the small subunit ribosomal RNA, ssu rRNA (Hopkins et al., 1997Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, Thompson RC. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol 1997; 83(1): 44-51. http://dx.doi.org/10.2307/3284315. PMid:9057695.
http://dx.doi.org/10.2307/3284315...
; Appelbee et al., 2003Appelbee AJ, Frederick LM, Heitman TL, Olson ME. Prevalence and genotyping of Giardia duodenalis from beef calves in Alberta, Canada. Vet Parasitol 2003; 112(4): 289-294. http://dx.doi.org/10.1016/S0304-4017(02)00422-3. PMid:12623208.
http://dx.doi.org/10.1016/S0304-4017(02)...
), beta-giardin, bg (Lalle et al., 2005Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Cacciò SM. Genetic heterogeneity at the β-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol 2005; 35(2): 207-213. http://dx.doi.org/10.1016/j.ijpara.2004.10.022. PMid:15710441.
http://dx.doi.org/10.1016/j.ijpara.2004....
), glutamate dehydrogenase, gdh (Cacciò et al., 2008Cacciò SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J Parasitol 2008; 38(13): 1523-1531. http://dx.doi.org/10.1016/j.ijpara.2008.04.008. PMid:18571176.
http://dx.doi.org/10.1016/j.ijpara.2008....
), and triosephosphate isomerase, tpi (Sulaiman et al., 2003Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 2003; 9(11): 1444-1452. http://dx.doi.org/10.3201/eid0911.030084. PMid:14718089.
http://dx.doi.org/10.3201/eid0911.030084...
) genes. The changes made to the protocols will be made available in a Supplementary Material (Suppl 01-Tables S1, S2, S3, and S4). nPCR amplicons were electrophoresed on 1 to 1.5% agarose, stained with SYBR® Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), and bands visualized using a UV transilluminator. Amplicons that produced strong bands were purified (IllustraTM GFXTM PCR DNA and Gel Band Purification Kit, Millipore Sigma) prior to Sanger-sequencing in both directions, with primers used for the secondary PCR. CodonCode Aligner software version 4.0.1 (CodonCode Corporation Dedham®, MA, USA) was used to determine a consensus sequence. Alignment was made using the Clustal W method (Thompson et al., 1997Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997; 25(24): 4876-4882. http://dx.doi.org/10.1093/nar/25.24.4876. PMid:9396791.
http://dx.doi.org/10.1093/nar/25.24.4876...
) in BioEdit (Hall, 1999Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98.), using homologous sequences downloaded from GenBank as references. Sequences were deposited in GenBank under accession numbers MW929203-MW929205 (bg) and MW929206-MW929208 (tpi).

Phylogenetic analysis

Phylogenetic analysis was made using the Neighbor-Joining method and the Kimura 2-parameter model (Kimura, 1980Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16(2): 111-120. http://dx.doi.org/10.1007/BF01731581. PMid:7463489.
http://dx.doi.org/10.1007/BF01731581...
). The tree was constructed using MEGA X software. Evaluation of the reliability of clusters was confirmed using Bootstrap values from 1000 replicates.

Statistical analysis

The global prevalence of giardiasis was determined by the sum of positive cases in at least one method. Only samples analyzed by both methods were used in the comparison between microscopy and nPCR in both populations. The absolute and relative frequencies of amplification per gene and positive and negative cases in the parasitological diagnosis by the categorical variable are shown in Table 1. The agreement between methods was determined by the Kappa (k) index, statistically testing the consistency of k and ranking it as poor (0 to 0.20); low (0.21 to 0.40); moderate (0.41 to 0.60); substantial (0.61 to 0.80); and almost perfect (0.80 to 1.0) (Landis & Koch, 1977Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33(1): 159-174. http://dx.doi.org/10.2307/2529310. PMid:843571.
http://dx.doi.org/10.2307/2529310...
). All statistical analyses were conducted using Epi Info 7 (Centers for Disease Control and Prevention—CDC, Atlanta, USA).

Table 1
Results from parasitological screening of Giardia spp. and PCR protocols targeting the SSU-rRNA, bg, tpi, and gdh genes. Population: children aged up to 5 years (n= 141) and dogs (n= 92) from rural areas of Ilhéus, Bahia, Brazil, 2016.

Results

Although collector flasks were delivered to collect feces from 350 children and 359 dogs, we received samples from only 193 children and 143 dogs, considering samples for both parasitological and molecular analysis.

Giardiasis parasitological diagnosis

In the population of children, 52/192 (27.1%) positive samples were detected by the CF method and 51/192 positive samples (26.6%) by the TFC method. In the canine population, 20/139 positive samples (14.4%) were detected by the CF method and 12/139 positive samples (8.6%) by the TFC method. Considering the positivity in at least one test, giardiasis was detected in 72 children (37.5%; 95% CI: 30.6%-44.7%) and 24 dogs (17.3%; 95% CI: 11.4%-24.6%).

Giardia PCR results and sequencing

G. duodenalis DNA was detected in 60 fecal samples from children (60/141; 42.5%; 95% CI: 34.3%-51.2%), with different detection rates between genes (bg: 29/60, 48.3%; tpi: 24/60, 40%; ssu rRNA: 17/60, 28.3%; gdh: 13/60, 21.7%). Among the dogs, 26 samples were positive (26/92; 28.3%; 95% CI: 19.4%-38.6%), with also differences in detection rates (ssu rRNA: 13/16, 50%; bg: 12/ 26, 46.1%; tpi: 4/26, 15.4%; gdh: 3/26, 11.5%).

