Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes.

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.

Anti-Plasmodium falciparum invasion ligand antibodies in a low malaria transmission region, Loreto, Peru.

BACKGROUND: Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals. METHODS: ELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8). RESULTS: Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. CONCLUSIONS: These data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.

Frecuencia de mutaciones encontradas en niños con enfermedad renal crónica por síndrome nefrótico cortico-resistente / Frequency of mutations found in children with chronic kidney disease due to steroid-resistant nephrotic syndrome

RESUMEN La enfermedad renal crónica en niños puede tener como etiología un grupo cuya causa genética, incluye las anomalías congénitas del riñón y las vías urinarias y las nefropatías hereditarias. Objetivo: Describir la frecuencia de mutaciones en niños con síndrome nefrótico corticoresistente (SNRE). Material y métodos: Estudio multicéntrico, tipo serie de casos, realizado en niños con SNRE, mediante el resultado de secuenciamiento directo de los genes; NPHS1, NPHS2, NPHP1 y WT1. Resultados: Se enrolaron 33 pacientes pediátricos con enfermedad renal crónica, 45,5% mujeres, edad promedio 13±7 años, 78,8% de raza mestiza, 24,2% consanguíneos, 60,6% se encontraban en hemodiálisis, 72,7% presentaban síndrome nefrótico cortico resistente y de ellos 8/24 (33,3%) presentó al menos una mutación en los genes; WT1, NPHS1, NPHP1y NPHS2 siendo la frecuencia de estas mutaciones de 37,5% (3/8), 25% (2/8), 25% (2/8) y 12,5% (1/8), respectivamente. Conclusiones: El 33,3% de los pacientes pediátricos con enfermedad renal crónica con síndrome nefrótico corticoresistente (SNRE) presentaron mutaciones, la más frecuente fue en el gen WT1.

SUMMARY Chronic kidney disease in children may be caused by a group of genetic abnormalities of the kidney, urinary tract and hereditary nephropathies. Objective: To report the frequency of mutations in children with steroid-resistant nephrotic syndrome (SRNS). Methods: A multicentric case series among children with SRNS identified through direct sequencing of NPHS1, NPHS2, NPHP1 and WT1 genes. Results: 33 children were enrolled; 45.5% were females; mean age was 13±7 years; 78.8% were mestizo: 24.2% consanguineous; 60.6% were receiving dialysis: 72.7% had SRNS and 8/24 (33.3%) of them presented at least one mutation to WT1, NPHS1, NPHP1 and NPHS2 genes. Corresponding values for these mutations were 37.5% (3/8), 25% (2/8), 25% (2/8) and 12.5% (1/8), respectively. Conclusions: 33% of pediatric patients with SRNS presented gene mutations, the most frequent of these mutations was WT1.

Evaluation of Fas2-ELISA for the serological detection of Fasciola hepatica infection in humans.

The performance of Fas2-ELISA for the diagnosis of Fasciola hepatica infection in children living in areas of high endemicity for fascioliasis in the Peruvian Andes is analyzed. Fas2-ELISA is based on the detection of circulating IgG antibodies elicited in infected individuals against a F. hepatica antigen termed Fas2. The study was conducted in three Andean localities, Huertas-Julcan in Junin, Asillo in Puno, and Cajamarca, with a total population of 634 children in an age range 1 to 16 years old. Child fascioliasis prevalence was 21.1% in Huertas-Julcan, 25.4% in Asillo, and 24% in Cajamarca, estimated by coprological inspection. The seroprevalence of F. hepatica infection, determined by Fas2-ELISA, was 27.8% in Huertas-Julcan, 44.6% in Asillo, and 29.1% in Cajamarca. The overall sensitivity of Fas2-ELISA was 92.4%, the specificity 83.6%, and the negative predictive value 97.2%. No association between OD(450) Fas2-ELISA and infection intensity measured by egg counting was observed. Results show that Fas2-ELISA is a highly sensitive immunodiagnostic test for the detection of F. hepatica infection in children living in human fascioliasis endemic areas.

Síndrome hemolítico urémico atípico en lactantes con mutaciones genéticas: Reporte de 3 casos / Atypical hemolytic uremic syndrome (aHUS) in infants, with genetic mutations, about three cases

RESUMEN El síndrome hemolítico urémico atípico (SHUa) es una entidad clínica considerada rara; sin embargo, es la causa más común de insuficiencia renal aguda en niños. Esta enfermedad se acompaña de anemia hemolítica microangiopática, trombocitopenia, retención nitrogenada y afectación de la función renal, por lo que representa alta morbilidad y compromiso sistémico. Se reportan tres casos de SHUa en lactantes que presentaron pródromos respiratorios, diarrea, anemia hemolítica y trombocitopenia, con pérdida de función renal. Estos casos mostraron que dicha patología está asociada a mutaciones en los genes: CFH (Complemento Factor H), MCP (Membrana Cofactor Proteín), CFHR1 (Complemento Factor H-Related Proteín1), CFHR5 (Complemento factor H-Related Protein 5) y el gen C3 (Complemento component 3). Los genes CFH y MCP se encontraron afectados en dos de los casos, mientras que el tercer caso mostró una mutación nueva no reportada en el gen C3. Estos resultados evidencian que estas mutaciones están presentes en el Perú, por lo que se debe investigar y establecer medidas de prevención para reducir el alto riesgo de morbilidad y mortalidad que presentan los niños portadores SHUa.

