RESUMO
Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five "single-copy" probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.
Assuntos
Entamoeba/genética , Entamebíase/epidemiologia , Actinas/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Quitinases/genética , Demografia , Diploide , Entamoeba/citologia , Entamoeba histolytica/citologia , Entamoeba histolytica/genética , Humanos , Hibridização in Situ Fluorescente , Índia/epidemiologia , Íntrons , México/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SerinaAssuntos
Disenteria Amebiana/parasitologia , Entamoeba histolytica/isolamento & purificação , Entamoeba/isolamento & purificação , Fezes/parasitologia , Animais , Sequência de Bases , Criança , Disenteria Amebiana/epidemiologia , Humanos , México/epidemiologia , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
We used the polymerase chain reaction (PCR) to study the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica in a rural community in Mexico. Formalin-fixed stool samples were used for extraction of DNA. The PCR amplifications were performed using two sets of primers that discriminate between pathogenic or non-pathogenic E. histolytica. A total of 201 randomly selected individuals were studied. Among them, 25 (12%) were diagnosed to be infected with E. histolytica by microscopy; PCR identified 24 of these as positive (sensitivity = 0.96) and of 176 negative individuals, only three were identified as positive (specificity = 0.98). The PCR analysis defined three populations: 14 cases were positive for both pathogenic and nonpathogenic E. histolytica, nine cases were positive for pathogenic and negative for nonpathogenic E. histolytica, and only one case was negative for pathogenic and positive for nonpathogenic E. histolytica. Infection by E. histolytica was strongly associated to infection with Entamoeba coli (odds ratio [OR] = 9.41, 95% confidence interval [CI] = 3.09, 28.65, P < 0.0004) and Endolimax nana (OR = 6.15, 95% CI = 2.03, 18.17, P < 0.0004). This new technique has high specificity and sensitivity; it is simple, reproducible, fast, avoids the need to culture trophozoites, and can be applied in the field for epidemiologic studies.
Assuntos
DNA de Protozoário/análise , Disenteria Amebiana/epidemiologia , Entamoeba histolytica/patogenicidade , Adolescente , Adulto , Amebíase/complicações , Animais , Sequência de Bases , Criança , Pré-Escolar , DNA de Protozoário/química , Disenteria Amebiana/complicações , Disenteria Amebiana/parasitologia , Endolimax/isolamento & purificação , Entamoeba histolytica/genética , Fezes/parasitologia , Feminino , Humanos , Lactente , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , População Rural , Sensibilidade e EspecificidadeRESUMO
E. histolytica parasites in Mexican children's stools were identified and typed as pathogenic or non-pathogenic using the polymerase chain reaction (PCR) and nonradioactive probes. PCRs were performed with primers specific for 145 base pair (bp) pathogenic or 133 bp non-pathogenic DNA sequences, which are highly repeated in E. histolytica parasites with pathogenic or non-pathogenic isoenzyme patterns, respectively. Dot-blotted PCR products were identified with a horseradish peroxidase-conjugated oligonucleotide probe specific for either the 145 bp pathogenic or 133 bp non-pathogenic sequences. The PCR and the 145 bp pathogenic probe correctly identified eight cultures with pathogenic isoenzyme types and none of nine cultures with non-pathogenic isoenzyme. The PCR and 133 bp non-pathogenic probe identified all of the non-pathogenic cultures, none of the axenized pathogenic cultures, and three of five xenic cultures with pathogenic isoenzymes. The two probes together identified all 49 stools containing E. histolytica by light microscopy (sensitivity = 1.0), which represented the entire set of the E. histolytica-positive stools diagnosed at the Hospital Infantil over a 10 week period. Most patient isolates were positive with both 145 bp pathogenic and 133 bp non-pathogenic probes, suggesting that these children, 60% of whom were dysenteric, are infected with mixed populations of amebas.
Assuntos
Sondas de DNA , Disenteria Amebiana/diagnóstico , Entamoeba histolytica/isolamento & purificação , Reação em Cadeia da Polimerase , Animais , Sequência de Bases , Países em Desenvolvimento , Disenteria Amebiana/epidemiologia , Disenteria Amebiana/parasitologia , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Fezes/parasitologia , Humanos , Incidência , Isoenzimas/análise , México/epidemiologia , Dados de Sequência Molecular , Prevalência , Proteínas de Protozoários/análise , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
To development a reliable murine model of Leishmania braziliensis braziliensis infection, parasites were injected into BALB/c mice in the presence of phlebotomine sand fly salivary gland lysates, which have previously been shown to greatly increase the infectivity of L. major in mice. When injected with salivary gland lysates, L. braziliensis braziliensis produced progressively enlarging cutaneous nodules, containing many macrophages filled with Leishmania amastigotes. In contrast, L. braziliensis injected without gland extracts produced small and rapidly regressing lesions. Isoenzyme analysis, monoclonal antibodies, and the polymerase chain reaction with L. braziliensis-specific oligonucleotide primers and probes confirmed that parasites causing the lesions were L. braziliensis.