Only samples that had good DNA amplification (strong bands) were sequenced. Assemblages A, B, and E were identified in children and assemblage E in dogs (Table 2). Regarding bg sequences, three of them were identical to sequences obtained from cattle (KY769092, MH079430) and sheep (KY633473, MK573342) identified as assemblage E. One bg sequence, identified as assemblage A, was identical to sequences obtained from children (MG845549, MG845542) in Salvador, Brazil (Figueiredo Pacheco et al., 2020Figueiredo Pacheco FT, Novaes Rodrigues Silva RK, Souza de Carvalho S, Carvalho Rocha F, Trindade das Chagas GM, Chagas Gomes D, et al. Predominance of Giardia duodenalis AII sub-assemblage in young children from Salvador, Bahia, Brazil. Biomedica 2020; 40(3): 557-568. http://dx.doi.org/10.7705/biomedica.5161. PMid:33030834.
http://dx.doi.org/10.7705/biomedica.5161...
). All tpi sequences identified as assemblage A were identical to sequences obtained from humans (MH673818). In addition, two sequences identified as assemblage B were identical to sequences obtained from water (AY368171) in the USA and humans (KX468986) in Spain.

Table 2
Results from PCR and genetic sequencing and profile of clinical-epidemiological cases of infection by Giardia duodenalis.

The evolutionary relationship of assemblages found in this study in both populations is shown in Figure 2.

Figure 2
Phylogenetic tree between Giardia duodenalis obtained from this study and previous studies based on bg (A) and tpi (B) genes. The evolutionary history was inferred using the Neighbor-Joining method using the Kimura 2-parameter model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Reference sequences with their accession numbers in GenBank are provided.

Clinical-epidemiological profile of children and dogs that had their infections confirmed by sequencing is described in Table 2. Children 145,150,162, and 182 were from the same community.

Comparison between parasitological and molecular diagnosis of Giardia

Considering positive samples in at least one parasitological method or loci, the overall prevalence was 54.9% in children (106/193; 95% CI: 47.1%-61.6%), and 32.9% in dogs (47/143; 95% CI: 25.2%-41.2%). Infection was present in all districts of Ilhéus (Figure 3).

Figure 3
Location and distribution map of giardiasis cases in the Municipality of Ilhéus, State of Bahia, Brazil. This map was prepared using the QGIS program (version 2.18).

In the population of children, twenty-six samples were positive with both parasitological and molecular techniques, but the agreement was poor (index k= 0.07). Of the 86 samples negative by microscopy, 34 were positive (39.5%) by PCR, and of the 81 samples negative by PCR, 29 were positive (35.8%) by microscopy. The agreement between the parasitological analysis and the bg (k= 0.02) and tpi (k= 0.15) genes was poor, and there was no agreement between the SSU-rRNA and gdh genes with the result of the parasitological analysis (k < 0).

In the population of dogs, three samples were positive with both techniques, which had no agreement between their results (k= -0.14). Of the 73 samples negative by microscopy, 23 (31.5%) were positive by PCR, and of the 66 samples negative by PCR, 16 (24.2%) were positive by microscopy. The agreement between the parasitological analysis and the SSU-rRNA gene (k= 0.09) was poor, and there was no agreement between the other genes with the result of the parasitological analysis (k < 0).

Kappa agreement between the techniques for both populations is shown in Table 3. The distribution of giardiasis cases detected by parasitological and molecular techniques is shown in Table 1.

Table 3
Kappa agreement (k) between parasitological (OM) and molecular (Mol) methods used for diagnosis of Giardia infections.