SUMMARY The atypical hemolytic uremic syndrome (aHUS) is a rare clinical entity, but it is the most common cause of acute kidney failure in kids. The disease is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, and it is associated with high morbidity and systemic involvement. We report here three cases of aHUS in infants presenting with prodromal respiratory symptoms, diarrhea, hemolytic anemia, thrombocytopenia and acute renal failure. aHUS cases depict mutations in several genes: membrane cofactor protein (MCP) and complement factor H related proteins 1 and 5 (CFH, RP1 and PR5. Two our patients showed mutations in the genes CFH and MCP, and one presented a new non-previously reported mutation in the gen C3. Our results emphasize the existence of these aHUS mutations and underscore the need to study them to prevent morbidity and mortality.

Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos).

Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.

Lack of molecular correlates of Plasmodium vivax ookinete development.

Previous studies of Plasmodium vivax transmission to Anopheles spp. mosquitoes have not been able to predict mosquito infectivity on the basis of microscopic or molecular quantification of parasites (total parasites in the sample or total number of gametocytes) in infected blood. Two methods for production of P. vivax ookinete cultures in vitro, with yields of 10(6) macrogametocytes, 10(4) zygotes, and 10(3) ookinetes, respectively, per 10 mL of P. vivax-infected patient blood with approximately 0.01% parasitemia, were used to study P. vivax sexual stage development. The quantity of gametocytes, determined by counting Giemsa-stained blood smears, and quantity and type of gametocyte as determined by quantitative reverse transcriptase-polymerase chain reaction for Pvalpha tubulin II and macrogametocyte-specific pvg377 did not predict ookinete yield. Factors that affect the efficiency of in vitro P. vivax ookinete transformation remain poorly understood.

Optimized in vitro production of Plasmodium vivax ookinetes.

Previous reports have described obtaining mature Plasmodium vivax ookinetes in vitro using blood from infected patients using a simplified, field-based protocol. Here, we report protocols that produce improved P. vivax ookinete yields and morphological development. Optimal conditions included induction of gametogenesis using 10 mM Tris, 170 mM NaCl, 10 mM glucose, 25 mM NaHCO(3), and 100 µM xanthurenic acid for 90 minutes at pH 8.0-8.2, followed by culture in RPMI-1640, 50 mg/mL hypoxanthine, 25 mM HEPES, 29 mM NaHCO(3), 2 mM L-glutamine, and 20% fetal bovine serum at pH 8.4 for 36 hours. Ookinetes were produced in 86% (18/21) of optimized in vitro cultures; yields ranged from 6.5 × 10(4) to 2.8 × 10(6); percent gametocyte conversion ranged from 1.4% to 4.7%. This improved method is suitable for preparation of P. vivax ookinetes in quantities sufficient for biochemical, molecular, and cell biological analysis where basic laboratory facilities are in proximity to patients with vivax malaria.

Plasmodium falciparum and Plasmodium vivax infections in the Peruvian Amazon: propagation of complex, multiple allele-type infections without super-infection.

Outcrossing potential between Plasmodium parasites is defined by the population-level diversity (PLD) and complexity of infection (COI). There have been few studies of PLD and COI in low transmission regions. Since the 1995-1998 Peruvian Amazon epidemic, there has been sustained transmission with < 0.5 P. falciparum and < 1.6 P. vivax infections/person/year. Using weekly active case detection, we described PLD by heterozygosity (H(e)) and COI using P. falciparum Pfmsp1-B2 and P. vivax Pvmsp3alpha. Not being homologous genes, we limited comparisons to within species. P. falciparum (N = 293) had low (H(e) = 0.581) and P. vivax (N = 186) had high (H(e) = 0.845) PLD. A total of 9.5% P. falciparum infections and 26.3% P. vivax infections had COI > 1. Certain allele types were in more mixed infections than expected by chance. The few appearances of new alleles could be explained by stochastic polymerase chain reaction detection or synchronization/sequestration. The results suggest propagation of mixed infections by multiple inocula, not super-infection, implying decade-long opportunity for outcrossing in these mixed infections.

Caracterización de las mutaciones en el gen rpoß asociadas a la resistencia a rifampicina en pacientes con tuberculosis pulmonar / Description of gene rpoß mutations associated with rifampicin resistance in patients with pulmonary tuberculosis

Antecedentes: la resistencia a rifampicina en M. tuberculosis involucra mutaciones en el gen rpoß que codifica a la subunidad ß de la ARN polimerasa. Objetivo: Identificar las mutaciones del gen rpoß, en cepas de M. tuberculosis asosciadas con resistencia a rifampicina aisladas de la Subregión de Salud Lima Norte, Perú. Materiales y métodos: Se cultivó en Lowestein - Jenseen 73 muestras de esputo de pacientes con tuberculosis pulmonar. A 62, con más de 10 colonias por tubo, se les comprobó susceptibilidad a isoniazida, rifampicina, estreptomicina y etambutol. Se realizó la extracción de ADN por PCR, clonación en el vector pGEM-T, transformación, selección de clonas recombinantes y secuenciamiento del ADN plasmídico para la determinación de los polimorfismos del gen rpoß. Resultados: 52 (83,9 por ciento) cepas fueron resistentes a rifampicina (Rif) y 10 (16,1 por ciento) susceptibles (Rif). Se encontró alteraciones en el gen rpoß en 51 de 52 cepas Rif. Se identificaron 20 mutaciones. Las mutaciones más frecuentes fueron encontradas en los codones Ser-531 (62,7 por ciento), His-526 (15,7 por ciento), Asp-516 (11,8 por ciento) y Gln-513 (5,9 por ciento). No se observó mutación alguna en las 10 cepas Rif. 94,2 por ciento de nuestras cepas Rif fueron también resistentes a isoniazida. Conclusiones: Se encontraron mutaciones en el gen rpoß de casi todas las cepas Rif; asimismo, casi todas las cepas Rif fueron también resistentes a isoniazida.