The Genetic Architecture of Chronic Mountain Sickness in Peru.

Chronic mountain sickness (CMS) is a pathological condition resulting from chronic exposure to high-altitude hypoxia. While its prevalence is high in native Andeans (>10%), little is known about the genetic architecture of this disease. Here, we performed the largest genome-wide association study (GWAS) of CMS (166 CMS patients and 146 controls living at 4,380 m in Peru) to detect genetic variants associated with CMS. We highlighted four new candidate loci, including the first CMS-associated variant reaching GWAS statistical significance (rs7304081; P = 4.58 × 10-9). By looking at differentially expressed genes between CMS patients and controls around these four loci, we suggested AEBP2, CAST, and MCTP2 as candidate CMS causal genes. None of the candidate loci were under strong natural selection, consistent with the observation that CMS affects fitness mainly after the reproductive years. Overall, our results reveal new insights on the genetic architecture of CMS and do not provide evidence that CMS-associated variants are linked to a strong ongoing adaptation to high altitude.

Anticuerpos de cadena única de alpaca para la detección de antígenos de Fasciola hepática / Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens

RESUMEN Objetivo. Producir anticuerpos recombinantes de cadena única de alpaca que se unan con alta afinidad y especificidad al antígeno excretado-secretado (ES) de Fasciola hepatica para el desarrollo de tecnologías nuevas de diagnóstico de fascioliasis humana y animal. Materiales y métodos. Se ha construido una genoteca de cADNde los dominios variables de anticuerpos de cadena única pesada, conocidos como VHH, a partir de células mononucleares de sangre periférica de una alpaca inmunizada con el antígeno ES de F. hepatica. La genoteca fue tamizada con el antígeno ES por despliegue diferencial de fagos (phage display), seleccionando diez VHH que se unen específicamente a ES. El VHH anti ES fue clonado en un vector de expresión, la proteína recombinante (VHH-ES1) de 15,3 kDa fue producida por fermentación en E. coli y purificada a homogeneidad por cromatografía de afinidad. La unión del VHH-ES1 al antígeno ES fue evaluada por ELISA usando VHH-ES1 como anticuerpo de captura, antisuero policlonal anti-ES de conejo y conjugado anti IgG de conejo con peróxidasa de rábano. Resultados. Se ha identificado y producido un VHH-ES1 recombinante que se une al antígeno ES (VHH-ES1) que correspondía a un anticuerpo de la subclase IgG2 de bisagra larga. La unión del anticuerpo VHH-ES1 al antígeno muestra linealidad respecto a la concentración de ES en el rango de 50-5000 ng/mL y el valor límite de detección del antígeno está en el rango de 30-170 ng/mL de ES (R2=0,99). Conclusión . El VHH-ES1 se une con afinidad y especificidad al antígeno ES de F. hepatica y es un anticuerpo promisorio a evaluar para el desarrollo de nuevas tecnologías de diagnóstico de fascioliasis.

ABSTRACT Objectives. To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. Materials and Methods. A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. Results. A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). Conclusions. The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

[Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens]. / Anticuerpos de cadena única de alpaca para la detección de antígenos de Fasciola hepática.

OBJECTIVES.: To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. MATERIALS AND METHODS.: A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. RESULTS.: A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). CONCLUSIONS.: The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

OBJETIVO.: Producir anticuerpos recombinantes de cadena única de alpaca que se unan con alta afinidad y especificidad al antígeno excretado-secretado (ES) de Fasciola hepatica para el desarrollo de tecnologías nuevas de diagnóstico de fascioliasis humana y animal. MATERIALES Y MÉTODOS.: Se ha construido una genoteca de cADNde los dominios variables de anticuerpos de cadena única pesada, conocidos como VHH, a partir de células mononucleares de sangre periférica de una alpaca inmunizada con el antígeno ES de F. hepatica. La genoteca fue tamizada con el antígeno ES por despliegue diferencial de fagos (phage display), seleccionando diez VHH que se unen específicamente a ES. El VHH anti ES fue clonado en un vector de expresión, la proteína recombinante (VHH-ES1) de 15,3 kDa fue producida por fermentación en E. coli y purificada a homogeneidad por cromatografía de afinidad. La unión del VHH-ES1 al antígeno ES fue evaluada por ELISA usando VHH-ES1 como anticuerpo de captura, antisuero policlonal anti-ES de conejo y conjugado anti IgG de conejo con peróxidasa de rábano. RESULTADOS.: Se ha identificado y producido un VHH-ES1 recombinante que se une al antígeno ES (VHH-ES1) que correspondía a un anticuerpo de la subclase IgG2 de bisagra larga. La unión del anticuerpo VHH-ES1 al antígeno muestra linealidad respecto a la concentración de ES en el rango de 50-5000 ng/mL y el valor límite de detección del antígeno está en el rango de 30-170 ng/mL de ES (R2=0,99). CONCLUSIÓN: El VHH-ES1 se une con afinidad y especificidad al antígeno ES de F. hepatica y es un anticuerpo promisorio a evaluar para el desarrollo de nuevas tecnologías de diagnóstico de fascioliasis.