Discussion

The greater number of cases of giardiasis detected from the combination of diagnostic techniques in both populations demonstrates and reinforces the need for the prevalence rates of this infection to be investigated using more than one parasitological technique or more than one genetic marker (Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
; Hooshyar et al., 2019Hooshyar H, Rostamkhani P, Arbabi M, Delavari M. Giardia lamblia infection: review of current diagnostic strategies. Gastroenterol Hepatol Bed Bench 2019; 12(1): 3-12. PMid:30949313.). In children, the overall prevalence of infection (54.4%) was higher than that reported in other Brazilian studies (Santos et al., 2012Santos CKS, Grama DF, Limongi JE, Costa FC, Couto TR, Soares RM, et al. Epidemiological, parasitological and molecular aspects of Giardia duodenalis infection in children attending public daycare centers in southeastern Brazil. Trans R Soc Trop Med Hyg 2012; 106(8): 473-479. http://dx.doi.org/10.1016/j.trstmh.2012.05.011. PMid:22739043.
http://dx.doi.org/10.1016/j.trstmh.2012....
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Mariano et al., 2015Mariano APM, Santos EN, Santos TN, Mota TN, Silva JA, Carvalho SMS, et al. Parasites in South Bahia: focus on giardiasis and ascariasis among preschoolers of Itabuna. Int J Health Sci (Qassim) 2015; 3(1): 61-75. http://dx.doi.org/10.15640/ijhs.v3n1a4.
http://dx.doi.org/10.15640/ijhs.v3n1a4...
; Nunes et al., 2016Nunes BC, Pavan MG, Jaeger LH, Monteiro KJL, Xavier SCC, Monteiro FA, et al. Spatial and molecular epidemiology of Giardia intestinalis deep in the Amazon, Brazil. PLoS One 2016; 11(7): e0158805. http://dx.doi.org/10.1371/journal.pone.0158805. PMid:27392098.
http://dx.doi.org/10.1371/journal.pone.0...
). In dogs, the prevalence was similar to previous reports (Mundim et al., 2003Mundim MJS, Souza SZ, Hortêncio SM, Cury MC. Freqüência de Giardia spp. por duas técnicas de diagnóstico em fezes de cães. Arq Bras Med Vet Zootec 2003; 55(6): 770-773. http://dx.doi.org/10.1590/S0102-09352003000600016.
http://dx.doi.org/10.1590/S0102-09352003...
; Labruna et al., 2006Labruna MB, Pena HFJ, Souza SLP, Pinter A, Silva JCR, Ragozo AMA, et al. Prevalência de endoparasitas em cães da área urbana do município de Monte Negro, Rondônia. Arq Inst Biol (Sao Paulo) 2006; 73(2): 183-193. http://dx.doi.org/10.1590/1808-1657v73p1832006.
http://dx.doi.org/10.1590/1808-1657v73p1...
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Coelho et al., 2017Coelho CH, Durigan M, Leal DAG, Schneider AB, Franco RMB, Singer SM. Giardiasis as a neglected disease in Brazil: systematic review of 20 years of publications. PLoS Negl Trop Dis 2017; 11(10): e0006005. http://dx.doi.org/10.1371/journal.pntd.0006005. PMid:29065126.
http://dx.doi.org/10.1371/journal.pntd.0...
).

Within the scope of parasitological analysis, the individual performance of centrifugal-flotation and centrifugal-sedimentation was not as efficient as their parallel association in this study. In addition to reiterating the recommendations for the use of microscopy in the diagnosis of Giardia (Faria et al., 2016Faria CP, Zanini GM, Dias GS, Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One 2016; 11(8): e0160762. http://dx.doi.org/10.1371/journal.pone.0160762. PMid:27517469.
http://dx.doi.org/10.1371/journal.pone.0...
; Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
; Hooshyar et al., 2019Hooshyar H, Rostamkhani P, Arbabi M, Delavari M. Giardia lamblia infection: review of current diagnostic strategies. Gastroenterol Hepatol Bed Bench 2019; 12(1): 3-12. PMid:30949313.), our study reinforces the need for parallel use of parasitological techniques in protozoan research, especially in the screening of Giardia samples, prior to application of PCR (Volotão et al., 2007Volotão AC, Costa-Macedo LM, Haddad FSM, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using β-giardin gene: a phylogenetic analysis. Acta Trop 2007; 102(1): 10-19. http://dx.doi.org/10.1016/j.actatropica.2007.02.010. PMid:17428432.
http://dx.doi.org/10.1016/j.actatropica....
; David et al., 2011David EB, Coradi ST, Oliveira-Sequeira TCG, Ribolla PEM, Katagiri S, Guimarães S. Diagnosis of Giardia infections by PCR-based methods in children of an endemic area. J Venom Anim Toxins Incl Trop Dis 2011; 17(2): 209-215. http://dx.doi.org/10.1590/S1678-91992011000200012.
http://dx.doi.org/10.1590/S1678-91992011...
, 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Quadros et al., 2016Quadros RM, Weiss PHE, Marques SMT, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo 2016; 58(0): 66. http://dx.doi.org/10.1590/S1678-9946201658066. PMid:27680171.
http://dx.doi.org/10.1590/S1678-99462016...
; Faria et al., 2016Faria CP, Zanini GM, Dias GS, Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One 2016; 11(8): e0160762. http://dx.doi.org/10.1371/journal.pone.0160762. PMid:27517469.
http://dx.doi.org/10.1371/journal.pone.0...
). The presence of false-negative cases on microscopy was probably related to the low number of cysts observed in most of our samples, especially in canine fecal samples, in which the number of cysts was even lower (Katagiri & Oliveira-Sequeira, 2007Katagiri S, Oliveira-Sequeira TCG. Zoonoses causadas por parasitas intestinais de cães e o problema do diagnóstico. Arq Inst Biol (Sao Paulo) 2007; 74(2): 175-184. http://dx.doi.org/10.1590/1808-1657v74p1752007.
http://dx.doi.org/10.1590/1808-1657v74p1...
; Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
).

