• View in gallery

    PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (CO1): Lane 1, size marker; lane 2, HP1; lane 3, HP2; lane 4, HP3; lane 5, HP4; lane 6, HP5; lane 7, HP6; lane 8, HP7; lane 9, HP8; lane 10, HP9; lane 11, positive control; lane 12, negative control.

  • View in gallery

    Scheme of CO1 and DCO1 attach primers site. This figure appears in color at www.ajtmh.org.

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    • Search Google Scholar
    • Export Citation
  • 2

    Khuroo MS, 2002. Hydatid disease: current status and recent advances. Ann Saudi Med 22 :56–64.

  • 3

    Xiao N, Qiu J, Nakao M, Li T, Yang W, Chen X, Schantz PM, Craig PS, Ito A, 2006. Echinococcus shiquicus, a new species from the Qinghai–Tibet plateau region of China: discovery and epidemiological implications. Parasitol Int 55 (Suppl):S233–S236.

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    • Export Citation
  • 4

    McManus DP, Zhang W, Li J, Bartley PB, 2003. Echinococcosis. Lancet 362 :1295–1304.

  • 5

    Moro PL, Gilman RH, Verastegui M, Bern C, Silva B, Bonilla JJ, 1999. Human hydatidosis in the central Andes of Peru: evolution of the disease over 3 years. Clin Infect Dis 29 :807–812.

    • Search Google Scholar
    • Export Citation
  • 6

    Moro PL, McDonald J, Gilman RH, Silva B, Verastegui M, Malqui V, Lescano G, Falcon N, Montes G, Bazalar H, 1997. Epidemiology of Echinococcus granulosus infection in the central Peruvian Andes. Bull World Health Organ 75 :553–561.

    • Search Google Scholar
    • Export Citation
  • 7

    Moro PL, Schantz PM, 2006. Echinococcosis: historical landmarks and progress in research and control. Ann Trop Med Parasitol 100 :703–714.

    • Search Google Scholar
    • Export Citation
  • 8

    Daniel Mwambete K, Ponce-Gordo F, Cuesta-Bandera C, 2004. Genetic identification and host range of the Spanish strains of Echinococcus granulosus. Acta Trop 91 :87–93.

    • Search Google Scholar
    • Export Citation
  • 9

    McManus DP, Rishi AK, 1989. Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes. Parasitology 99 :17–29.

    • Search Google Scholar
    • Export Citation
  • 10

    McManus DP, Thompson RC, 2003. Molecular epidemiology of cystic echinococcosis. Parasitology 127 (Suppl):S37–S51.

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  • 12

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    • Search Google Scholar
    • Export Citation
  • 13

    Guarnera EA, Parra A, Kamenetzky L, Garcia G, Gutierrez A, 2004. Cystic echinococcosis in Argentina: evolution of meta-cestode and clinical expression in various Echinococcus granulosus strains. Acta Trop 92 :153–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Haag KL, Ayala FJ, Kamenetzky L, Gutierrez AM, Rosenzvit M, 2004. Livestock trade history, geography, and parasite strains: the mitochondrial genetic structure of Echinococcus granulosus in Argentina. J Parasitol 90 :234–239.

    • Search Google Scholar
    • Export Citation
  • 15

    Kamenetzky L, Gutierrez AM, Canova SG, Haag KL, Guarnera EA, Parra A, Garcia GE, Rosenzvit MC, 2002. Several strains of Echinococcus granulosus infect livestock and humans in Argentina. Infect Genet Evol 2 :129–136.

    • Search Google Scholar
    • Export Citation
  • 16

    Rosenzvit MC, Zhang LH, Kamenetzky L, Canova SG, Guarnera EA, McManus DP, 1999. Genetic variation and epidemiology of Echinococcus granulosus in Argentina. Parasitology 118 :523–530.

    • Search Google Scholar
    • Export Citation
  • 17

    Cruz-Reyes A, Constantine CC, Boxell AC, Hobbs RP, Thompson RC, 2007. Echinococcus granulosus from Mexican pigs is the same strain as that in Polish pigs. J Helminthol 81 :287–292.

