Assuntos
Coinfecção , Vírus da Hepatite B , Hepatite B/diagnóstico , Hepatite D/diagnóstico , Vírus Delta da Hepatite , Biomarcadores/sangue , DNA Viral/sangue , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática , Hepatite B/sangue , Hepatite B/etnologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/sangue , Anticorpos Anti-Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite D/sangue , Hepatite D/etnologia , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/imunologia , Humanos , Indígenas Sul-Americanos , Mutação , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos TestesRESUMO
Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Hepatite C/virologia , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Nucleosídeos/análogos & derivadosRESUMO
The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Proteínas de Neoplasias/genética , Tensoativos/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Aminas/química , Aminas/farmacologia , Linhagem Celular Tumoral , Humanos , Ácido Oxâmico/química , Ácido Oxâmico/farmacologia , Reação em Cadeia da PolimeraseRESUMO
The hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in humans. Approximately 5% of the infected people die from cirrhosis or hepatocellular carcinoma. The current standard therapy comprises a combination of pegylated-interferon alpha and ribavirin. Due to the relatively low effectiveness, the prohibitive costs and the extensive side effects of the treatment, an intense research for new direct-acting anti-HCV agents is taking place. Furthermore, NS3 protease inhibitors recently introduced into the market are not effective against all HCV subgenotypes. Thiosemicarbazones (TSCs) have shown antiviral activity against a wide range of DNA and RNA viruses. However, their extremely low aqueous solubility and high self-aggregation tendency often preclude their reliable biological evaluation in vitro. In this work, we investigated and compared for the first time the anti-HCV activity of two 1-indanone TSCs, namely 5,6-dimethoxy-1-indanone TSC and 5,6-dimethoxy-1-indanone N4-allyl TSC, and their inclusion complexes with hydroxypropyl-ß-cyclodextrin (HPß-CD) in Huh-7.5 cells containing the full-length and the subgenomic subgenotype 1b HCV replicon system. Studies of physical stability in culture medium showed that free TSCs precipitated rapidly and formed submicron aggregates. Conversely, TSC complexation with HPß-CD led to more stable systems with minimal size growth and drug concentration loss. More importantly, both TSCs and their inclusion complexes displayed a potent suppression of the HCV replication in both cell lines with no cytotoxic effects. The mechanism likely involves the inhibition of non-structural proteins of the virus. In addition, findings suggested that the cyclodextrin released the drug to the culture medium over time. This platform could be exploited for the study of the drug toxicity and pharmacokinetics animal models.
Assuntos
Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Tiossemicarbazonas/administração & dosagem , beta-Ciclodextrinas/administração & dosagem , 2-Hidroxipropil-beta-Ciclodextrina , Antivirais/química , Linhagem Celular Tumoral , Hepacivirus/fisiologia , Humanos , RNA Viral/análise , Tiossemicarbazonas/química , Replicação Viral/efeitos dos fármacos , beta-Ciclodextrinas/químicaRESUMO
BACKGROUND: Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) polymerase chain reaction (PCR). OBJECTIVES: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. STUDY DESIGN: Forty-six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110-195) and HDAg-L (aa110-214) was performed. RESULTS: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc-only positive, 2 of them identified as cases of occult hepatitis B infection (OBI). The 3 cases were HBeAg-negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 - reportedly known to promote in vitro an unsuitable interaction with HBsAg - were observed. CONCLUSIONS: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.
Assuntos
Hepatite B/complicações , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/sangue , Antígenos da Hepatite delta/genética , Adolescente , Adulto , Idoso , Argentina , Doenças Assintomáticas , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Anticorpos Anti-Hepatite/sangue , Humanos , Imunoglobulina G/sangue , Indígenas Sul-Americanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Adulto JovemRESUMO
BACKGROUND: Hepatitis B virus infection is frequent among Amerindians. In Argentina HBV genotypes A, B, C, D, E, F and H were described in different populations, while some cases of occult hepatitis B infection (OBI) were reported in human immunodeficiency virus (HIV) coinfected patients. OBJECTIVE: To determine the prevalence, genetic diversity of HBV and to analyze the deduced amino acid sequence of both S and viral polymerase (P) genes among Amerindians of Argentina. STUDY DESIGN: A cross-sectional study including 561 individuals belonging to distinct ethnic groups, the Mbyá-guaraní (MG), the Kolla (K), the Sagua-Huarpe (SH) and the Wichi (W) was performed. RESULTS: The prevalence of HBsAg was 1.7% and 1.4% for the MG and SH, respectively, while anti-HBc was detected in all communities. HBV DNA of S/P and preCore-Core genomic regions were amplified by nested polymerase chain reaction in 59 reactive samples for anti-HBc total Ig and/or HBsAg. Of them, thirteen exhibited detectable HBV DNA, eleven of which were identified as OBI. Genotype F was predominant in the MG community with co-circulation of subgenotypes F4, F1b, A2 and D3, while subgenotype C2 prevailed within the SH community. All cases exhibited the polymorphism rtL217R within the RT domain associated to resistance to adefovir. Mutations rtD206E and rtV207I associated with lamivudine resistance were found in two MG and three SH respectively. Other new substitutions were described within the P sequence. CONCLUSIONS: This study shows for the first time the predominance of OBI, HBV subgenotypes and natural variants in Amerindians from Argentina.