Several studies have shown greater sensitivity of molecular methods to detect Giardia (Bouzid et al., 2015Bouzid M, Halai K, Jeffreys D, Hunter PR. The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples. Vet Parasitol 2015; 207(3-4): 181-202. http://dx.doi.org/10.1016/j.vetpar.2014.12.011. Pmid:25583357.
http://dx.doi.org/10.1016/j.vetpar.2014....
; Ramírez et al., 2015Ramírez JD, Heredia RD, Hernández C, León CM, Moncada LI, Reyes P, et al. Molecular diagnosis and genotype analysis of Giardia duodenalis in asymptomatic children from a rural area in central Colombia. Infect Genet Evol 2015; 32: 208-213. http://dx.doi.org/10.1016/j.meegid.2015.03.015. PMid:25795384.
http://dx.doi.org/10.1016/j.meegid.2015....
). However just as the use of only one parasitological technique underestimated the prevalence of giardiasis in our study, the individual use of PCR can also underestimate cases in human and animal populations (Arroyo-Salgado et al., 2014Arroyo-Salgado B, Buelvas-Montes Y, Villalba-Vizcaíno V, Salomón-Arzuza O. Caracterización genética por reacción en cadena de la polimerasa de Giardia intestinalis en muestras de humanos y perros del Caribe colombiano. Enferm Infecc Microbiol Clin 2014; 32(7): 424-427. http://dx.doi.org/10.1016/j.eimc.2013.07.016. PMid:24144785.
http://dx.doi.org/10.1016/j.eimc.2013.07...
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
). It should also be noted that the PCR technique can have varied sensitivity because, in addition to the presence of inhibitors, the extraction of DNA from the cysts is variable, with amplification rates from 36% to 85.7% (Babaei et al., 2011Babaei Z, Oormazdi H, Rezaie S, Rezaeian M, Razmjou E. Giardia intestinalis: DNA extraction approaches to improve PCR results. Exp Parasitol 2011; 128(2): 159-162. http://dx.doi.org/10.1016/j.exppara.2011.02.001. PMid:21315715.
http://dx.doi.org/10.1016/j.exppara.2011...
; Silva et al., 2022Silva ACS, Martins FDC, Ladeia WA, Kakimori MTA, Lucas JI, Sasse JP, et al. First report of Giardia duodenalis assemblage F in humans and dogs in southern Brazil. Comp Immunol Microbiol Infect Dis 2022; 89: 101878. http://dx.doi.org/10.1016/j.cimid.2022.101878. PMid:36108583.
http://dx.doi.org/10.1016/j.cimid.2022.1...
). Prevalence and diagnostic efficiency of molecular methods depend on the type and number of loci analyzed, and their applicability has been mainly restricted to the epidemiological purpose due to their high cost of execution (Feng & Xiao, 2011Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011; 24(1): 110-140. http://dx.doi.org/10.1128/CMR.00033-10. PMid:21233509.
http://dx.doi.org/10.1128/CMR.00033-10...
; Ryan & Cacciò, 2013Ryan U, Cacciò SM. Zoonotic potential of Giardia. Int J Parasitol 2013; 43(12-13): 943-956. http://dx.doi.org/10.1016/j.ijpara.2013.06.001. PMid:23856595.
http://dx.doi.org/10.1016/j.ijpara.2013....
). Ryan & Cacciò (2013)Ryan U, Cacciò SM. Zoonotic potential of Giardia. Int J Parasitol 2013; 43(12-13): 943-956. http://dx.doi.org/10.1016/j.ijpara.2013.06.001. PMid:23856595.
http://dx.doi.org/10.1016/j.ijpara.2013....
recommend using the loci bg, tpi, and gdh, at a minimum. The presence of false-negative cases in PCR, the difference between the amplification rates of the genes and the failure in the typing of the assemblages can result from many factors such as DNA degradation, ineffective DNA extraction, presence of PCR inhibitors, and the amount of non-specific amplified DNA (Arroyo-Salgado et al., 2014Arroyo-Salgado B, Buelvas-Montes Y, Villalba-Vizcaíno V, Salomón-Arzuza O. Caracterización genética por reacción en cadena de la polimerasa de Giardia intestinalis en muestras de humanos y perros del Caribe colombiano. Enferm Infecc Microbiol Clin 2014; 32(7): 424-427. http://dx.doi.org/10.1016/j.eimc.2013.07.016. PMid:24144785.
http://dx.doi.org/10.1016/j.eimc.2013.07...
; Faria et al., 2016Faria CP, Zanini GM, Dias GS, Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One 2016; 11(8): e0160762. http://dx.doi.org/10.1371/journal.pone.0160762. PMid:27517469.
http://dx.doi.org/10.1371/journal.pone.0...
; Calegar et al., 2022Calegar DA, Nunes BC, Monteiro KJL, Bacelar PAA, Evangelista BBC, Almeida MM, et al. Genotypic and epidemiologic profiles of Giardia duodenalis in four Brazilian biogeographic regions. Microorganisms 2022; 10(5): 940. http://dx.doi.org/10.3390/microorganisms10050940. PMid:35630389.
http://dx.doi.org/10.3390/microorganisms...
). In this study, it should be considered that the DNA of the samples for PCR was extracted a few months after collection, which may have caused degradation. In addition, it is known that the deterioration of genetic material can limit the efficiency of PCR, especially for single-copy genes such as bg, gdh, and tpi (Peng et al., 2003Peng MM, Wilson ML, Holland RE, Meshnick SR, Lal AA, Xiao L. Genetic diversity of Cryptosporidium spp. in cattle in Michigan: implications for understanding the transmission dynamics. Parasitol Res 2003; 90(3): 175-180. http://dx.doi.org/10.1007/s00436-003-0834-5. PMid:12783304.
http://dx.doi.org/10.1007/s00436-003-083...
). These factors may have determined the disagreement between the amplification rates of the ssu-rRNA, bg, gdh, and tpi loci in our study, which was also evidenced in other studies with populations of children (David et al., 2011David EB, Coradi ST, Oliveira-Sequeira TCG, Ribolla PEM, Katagiri S, Guimarães S. Diagnosis of Giardia infections by PCR-based methods in children of an endemic area. J Venom Anim Toxins Incl Trop Dis 2011; 17(2): 209-215. http://dx.doi.org/10.1590/S1678-91992011000200012.
http://dx.doi.org/10.1590/S1678-91992011...
, 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Ramírez et al., 2015Ramírez JD, Heredia RD, Hernández C, León CM, Moncada LI, Reyes P, et al. Molecular diagnosis and genotype analysis of Giardia duodenalis in asymptomatic children from a rural area in central Colombia. Infect Genet Evol 2015; 32: 208-213. http://dx.doi.org/10.1016/j.meegid.2015.03.015. PMid:25795384.
http://dx.doi.org/10.1016/j.meegid.2015....
; Scalia et al., 2016Scalia LAM, Fava NMN, Soares RM, Limongi JE, Cunha MJR, Pena IF, et al. Multilocus genotyping of Giardia duodenalis in Brazilian children. Trans R Soc Trop Med Hyg 2016; 110(6): 343-349. http://dx.doi.org/10.1093/trstmh/trw036. PMid:27317756.
http://dx.doi.org/10.1093/trstmh/trw036...
; Jerez Puebla et al., 2017Jerez Puebla LE, Núñez FA, Santos LP, Rivero LR, Silva IM, Valdés LA, et al. Molecular analysis of Giardia duodenalis isolates from symptomatic and asymptomatic children from La Habana, Cuba. Parasite Epidemiol Control 2017; 2(3): 105-113. http://dx.doi.org/10.1016/j.parepi.2017.05.003. PMid:29774288.
http://dx.doi.org/10.1016/j.parepi.2017....
) and dogs (Scorza et al., 2012Scorza AV, Ballweber LR, Tangtrongsup S, Panuska C, Lappin MR. Comparisons of mammalian Giardia duodenalis assemblages based on the β-giardin, glutamate dehydrogenase and triose phosphate isomerase genes. Vet Parasitol 2012; 189(2-4): 182-188. http://dx.doi.org/10.1016/j.vetpar.2012.04.032. PMid:22652427.
http://dx.doi.org/10.1016/j.vetpar.2012....
; Gil et al., 2017Gil H, Cano L, Lucio A, Bailo B, Mingo MH, Cardona GA, et al. Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain. Infect Genet Evol 2017; 50: 62-69. http://dx.doi.org/10.1016/j.meegid.2017.02.013. PMid:28219812.
http://dx.doi.org/10.1016/j.meegid.2017....
). In this context, our study reinforces the need to use multiple genes for giardiasis diagnosis in human and animal populations (Faria et al., 2016Faria CP, Zanini GM, Dias GS, Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One 2016; 11(8): e0160762. http://dx.doi.org/10.1371/journal.pone.0160762. PMid:27517469.
http://dx.doi.org/10.1371/journal.pone.0...
; Soares & Tasca, 2016Soares R, Tasca T. Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods 2016; 129: 98-102. http://dx.doi.org/10.1016/j.mimet.2016.08.017. PMid:27546714.
http://dx.doi.org/10.1016/j.mimet.2016.0...
) to avoid diagnostic bias and the underestimation of prevalence. It is important to consider that in the absence of DNA degradation or inhibition during storage and/or processing, the use of a multicopy gene such as ssu-RNA would be expected to be sufficiently more sensitive than parasitological techniques or single-copy gene targeted PCRs. Thus, there would be no need for the parallel use of diagnostic techniques. However, in this study, it is reasonable to infer that the lack of sensitivity of the ssu-RNA gene was probably the result of DNA degradation, which may have significantly impacted the PCR sensitivity compared to the parasitological technique. We also emphasize the use of only one fecal sample per individual/dog in the molecular investigation of Giardia may also have underestimated the prevalence detected by this method (Scorza et al., 2012Scorza AV, Ballweber LR, Tangtrongsup S, Panuska C, Lappin MR. Comparisons of mammalian Giardia duodenalis assemblages based on the β-giardin, glutamate dehydrogenase and triose phosphate isomerase genes. Vet Parasitol 2012; 189(2-4): 182-188. http://dx.doi.org/10.1016/j.vetpar.2012.04.032. PMid:22652427.
http://dx.doi.org/10.1016/j.vetpar.2012....
). Our finding also serves as a warning to researchers who usually use the ssu-rRNA gene in screening samples for Giardia before using other genes (Silva et al., 2012Silva FMP, Monobe MM, Lopes RS, Araujo JP Jr. Molecular characterization of Giardia duodenalis in dogs from Brazil. Parasitol Res 2012; 110(1): 325-334. http://dx.doi.org/10.1007/s00436-011-2492-3. PMid:21695567.
http://dx.doi.org/10.1007/s00436-011-249...
; Gasparinho et al., 2017Gasparinho C, Ferreira FS, Mayer AC, Mirante MC, Vaz Nery S, Santos-Reis A, et al. Molecular characterization of Giardia lamblia in children less than 5 years of age with diarrhoea attending the Bengo General Hospital, Angola. Trans R Soc Trop Med Hyg 2017; 111(11): 497-503. http://dx.doi.org/10.1093/trstmh/try004. PMid:29438541.
http://dx.doi.org/10.1093/trstmh/try004...
).