    • Search Google Scholar
    • Export Citation
  • 18

    Bartholomei-Santos ML, Heinzelmann LS, Oliveira RP, Chemale G, Gutierrez AM, Kamenetzky L, Haag KL, Zaha A, 2003. Isolation and characterization of microsatellites from the tapeworm Echinococcus granulosus. Parasitology 126 :599–605.

    • Search Google Scholar
    • Export Citation
  • 19

    Cabrera M, Canova S, Rosenzvit M, Guarnera E, 2002. Identification of Echinococcus granulosus eggs. Diagn Microbiol Infect Dis 44 :29–34.

    • Search Google Scholar
    • Export Citation
  • 20

    da Silva CM, Ferreira HB, Picon M, Gorfinkiel N, Ehrlich R, Zaha A, 1993. Molecular cloning and characterization of actin genes from Echinococcus granulosus. Mol Biochem Parasitol 60 :209–219.

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Human Hydatid Disease in Peru Is Basically Restricted to Echinococcus granulosus Genotype G1

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  • 1 Department of Microbiology, School of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru; Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, “ANLIS Dr. Carlos G. Malbrán”, Buenos Aires, Argentina; Cysticercosis Unit, Instituto Nacional de Ciencias Neurológicas, Lima, Peru; Thoracic and Cardiovascular Surgery Program, Hospital Nacional Dos de Mayo, Lima, Peru; School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru; Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland

A molecular PCR study using DNA from 21 hydatid cysts was performed to determine which strain type is responsible for human infection in Peru. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was amplified in 20 out of 21 samples, revealing that all but 1 sample (19/20, 95%) belonged to the common sheep strain (G1). The remaining samples belonged to the camel strain (G6). The G1 genotype was most frequently found in human cases of cystic hydatid disease (CHD) in Peru. Local control measures should focus primarily on decreasing dog and sheep infection rather than intermediate reservoirs.

INTRODUCTION

All the 5 recognized species within the genus Echinococcus require 2 hosts to perpetuate their life cycle: a carnivore as the definitive host, which carries the adult egg-producing tapeworm, and a herbivore as the intermediate host in which larval metacestode stages establish and develop, causing hydatid disease. Echinococcus granulosus causes cystic hydatid disease (CHD), Echinococcus multilocularis causes alveolar hydatid disease, Echinococcus oligarthus and Echinococcus vogeli both cause polycystic hydatid disease, and Echinococcus shiquicus causes unilocular minicyst hydatid disease.13 Humans can act as intermediary hosts of the first 4 species, with diverse clinical presentations depending on the affected organ and type of larvae.

Cystic hydatid disease is an important and widespread zoonosis, especially in sheep-raising areas of Europe (Mediterranean countries), Asia (Russia, China), North and East Africa, Australia, and South America (Peru, Bolivia, Argentina, Chile, Uruguay, and Rio Grande do Sul state in Brazil). It affects the liver (52–77% of cases), lung (9–44%), and other organs such as brain, heart, and bones.46 CHD is a major public health problem in Peru, with a prevalence of 6–9% in many areas of the country and numerous human cases reported every year.6,7

Around the world, strain-typing surveys have shown that human infection is mostly often by the common sheep strain (G1) in mainland Australia, Tasmania, Jordan, Lebanon, Holland, Kenya, China, and Spain.811 G1 may coexist with other strains, such as cattle strain (G5) in Holland; camel strain (G6) in Nepal, Iran, and Mauritania; porcine strain (G7) in Poland and Slovakia; and cervid strain (G8) in the United States. When multiple strains are present, they may infect atypical intermediate hosts; e.g., G5 infection in sheep and goats in Nepal and G7 beaver infection in Poland.10,12 In Argentina, human infections are caused by strains G1, G2, G5, and G6.1316 There is little information available on strain composition of hydatid disease in other Latin American countries.17,18 We carried out a survey using a PCR analysis and CO1 sequencing of E. granulosus isolates collected from humans to determine the E. granulosus strains that infect humans in Peru.