Assuntos
DNA Viral/sangue , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B/virologia , Adolescente , Adulto , Idoso , Argentina , Análise por Conglomerados , Estudos Transversais , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Variação Genética , Genótipo , Vírus da Hepatite B/isolamento & purificação , Humanos , Indígenas Sul-Americanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteínas Virais/genética , Adulto JovemRESUMO
Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Polímeros/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Etilenodiaminas/química , Humanos , Peso Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Polietilenoglicóis/química , Polímeros/química , Propídio/metabolismo , Propilenoglicóis/química , Rodamina 123/metabolismo , Vimblastina/metabolismoRESUMO
Hepatitis B virus (HBV) variants may either emerge in patients with chronic hepatitis B (CHB) as a result of positive selection pressure exerted by their own immune response, or during therapy with nucleos(t)ide analogues (NAs). Naturally occurring HBV variants with primary antiviral resistance are rarely observed. The aim of this study was to retrospectively analyze the (eventual) circulation of HBV variants with natural resistance to NAs currently used as therapy for CHB in Argentina. This study reports 13 cases of CHB-infected patients with natural antiviral resistance to at least one NA. Five of them were also carriers of S-variants that might escape the humoral immune system recognition with potential resistance to adefovir. In addition to the already reported A2 HBV subgenotype association to NAs natural resistance, E and F genotypes association to such resistance is described for the first time. These findings suggest that sequence analysis of the HBV reverse transcriptase might be an essential tool before starting antiviral therapy, in order to choose the proper NAs for optimizing the therapeutic management of chronically infected patients. Moreover, the circulation and transmission of S-mutants with resistance to such antiviral drugs should be of public health concern as they may represent an additional risk for the community.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Farmacorresistência Viral , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Mutação de Sentido Incorreto , Organofosfonatos/farmacologia , Adenina/farmacologia , Adolescente , Adulto , Argentina , Criança , Pré-Escolar , Feminino , Genótipo , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Genomic heterogeneity and quasispecies composition of GB virus C (GBV-C) within plasma and lymphocyte subsets in a naturally infected blood donor were investigated. For this purpose, fragments from the 5' untranslated region (5' UTR) and the E2 gene recovered from plasma, B and T lymphocytes, were cloned and sequenced. A total of 63 clones was analysed: 95.2 % of them (n=60) - obtained from plasma and cells - were assigned to genotype 2b, while only three derived from plasma corresponded to genotyope 3. The G215A transition within this region was present in 90.9 % of the clones from B lymphocytes, but absent in the remaining cell compartments (P<0.01). Apparently, most of the circulating GBV-C quasispecies in this blood donor were related to the viral population infecting CD8(+) T cells, and B cells to a lesser extent. This is the first report showing the quasispecies nature of GBV-C in lymphocyte subsets within peripheral blood mononuclear cells.
Assuntos
Infecções por Flaviviridae/virologia , Vírus GB C/classificação , Vírus GB C/genética , Hepatite Viral Humana/virologia , Subpopulações de Linfócitos/virologia , Sequência de Bases , Doadores de Sangue , Especiação Genética , Variação Genética , Humanos , Dados de Sequência Molecular , Filogenia , Proteínas ViraisRESUMO
In spite of the progress made in vaccine and antiviral therapy development, hepatitis B virus (HBV) infection is still the most common cause of liver cirrhosis and hepatocellular carcinoma, with more than 400 million people chronically infected worldwide. Antiviral therapy with nucleos(t)ide analogues and/or immunomodulating peptides is the only option to control and prevent the progression of the disease in chronic hepatitis B (CHB)-infected patients. So far, the current antiviral monotherapy remains unsatisfactory because of the low efficacy and the development of drug resistance mutants. Moreover, viral rebound is frequently observed following therapy cessation, since covalent closed circular DNA (cccDNA) is not removed from hepatocytes by antiviral therapy. First, this review describes the current pharmacotherapy for the management of CHB and the new drug candidates being investigated. Then, the challenges in the development of drug delivery systems for the targeting of antiviral drugs to the liver parenchyma are discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to eradicate the virus from the host are addressed.