Giardia genotyping studies in children and dogs have been restricted to both South and Southeast regions of Brazil (Volotão et al., 2007Volotão AC, Costa-Macedo LM, Haddad FSM, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using β-giardin gene: a phylogenetic analysis. Acta Trop 2007; 102(1): 10-19. http://dx.doi.org/10.1016/j.actatropica.2007.02.010. PMid:17428432.
http://dx.doi.org/10.1016/j.actatropica....
; David et al., 2015David ÉB, Guimarães S, Oliveira AP, Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors 2015; 8(1): 103. http://dx.doi.org/10.1186/s13071-015-0714-8. PMid:25889093.
http://dx.doi.org/10.1186/s13071-015-071...
; Quadros et al., 2016Quadros RM, Weiss PHE, Marques SMT, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo 2016; 58(0): 66. http://dx.doi.org/10.1590/S1678-9946201658066. PMid:27680171.
http://dx.doi.org/10.1590/S1678-99462016...
; Scalia et al., 2016Scalia LAM, Fava NMN, Soares RM, Limongi JE, Cunha MJR, Pena IF, et al. Multilocus genotyping of Giardia duodenalis in Brazilian children. Trans R Soc Trop Med Hyg 2016; 110(6): 343-349. http://dx.doi.org/10.1093/trstmh/trw036. PMid:27317756.
http://dx.doi.org/10.1093/trstmh/trw036...
; Coelho et al., 2017Coelho CH, Durigan M, Leal DAG, Schneider AB, Franco RMB, Singer SM. Giardiasis as a neglected disease in Brazil: systematic review of 20 years of publications. PLoS Negl Trop Dis 2017; 11(10): e0006005. http://dx.doi.org/10.1371/journal.pntd.0006005. PMid:29065126.
http://dx.doi.org/10.1371/journal.pntd.0...
). And in the Midwest and Northeast regions, which concentrate the largest series of acute diarrheal diseases in the country (Brasil, 2018Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância de Doenças e Agravos não Transmissíveis e Promoção da Saúde. Saúde Brasil 2017: uma análise da situação de saúde e os desafios para o alcance dos objetivos de desenvolvimento sustentável [online]. 2018 [cited 2022 Aug 20]. Available from: http://bvsms.saude.gov.br/bvs/publicacoes/saude_brasil_2017_analise_situacao_saude_desafios_objetivos_desenvolvimento_sustetantavel.pdf
http://bvsms.saude.gov.br/bvs/publicacoe...
), the epidemiology of this infection has still been poorly understood. Zoonotic assemblages A and B were detected in Northeastern children in a resource-poor community, in Ceará (Kohli et al., 2008Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 2008; 102(7): 718-725. http://dx.doi.org/10.1016/j.trstmh.2008.03.002. PMid:18485429.
http://dx.doi.org/10.1016/j.trstmh.2008....
) and in daycare centers in Bahia (Figueiredo Pacheco et al., 2020Figueiredo Pacheco FT, Novaes Rodrigues Silva RK, Souza de Carvalho S, Carvalho Rocha F, Trindade das Chagas GM, Chagas Gomes D, et al. Predominance of Giardia duodenalis AII sub-assemblage in young children from Salvador, Bahia, Brazil. Biomedica 2020; 40(3): 557-568. http://dx.doi.org/10.7705/biomedica.5161. PMid:33030834.
http://dx.doi.org/10.7705/biomedica.5161...
). Our study brings the first report of infection by these assemblages in children from rural areas, including tourist communities, in this region. We observed the presence of diarrhea in the infection with both assemblages. In contrast, Figueiredo Pacheco et al. (2020)Figueiredo Pacheco FT, Novaes Rodrigues Silva RK, Souza de Carvalho S, Carvalho Rocha F, Trindade das Chagas GM, Chagas Gomes D, et al. Predominance of Giardia duodenalis AII sub-assemblage in young children from Salvador, Bahia, Brazil. Biomedica 2020; 40(3): 557-568. http://dx.doi.org/10.7705/biomedica.5161. PMid:33030834.
http://dx.doi.org/10.7705/biomedica.5161...
detected cases of diarrhea only in children infected with the AI sub-assemblage. They also found that 91.8% of children with giardiasis had no diarrhea or any other relevant symptoms. Unfortunately, it was not possible to investigate the association between these assemblages and clinical signs due to the low number of samples that had their assemblages detected by sequencing. However, Kohli et al. (2008)Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 2008; 102(7): 718-725. http://dx.doi.org/10.1016/j.trstmh.2008.03.002. PMid:18485429.
http://dx.doi.org/10.1016/j.trstmh.2008....
did not find an association between diarrhea and assemblages A and B in single or mixed infections in children from Ceará.