MATERIALS AND METHODS

This study was performed in Lima, Peru, at the Hospital Nacional Dos de Mayo (a government referral center for treatment of hydatid disease), using cyst material excised from patients who had surgery for CHD during the period March 2006–January 2007. Immediately after excision, the specimen was placed in ethanol (70%), stored at 4°C, and processed within 2 days of collection.

Macroscopic information on the appearance, size, and status of the larvae was collected from surgical reports. The nature and fertility of the sample were confirmed by microscopic observation of E. granulosus protoscoleces. Each cyst was separated into membrane and intracystic fluid with protoscoleces (hydatid sand). The germinal layer was washed 3 times in ethanol to remove any contaminant (debris, blood, host tissue), and both membrane and hydatid sand were preserved submerged in 70% ethanol and stored at −20°C. Samples were sent to Departamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, ANLIS, in Buenos Aires, Argentina, for strain identification. There, total E. granulosus DNA was extracted using the DNeasy Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Purified DNA samples were stored at −20°C until their use in PCR reactions. E. granulosus genotype was determined by mitochondrial cytochrome c oxidase subunit 1 (CO1) sequencing, as previously described.15 The sequences were determined at the Facultad de Ciencias Exactas y Naturales, UBA, in Buenos Aires (USFCEyN).

Additional PCR reactions performed were amplification of the DCO1 mitochondrial fragment using the set of primers DCO1F and DCO1R as previously described by Cabrera and others19; amplification of the E. granulosus actin gene as described by da Silva and others20; and amplification of an E. granulosus repetitive DNA element as described by Abbasi and others.21

RESULTS

We analyzed a total of 21 cysts from 21 individuals. The majority of individuals (N = 18) came from villages in the Central Peruvian Highlands, with altitudes varying between 3000 and 4500 m above sea level. Villages in the area have similar ecology, agriculture, and livestock. Of the 21 cysts, 19 were lung cysts and 2 were liver cysts. Seven cysts showed evidences of complication (2 infected and 5 ruptured), and 4 cysts had daughter cysts. The mean volume was 586.68 ± 627.46 mL (range 8–2250 mL) (Table 1). Preserved protoscoleces were seen under the microscope in 8 cysts. In the other 13, parasite cells, degenerated protoscoleces, and/or parasite structures—e.g., hooks—were observed. The CO1 gene was amplified in 20 out of 21 samples (Figure 1).

A second reaction of PCR-CO1 with addition of an internal E. granulosus DNA control was carried out in the nonamplifying sample. Because a control band of the expected size was obtained, we ruled out the presence of inhibitors in the sample. Also, a second reaction to amplify a more internal region of the cytochrome c oxidase subunit 1 gene was performed by using DCO1 primers to determine if the absence of amplification was produced by substitutions in the CO1 annealing primers site. Again, no amplification products were obtained. To confirm the identity and quality of the extracted DNA from this sample, 2 reactions using different primers were performed (1 for the constitutive gene actin and 1 for an E. granulosus-specific repetitive DNA element). In both cases, we obtained the expected amplification product (Figure 2). Details on these reactions are provided in the supplemental online material at www.ajtmh.org.

Sequencing of the mitochondrial CO1 gene confirmed that all the 20 cysts whose material was amplified were E. granulosus metacestodes. All but 1 sample (19; 95%) belonged to the common sheep strain (G1). The remaining sample belonged to the camel strain (G6) (Table 1).

DISCUSSION

Using sequencing of the mitochondrial CO1 gene, we demonstrated a clear predominance of the common sheep/dog strain (G1), with a single isolate of camel/dog strain (G6) of E. granulosus in Peruvian CHD human cases. We could not identify the reason why 1 sample did not amplify despite being confirmed as E. granulosus DNA by other molecular markers. Because inhibition was shown to be unlikely, a possible explanation would be the presence of a mutation in the CO1 gene.