Assuntos
Leishmania braziliensis/patogenicidade , Leishmaniose Mucocutânea/transmissão , Psychodidae/fisiologia , Animais , Anticorpos Monoclonais , Sequência de Bases , Sondas de DNA , DNA de Protozoário/análise , Modelos Animais de Doenças , Isoenzimas/análise , Leishmania braziliensis/genética , Leishmaniose Mucocutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Saliva/fisiologia , Glândulas Salivares/fisiologiaRESUMO
Although drug therapy is critical for control of amoebiasis, little is known about mechanisms of drug resistance by E. histolytica parasites. Here we tested the hypothesis that multidrug resistant (mdr) amoeba mutants, similar to mdr tumor cells, are drug resistant based upon overexpression of a P-glycoprotein pump that effluxes drugs from the cells. Using primers to conserved regions of the human P-glycoprotein and the polymerase chain reaction (PCR), we identified multiple 344 base par segments of amoeba DNA similar to the mammalian P glycoprotein. The amino acid sequences of amoeba mdr-like PCR products were from 53 to 97 identical with each other, 55 to identical to human mdr1 sequences, and 41-44% identical with P. falciparum mdr-like sequences. On northern blots, the mdr-like PCR products identified amoeba mRNAs 4.5-5 kilobases long, similar to the 5 kilobase mRNAs reported for the mammalian mdr gene. These mRNAs were increased at least seven times in emetine resistant mutant clone C2 amoebae versus wild-type clone A parasites. Further, the expression of the mdr-like mRNAs was increased three to four times when clone C2 mutants were grown under drug pressure versus the same parasites grown without emetine. In contrast, the number of genomic copies of the mdr-like DNA segments was not increased in the mutant clone C2 versus the wild-type clone A amoebae, and no rearrangements of the mdr-like DNA segments by the mutant were identified on Southern blots. In conclusion there appears to be a family of mdr-like genes in E. histolytica, which may be involved in drug resistance by the parasite.
Assuntos
Emetina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Resistência a Medicamentos , Entamoeba histolytica/genética , Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieAssuntos
Colchicina/farmacologia , Emetina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Verapamil/farmacologia , Animais , DNA de Protozoário/genética , Interações Medicamentosas , Resistência a Medicamentos , Entamoeba histolytica/genética , Entamoeba histolytica/crescimento & desenvolvimento , Amplificação de Genes , Hibridização de Ácido NucleicoRESUMO
To evaluate the safety and immunogenicity of the Haemophilus influenzae type b polysaccharide vaccine, PRP, and a new polysaccharide-diphtheria toxoid conjugate vaccine, PRP-D, a collaborative study was carried out in six centers in five states. Subjects were 585 infants 15 to 24 months of age. They were randomly assigned to receive a single dose of PRP or PRP-D vaccine. There were no significant differences in the rate of adverse reactions between the two vaccine groups. Minor local reactions occurred in 10.3% of PRP and 12.5% of PRP-D recipients, and fever in 27.4% of PRP and 23.8% of PRP-D recipients. All reactions resolved within 48 hours. Serum samples were obtained just before vaccination and after 1 month. Prevaccination antibody levels were similar for the PRP (0.035 micrograms/mL) and PRP-D (0.027 micrograms/mL) groups, with no differences in levels by age, sex, race, vaccine lot, or study site. Both groups had significant rises in geometric mean levels, but this difference was significantly greater for PRP-D (2.166 micrograms/mL) than for PRP (0.154 micrograms/mL). In addition, the percentage of responders as determined by three definitions (twofold titer rise, greater than 0.15 micrograms/mL, and greater than 1.0 micrograms/mL) was also significantly greater for PRP-D than PRP. In contrast to a marked age-related immunogenicity to PRP (P less than 0.001), there was no significant variation in immune response to PRP-D by age. PRP-D conjugate vaccine appears to be as safe and significantly more immunogenic than PRP vaccine for children vaccinated at 15 to 24 months of age.
Assuntos
Vacinas Bacterianas/imunologia , Toxoide Diftérico/imunologia , Haemophilus influenzae/imunologia , Vacinação , Fatores Etários , Vacinas Bacterianas/efeitos adversos , Ensaios Clínicos como Assunto , Toxoide Diftérico/efeitos adversos , Feminino , Humanos , Lactente , Masculino , Distribuição AleatóriaRESUMO
At approximately 2 years of age, 27 infants previously immunized at 9 to 15 months of age with two doses of polyribosylribitol phosphate-diphtheria toxoid conjugate vaccine (PRP-D) and 23 infants immunized with polyribosylribitol phosphate (PRP) vaccine were given a single injection of PRP-D. Pre- and post-immunization sera were obtained. No serious local or systemic reactions were observed. The PRP-D recipients had a geometric mean anti-PRP antibody level of 4.8 micrograms/ml 1 month after the second primary injection, retained 1.2 microgram/ml 1 year later, and had a level of 71 micrograms/ml after the booster immunization. In contrast, PRP recipients had a geometric mean level of 0.083 microgram/ml 1 month after the second primary injection, retained 0.042 microgram/ml 1 year later, and after a single dose of PRP-D at approximately 2 years of age had a geometric mean level of 8.6 micrograms/ml. The significantly higher antibody response in the prior PRP-D recipients suggests the recall of immunologic memory induced by the PRP-D vaccine.