Despite being considered livestock-specific, assemblage E has been detected inside and outside Brazil in humans (Foronda et al., 2008Foronda P, Bargues MD, Abreu-Acosta N, Periago MV, Valero MA, Valladares B, et al. Identification of genotypes of Giardia intestinalis of human isolates in Egypt. Parasitol Res 2008; 103(5): 1177-1181. http://dx.doi.org/10.1007/s00436-008-1113-2. PMid:18622625.
http://dx.doi.org/10.1007/s00436-008-111...
; Helmy et al., 2014Helmy YA, Klotz C, Wilking H, Krücken J, Nöckler K, Von Samson-Himmelstjerna G, et al. Epidemiology of Giardia duodenalis infection in ruminant livestock and children in the Ismailia province of Egypt: insights by genetic characterization. Parasit Vectors 2014; 7(1): 321. http://dx.doi.org/10.1186/1756-3305-7-321. PMid:25015671.
http://dx.doi.org/10.1186/1756-3305-7-32...
; Abdel-Moein & Saeed, 2016Abdel-Moein KA, Saeed H. The zoonotic potential of Giardia intestinalis assemblage E in rural settings. Parasitol Res 2016; 115(8): 3197-3202. http://dx.doi.org/10.1007/s00436-016-5081-7. PMid:27112756.
http://dx.doi.org/10.1007/s00436-016-508...
; Fantinatti et al., 2016Fantinatti M, Bello AR, Fernandes O, Da-Cruz AM. Identification of Giardia lamblia assemblage E in humans points to a new anthropozoonotic cycle. J Infect Dis 2016; 214(8): 1256-1259. http://dx.doi.org/10.1093/infdis/jiw361. PMid:27511898.
http://dx.doi.org/10.1093/infdis/jiw361...
; Zahedi et al., 2017Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland - first report of assemblage E. Parasitology 2017; 144(9): 1154-1161. http://dx.doi.org/10.1017/S0031182017000439. PMid:28482937.
http://dx.doi.org/10.1017/S0031182017000...
) and dogs (Feng & Xiao, 2011Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011; 24(1): 110-140. http://dx.doi.org/10.1128/CMR.00033-10. PMid:21233509.
http://dx.doi.org/10.1128/CMR.00033-10...
; Fava et al., 2016Fava NMN, Soares RM, Scalia LAM, Cunha MJR, Faria ESM, Cury MC. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp Parasitol 2016; 161: 1-5. http://dx.doi.org/10.1016/j.exppara.2015.12.003. PMid:26704664.
http://dx.doi.org/10.1016/j.exppara.2015...
) that interact with environments frequented by farm animals, as we also observed. At the clinical level, as reported in a few other studies (Helmy et al., 2014Helmy YA, Klotz C, Wilking H, Krücken J, Nöckler K, Von Samson-Himmelstjerna G, et al. Epidemiology of Giardia duodenalis infection in ruminant livestock and children in the Ismailia province of Egypt: insights by genetic characterization. Parasit Vectors 2014; 7(1): 321. http://dx.doi.org/10.1186/1756-3305-7-321. PMid:25015671.
http://dx.doi.org/10.1186/1756-3305-7-32...
; Abdel-Moein & Saeed, 2016Abdel-Moein KA, Saeed H. The zoonotic potential of Giardia intestinalis assemblage E in rural settings. Parasitol Res 2016; 115(8): 3197-3202. http://dx.doi.org/10.1007/s00436-016-5081-7. PMid:27112756.
http://dx.doi.org/10.1007/s00436-016-508...
; Zahedi et al., 2017Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland - first report of assemblage E. Parasitology 2017; 144(9): 1154-1161. http://dx.doi.org/10.1017/S0031182017000439. PMid:28482937.
http://dx.doi.org/10.1017/S0031182017000...
), we detected assemblage E in samples of diarrheal children, suggesting the existence of a risk of zoonotic transmission. To the best of our knowledge, this is the first report of giardiasis by assemblage E in Northeastern children and dogs.