To date, 10 distinct well-characterized genetic intraspecific variants are recognized within E. granulosus (genotypes G1–10), based on polymerase chain reaction (PCR) amplification by sequencing mitochondrial markers in cytochrome c oxidase 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase 1 (ND1) genes. Seven of them are infectious to humans2225 (Table 2). There appears to be very limited genetic variation within E. multilocularis, and there are no available data to assess sequencing variability in E. vogeli, E. oliganthus, or E. shiquicus. Intraspecific variants or “strains” may play an important role with regard not only to life-cycle patterns and host assemblages but also to transmission dynamics, control of disease, pathogenicity, fertility of developed cysts, and rate of growth.1,13,16,23,2631

Although the number of Peruvian isolates examined was not extensive, the G1 genotype was far more prevalent in humans than the G6 genotype. The common sheep strain, G1, is widely reported as cause of human infection in Southern and Eastern Europe, Northern and Eastern Africa, parts of Asia, Australia, and South America (Argentina). Although it predominantly affects sheep, in a few cases, G1 infection of other intermediary hosts, such as cattle and goat, has been described.13,15,16,27 On the other hand, G6, typically a camel strain, has also been reported in cattle.32,33 In Argentina, this strain may contribute for up to 37% of human CHD cases, second to G1 infection with 46%.13 Our examined samples came from the Peruvian Central Highlands, which comprise approximately 70% of the endemic areas for CHD in Peru. Although it is possible that samples from the Southern Highlands (Puno, Cusco) near Bolivia and Chile could have different patterns, we consider it unlikely given the high similarities in terms of ecology, altitude, behavior, and livestock raised.

G1 is the commonest strain in CHD human cases worldwide. Its predominance supports that the endemicity of E. granulosus in the Peruvian highlands is based on a sheep/dog cycle. This is highly consistent with its geographical pattern, overlapping major sheep raising areas between 3200 and 4500 meters of altitude. This information provides support to concentrate control measures in Peru to decrease dog and sheep infection rates in preference to working on other intermediate reservoirs.

Table 1

Localization and characteristics of the hydatid cysts related with Echinococcus granulosus strain

HPOrgan affectedGeographic locationTypeDaughter cystVolume (mL)Strain
LLL = left lower lobe; RHL = right hepatic lobe; RUL = right upper lobe; LUL = left upper lobe; RLL = right lower lobe; “—”= strain could not be determined.
* Patients without abdominal ultrasound or CT scan.
1Lung* (LLL)PascoHyalineNo810G1
2lung (LLL)JuninHyalineNo441G1
3Lung (LLL)AyacuchoBrokenYes2250G1
4Liver (RHL)PascoHyalineNo100G1
5Lung (RUL)JuninHyalineNo384G1
6Lung (LLL)HuancavelicaBrokenNo90G6
7Liver (RHL)JuninInfectedNo216G1
8Lung (RUL)LimaBrokenNo96G1
9Lung* (LLL)JuninHyalineNo595G1
10Lung (RUL)AyacuchoHyalineNo576G1
11Lung* (LUL)PascoInfectedYes420
12Lung (RLL)PascoHyalineNo2085G1
13Lung (LLL)LimaHyalineNo125G1
14Lung (RUL)PascoHyalineYes448G1
15Lung (LLL)HuancavelicaHyalineNo1500G1
16Lung (RUL)JuninBrokenNo770G1
17Lung (RLL)JuninBrokenYes80G1
18Lung (RLL)JuninHyalineNo576G1
19Lung (ML)LimaHyalineNo8G1
20Lung (LUL)JuninHyalineNo175G1
21Lung* (LLL)AyacuchoHyalineNo576G1
Table 2

Characteristics of different Echinococcus granulosus genotypes

Genotype (strain)*Definitive hostIntermediary hostHuman infectivityPrepatent period
* Genotype (strain), determined by molecular techniques; “?”, indetermined or low number of analyzed sample (see Refs. 1, 10, 16, 24, 26, and 3439).
G1 (common sheep strain)Dog, fox, dingo, wolf jackal, hyenaSheep, cattle, goat, buffalo, camel, pig, kangaroo.Yes45 days
G2 (Tasmanian sheep strain)DogSheep, cattleYes39 days
G3 (buffalo strain)Dog, fox?Buffalo, cattle???
G4 (horse strain)DogHorse, donkeysNoMore than G1
G5 (cattle strain)DogCattle, sheep, goat, buffaloYes33–35 days
G6 (camel strain)DogCamel, goat, cattle, sheepYes40 days
G7 (pig strain)Dog (fox?)Pig, wild boar, beaverYes34 days
G8 (cervid strain)Wolf, dogMooseYes?
G9?Pig?Yes?
G10 (Finland cervid strain)?Moose??
Figure 1.
Figure 1.

PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (CO1): Lane 1, size marker; lane 2, HP1; lane 3, HP2; lane 4, HP3; lane 5, HP4; lane 6, HP5; lane 7, HP6; lane 8, HP7; lane 9, HP8; lane 10, HP9; lane 11, positive control; lane 12, negative control.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 1; 10.4269/ajtmh.2008.79.89

Figure 2.
Figure 2.

Scheme of CO1 and DCO1 attach primers site. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 1; 10.4269/ajtmh.2008.79.89

*

Address correspondence to Hector H. Garcia, Department of Microbiology, Universidad Peruana Cayetano Heredia, Av. H. Delgado 430, SMP, Lima 31, Peru. E-mail: hgarcia@jhsph.edu

These authors contributed equally to this work.

Authors’ addresses: Saul J. Santivañez and Hector H. Garcia, Department of Microbiology, School of Sciences, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 31, Peru, Tel: 511-3287360, Fax: 511-3284038, E-mail: hgarcia@jhsph.edu. Mara C. Rosenzvit, Patricia M. Muzulin, and Ariana M. Gutierrez, Departmento de Parasitologia, Instituto Nacional de Enfermedades Infecciosas, “ANLIS Dr. Carlos G. Malbrán”, Av. Velez Sarsfield 563, 1281 Buenos Aires, Argentina. Mary L. Rodriguez and Silvia Rodriguez, Cysticercosis Unit, Instituto Nacional de Ciencias Neurológicas, Ancash 1271, Lima 01, Peru. Julio C. Vasquez, Thoracic and Cardiovascular Surgery Program, Hospital Nacional Dos de Mayo, Lima, Peru. Armando E. Gonzalez, School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru. Robert H. Gilman, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.

Acknowledgments: The authors thank the cooperation of medical personnel from Thoracic and Cardiovascular Surgery Program of the Hospital Nacional Dos de Mayo. We also appreciate the assistance and cooperation of personnel from The Cysticercosis Unit of Instituto Nacional de Ciencias Neurologicas.

Financial support: This work was partially supported by NIAID/NIH (grant P01AI051976), Fogarty/NIH (grants DW43001140 and DW43006581), and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto Nacional de Enfermedades Infecciosas (INEI, ANLIS) “Dr. Carlos G. Malbrán”, and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT).

REFERENCES

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    Eckert J, Thompson RC, 1997. Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans. Acta Trop 64 :19–34.

    • Search Google Scholar
    • Export Citation
  • 2

    Khuroo MS, 2002. Hydatid disease: current status and recent advances. Ann Saudi Med 22 :56–64.

  • 3

    Xiao N, Qiu J, Nakao M, Li T, Yang W, Chen X, Schantz PM, Craig PS, Ito A, 2006. Echinococcus shiquicus, a new species from the Qinghai–Tibet plateau region of China: discovery and epidemiological implications. Parasitol Int 55 (Suppl):S233–S236.

    • Search Google Scholar
    • Export Citation
  • 4

    McManus DP, Zhang W, Li J, Bartley PB, 2003. Echinococcosis. Lancet 362 :1295–1304.

  • 5

    Moro PL, Gilman RH, Verastegui M, Bern C, Silva B, Bonilla JJ, 1999. Human hydatidosis in the central Andes of Peru: evolution of the disease over 3 years. Clin Infect Dis 29 :807–812.

    • Search Google Scholar
    • Export Citation
  • 6

    Moro PL, McDonald J, Gilman RH, Silva B, Verastegui M, Malqui V, Lescano G, Falcon N, Montes G, Bazalar H, 1997. Epidemiology of Echinococcus granulosus infection in the central Peruvian Andes. Bull World Health Organ 75 :553–561.