Assuntos
Antivirais/administração & dosagem , Hepatite B/tratamento farmacológico , Fígado/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Sistemas de Liberação de Medicamentos , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Fígado/virologiaRESUMO
In this study we determined that the double mutant M460V/D605E in the UL97 gene of an HCMV isolate from an immunocompromised patient (MMT isolate) is related to resistance to ganciclovir (GCV) therapy. Our results suggest that the aspartic acid-to-glutamic acid substitution at codon 605 may be associated with a natural polymorphism of the UL97 gene, and not with positive selection pressure exerted by the antiviral drug. We also determined that GCV resistance due to the M460V mutation in the HCMV UL97 gene is not offset by a second mutation (D605E) at codon 605. Furthermore, we showed that when the two mutations related to GCV resistance were simultaneously detected in the same HCMV construct, virus-drug resistance might be enhanced in comparison to that of the single mutants studied separately. To our knowledge for the first time, seven of 12 amino acid changes (F102L, D118V, M330T, T400A, R507P and C511R and I533V) in the UL97 gene of an isolate are herein reported.
Assuntos
Antivirais/metabolismo , Citomegalovirus/enzimologia , Citomegalovirus/genética , Ganciclovir/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Antivirais/farmacologia , Linhagem Celular , Células Cultivadas , Citomegalovirus/efeitos dos fármacos , Farmacorresistência Viral/genética , Fibroblastos/virologia , Ganciclovir/farmacologia , Humanos , Hospedeiro Imunocomprometido , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polimorfismo GenéticoRESUMO
BACKGROUND: Hepatitis B virus (HBV) immune escape mutants with point mutations within the S gene may arise during the natural course of HBV infection, due to a positive selection pressure exerted by the host immune response. Mutations within the immunodominant B and T cell epitopes of hepatitis B surface antigen (HBsAg) allow the resulting S-mutants to propagate even in the presence of neutralizing anti-HBs antibodies and the HBV-specific T-cell immune response. AIM: To study the antiviral effect of Pegylated-interferon (Peg-IFN) in a patient with chronic hepatitis B carrying unusual S-(and P-) mutants in the presence of anti-HBs antibodies. PATIENTS, METHODS AND RESULTS: We report on a 43-year-old male chronically infected with a genotype A HBV strain, with cocirculation of both HBsAg and anti-HBs antibodies, who received treatment with 120 mug of Peg-IFN for 24 weeks. HBeAg seroconversion and clearance of both HBV DNA by polymerase chain reaction and HBsAg were successfully achieved. Improved histology was observed in a biopsy performed 44 weeks after Peg-IFN therapy was completed. It seems plausible that the ascribed genotype A could have contributed to the effective response to Peg-IFN, even though the treatment was provided only throughout a 24-week period. CONCLUSION: To our knowledge, this is the first report regarding the successful result obtained by using Peg-IFN as a treatment for a chronically HBV-infected patient carrying HBsAg immune escape mutants.
Assuntos
Antivirais/uso terapêutico , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Adulto , Vírus da Hepatite B/imunologia , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Humanos , Interferon alfa-2 , Masculino , Mutação , Polietilenoglicóis , Proteínas RecombinantesRESUMO
Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.
Assuntos
Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Adolescente , Adulto , Argentina , População Negra , Portador Sadio/virologia , Pré-Escolar , Feminino , Genótipo , Hepatite B/imunologia , Hepatite B/prevenção & controle , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B/genética , Humanos , Mutação , Filogenia , VacinaçãoRESUMO
Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of the S and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate to genotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside the major hydrophilic region, while the sixth clone exhibited a genotype A "wild-type" amino acid sequence. Observed replacements included both humoral and/or cellular (major histocompatibility complex class I [MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F, P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within a given clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymous mutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for the emergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the first loop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community, since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively prevent the liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence the accuracy of the results obtained with currently used diagnostic tests.
Assuntos
Epitopos , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutação Puntual , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Epitopos/genética , Produtos do Gene pol/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Masculino , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
The level of in vitro detection of viral genomes in mixes with two different hepatitis C virus (HCV) subtypes was investigated by artificially mixing previously measured subtype-specific HCV RNA genomes. The RNAs in these mixtures were reverse transcribed and then PCR amplified by using two sets of primers corresponding to the 5' untranslated region and digested with endonucleases to analyze the restriction fragment length polymorphism patterns. This approach facilitated detection of a wider range of type-specific HCV genomes than originally described, beyond equimolar concentrations of contributing HCV subtypes. Moreover, by using computerized image analysis, this study also demonstrated that the true contribution of each virus type-and consequently of mixed infections-may be underestimated when only visual observation is carried out. These results may be useful for comparing data obtained from this and other currently used methodologies.