In these communities, the high prevalence of giardiasis in children probably results from social and behavioral factors. Among them, it could be included inappropriate habits of food and personal hygiene, use and consumption of untreated water, inappropriate handling of pets and farm animals, and contact with environments contaminated with dog and farm animal feces. All these factors can favor regular interaction with different transmission cycles (Santos et al., 2010Santos SFO, Silva HD, Souza ES Jr, Anunciação CE, Silveira-Lacerda EP, Vilanova-Costa CAST, et al. Environmental monitoring of opportunistic protozoa in rivers and lakes in the neotropics based on yearly monitoring. Water Qual Expo Health 2010; 2(2): 97-104. http://dx.doi.org/10.1007/s12403-010-0027-2.
http://dx.doi.org/10.1007/s12403-010-002...
; Thompson & Ash, 2016Thompson RCA, Ash A. Molecular epidemiology of Giardia and Cryptosporidium infections. Infect Genet Evol 2016; 40: 315-323. http://dx.doi.org/10.1016/j.meegid.2015.09.028. PMid:26458528.
http://dx.doi.org/10.1016/j.meegid.2015....
; Ryan et al., 2019Ryan U, Hijjawi N, Feng Y, Xiao L. Giardia: an under-reported foodborne parasite. Int J Parasitol 2019; 49(1): 1-11. http://dx.doi.org/10.1016/j.ijpara.2018.07.003. PMid:30391227.
http://dx.doi.org/10.1016/j.ijpara.2018....
; Calegar et al., 2022Calegar DA, Nunes BC, Monteiro KJL, Bacelar PAA, Evangelista BBC, Almeida MM, et al. Genotypic and epidemiologic profiles of Giardia duodenalis in four Brazilian biogeographic regions. Microorganisms 2022; 10(5): 940. http://dx.doi.org/10.3390/microorganisms10050940. PMid:35630389.
http://dx.doi.org/10.3390/microorganisms...
).