    • Search Google Scholar
    • Export Citation
  • 7

    Moro PL, Schantz PM, 2006. Echinococcosis: historical landmarks and progress in research and control. Ann Trop Med Parasitol 100 :703–714.

    • Search Google Scholar
    • Export Citation
  • 8

    Daniel Mwambete K, Ponce-Gordo F, Cuesta-Bandera C, 2004. Genetic identification and host range of the Spanish strains of Echinococcus granulosus. Acta Trop 91 :87–93.

    • Search Google Scholar
    • Export Citation
  • 9

    McManus DP, Rishi AK, 1989. Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes. Parasitology 99 :17–29.

    • Search Google Scholar
    • Export Citation
  • 10

    McManus DP, Thompson RC, 2003. Molecular epidemiology of cystic echinococcosis. Parasitology 127 (Suppl):S37–S51.

  • 11

    Sadjjadi SM, 2006. Present situation of echinococcosis in the Middle East and Arabic North Africa. Parasitol Int 55 (Suppl):S197–S202.

  • 12

    Zhang LH, Joshi DD, McManus DP, 2000. Three genotypes of Echinococcus granulosus identified in Nepal using mitochondrial DNA markers. Trans R Soc Trop Med Hyg 94 :258–260.

    • Search Google Scholar
    • Export Citation
  • 13

    Guarnera EA, Parra A, Kamenetzky L, Garcia G, Gutierrez A, 2004. Cystic echinococcosis in Argentina: evolution of meta-cestode and clinical expression in various Echinococcus granulosus strains. Acta Trop 92 :153–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Haag KL, Ayala FJ, Kamenetzky L, Gutierrez AM, Rosenzvit M, 2004. Livestock trade history, geography, and parasite strains: the mitochondrial genetic structure of Echinococcus granulosus in Argentina. J Parasitol 90 :234–239.

    • Search Google Scholar
    • Export Citation
  • 15

    Kamenetzky L, Gutierrez AM, Canova SG, Haag KL, Guarnera EA, Parra A, Garcia GE, Rosenzvit MC, 2002. Several strains of Echinococcus granulosus infect livestock and humans in Argentina. Infect Genet Evol 2 :129–136.

    • Search Google Scholar
    • Export Citation
  • 16

    Rosenzvit MC, Zhang LH, Kamenetzky L, Canova SG, Guarnera EA, McManus DP, 1999. Genetic variation and epidemiology of Echinococcus granulosus in Argentina. Parasitology 118 :523–530.

    • Search Google Scholar
    • Export Citation
  • 17

    Cruz-Reyes A, Constantine CC, Boxell AC, Hobbs RP, Thompson RC, 2007. Echinococcus granulosus from Mexican pigs is the same strain as that in Polish pigs. J Helminthol 81 :287–292.

    • Search Google Scholar
    • Export Citation
  • 18

    Bartholomei-Santos ML, Heinzelmann LS, Oliveira RP, Chemale G, Gutierrez AM, Kamenetzky L, Haag KL, Zaha A, 2003. Isolation and characterization of microsatellites from the tapeworm Echinococcus granulosus. Parasitology 126 :599–605.

    • Search Google Scholar
    • Export Citation
  • 19

    Cabrera M, Canova S, Rosenzvit M, Guarnera E, 2002. Identification of Echinococcus granulosus eggs. Diagn Microbiol Infect Dis 44 :29–34.

    • Search Google Scholar
    • Export Citation
  • 20

    da Silva CM, Ferreira HB, Picon M, Gorfinkiel N, Ehrlich R, Zaha A, 1993. Molecular cloning and characterization of actin genes from Echinococcus granulosus. Mol Biochem Parasitol 60 :209–219.

    • Search Google Scholar
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Author Notes

Reprint requests: Hector H. Garcia, Department of Microbiology, Universidad Peruana Cayetano Heredia, Av. H. Delgado 430, SMP, Lima 31, Peru, Tel: 511-3287360, Fax: 511-3284038, E-mail: hgarcia@jhsph.edu.
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