We understand that the parallel use of parasitological techniques for the detection and screening of fecal samples of Giardia is a viable option for maintaining the diagnostic efficiency of giardiasis, both in private laboratories and in research. Likewise, when feasible, we reinforce the recommendation of using multiple genetic markers, especially in the context of research, avoiding the bias of prevalence that underpins the elaboration of plans for the prevention of diarrheal diseases. It is a fact that the association of methods makes the diagnostic process more expensive. However, the cost-effectiveness of these associations for controlling these diseases must be rationally evaluated. Accurate information on prevalence is essential for controlling giardiasis in highly endemic tourist areas, such as the municipality of Ilhéus, which has become a focus for the spread of diarrheal diseases, also beyond its borders.

Conclusion

The complementarity of parasitological and molecular methods proves to be a more effective strategy for detecting and controlling giardiasis in highly endemic areas, such as rural and semi-rural communities in the municipality of Ilhéus. However, given the high cost of implementing molecular techniques, especially when it is necessary to use several genetic markers, we recommend that the diagnosis of Giardia infection be made through the parallel use of parasitological techniques that use the principle of fluctuation. The zoonotic potential of assemblage E should be further investigated in this region.

Supplementary Material

Supplementary material accompanies this paper.

Suppl 01 Modifications made to established gene amplification protocols used in molecular research on Giardia. Table S1 Modifications of the bg gene amplification protocol established by Cacciò et al. (2002)Cacciò SM, De Giacomo M, Pozio E. Sequence Analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype giardia duodenalis cysts from human faecal samples. Int J Parasitol 2002; 32(1): 1023-1030. http://dx.doi.org/10.1016/S0020-7519(02)00068-1.
http://dx.doi.org/10.1016/S0020-7519(02)... (1st reaction) and by Lalle et al. (2005)Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Cacciò SM. Genetic heterogeneity at the β-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol 2005; 35(2): 207-213. http://dx.doi.org/10.1016/j.ijpara.2004.10.022. PMid:15710441.
http://dx.doi.org/10.1016/j.ijpara.2004....
(2nd reaction) for the molecular research of Giardia lamblia Table S2 Modifications of the gdh gene amplification protocol established by Cacciò et al. (2008)Cacciò SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J Parasitol 2008; 38(13): 1523-1531. http://dx.doi.org/10.1016/j.ijpara.2008.04.008. PMid:18571176.
http://dx.doi.org/10.1016/j.ijpara.2008.... (1st and 2nd reactions) for the molecular research of Giardia lamblia Table S3 Modifications of the ssu-rRNA gene amplification protocol established by Appelbee et al. (2003)Appelbee AJ, Frederick LM, Heitman TL, Olson ME. Prevalence and genotyping of Giardia duodenalis from beef calves in Alberta, Canada. Vet Parasitol 2003; 112(4): 289-294. http://dx.doi.org/10.1016/S0304-4017(02)00422-3. PMid:12623208.
http://dx.doi.org/10.1016/S0304-4017(02)... (1st reaction) and by Hopkins et al. (1997)Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, Thompson RC. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol 1997; 83(1): 44-51. http://dx.doi.org/10.2307/3284315. PMid:9057695.
http://dx.doi.org/10.2307/3284315...
(2nd reaction) for the molecular research of Giardia lamblia Table S4 Modifications of the tpi gene amplification protocol established by Sulaiman et al. (2003)Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 2003; 9(11): 1444-1452. http://dx.doi.org/10.3201/eid0911.030084. PMid:14718089.
http://dx.doi.org/10.3201/eid0911.030084... (1st and 2nd reactions) for the molecular research of Giardia lamblia

This material is available as part of the online article from https://doi.org/10.1590/S1984-29612023010

Acknowledgements

We would like to thank Ford Harvey for helping with the English version of this paper, Professor Marcelo V. Meireles from São Paulo State University for providing Cryptosporidium parvum positive control DNA, and Professor Giovanni Widmer, Tufts University for his help with the sequencing and the editing of the manuscript. Also, we would like to thank the City of Ilhéus for its collaboration with the collection of samples. Christiane M. B. M. da Rocha is a research fellow from the Brazilian National Council for Scientific and Technological Development (CNPq). This study was financed in part by the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001 and by the Bahia Research Foundation (FAPESB) - (APP0043/2016). George R. Albuquerque is a recipient of productivity fellowships from the National Council for Scientific and Technological Development (CNPq). (Grant 308243/2018-8).

  • How to cite: Harvey TV, Carvalho JPS, Aquino MCC, Oliveira BCM, Barros LD, Fehlberg HF, et al. Giardiasis in children and dogs, and the first report of assemblage E in dogs from northeastern Brazil. Braz J Vet Parasitol 2023; 32(1): e012222. https://doi.org/10.1590/S1984-29612023010

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Publication Dates

  • Publication in this collection
    20 Feb 2023
  • Date of issue
    2023

History

  • Received
    26 Aug 2022
  • Accepted
    15 Dec 2022
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br