Anti-Fas2 IgM antibodies in Fasciola hepatica infected patients with positive IgG serology.

Background: Fascioliasis is an infectious disease caused by parasites Fasciola hepatica and F. gigantica. Humans are infected by the consumption of vegetables and water contaminated with the infective form of the parasite. Materials and Methods: In this study, an IgM-ELISA with the cysteine proteinase Fas2 antigen was evaluated with sera from 76 patients infected with F. hepatica, 24 patients with other parasite infections and 84 healthy volunteers. Results: IgM-ELISA resulted in 43% positives in F. hepatica patients with positive serology to Fas2-ELISA, but no positives resulted from testing healthy volunteers and individuals infected with other parasites. The IgM-ELISA diagnostic parameters showed a sensitivity of 43.4% (95% CI 0.321-0.553), specificity of 100% (95% CI 0.957-1), and no cross-reactivity with other parasitic infection. Interference by rheumatoid factor in the IgM immunoassay was controlled by treating sera with rheumatoid factor absorbent before testing. Conclusions: Fas2 antigen is detected by circulating IgM in patients infected with F. hepatica and IgM-ELISA using Fas2 appears as a specific immunoassay to detect the acute phase of the acute phase of F. hepatica infection in humans.

Vascular endothelial growth factor-A is associated with chronic mountain sickness in the Andean population.

A study of chronic mountain sickness (CMS) with a candidate gene--vascular endothelial growth factor A (VEGFA)--was carried out in a Peruvian population living at high altitude in Cerro de Pasco (4380 m). The study was performed by genotyping of 11 tag SNPs encompassing 2.2 kb of region of VEGFA gene in patients with a diagnosis of CMS (n = 131; 49.1 ± 12.7 years old) and unrelated healthy controls (n = 84; 47.2 ± 13.4 years old). The VEGFA tag SNP rs3025033 was found associated with CMS (p < 0.05), individuals with AG genotype have 2.5 more risk of CMS compared to those with GG genotype (p < 0.02; OR, 2.54; 95% CI: 1.10-5.88). Pairwise Fst and Nei's distance indicate genetic differentiation between Cerro de Pasco population and HapMap3 population (Fst > 0.36, p < 0.01), suggesting selection is operating on the VEGF gene. Our results suggest that VEGFA is associated with CMS in long-term residents at high altitude in the Peruvian Andes.

Field evaluation of a coproantigen detection test for fascioliasis diagnosis and surveillance in human hyperendemic areas of Andean countries.

BACKGROUND: Emergence of human fascioliasis prompted a worldwide control initiative including a pilot study in a few countries. Two hyperendemic areas were chosen: Huacullani, Northern Altiplano, Bolivia, representing the Altiplanic transmission pattern with high prevalences and intensities; Cajamarca valley, Peru, representing the valley pattern with high prevalences but low intensities. Coprological sample collection, transport and study procedures were analyzed to improve individual diagnosis and subsequent treatments and surveillance activities. Therefore, a coproantigen-detection technique (MM3-COPRO ELISA) was evaluated, using classical techniques for egg detection for comparison. METHODOLOGY AND FINDINGS: A total of 436 and 362 stool samples from schoolchildren of Huacullani and Cajamarca, respectively, were used. Positive samples from Huacullani were 24.77% using the MM3-COPRO technique, and 21.56% using Kato-Katz. Positive samples from Cajamarca were 11.05% using MM3-COPRO, and 5.24% using rapid sedimentation and Kato-Katz. In Huacullani, using Kato-Katz as gold standard, sensitivity and specificity were 94.68% and 98.48%, respectively, and using Kato-Katz and COPRO-ELISA test together, they were 95.68% and 100%. In Cajamarca, using rapid sedimentation and Kato-Katz together, results were 94.73% and 93.58%, and using rapid sedimentation, Kato-Katz and copro-ELISA together, they were 97.56% and 100%, respectively. There was no correlation between coproantigen detection by optical density (OD) and infection intensity by eggs per gram of feces (epg) in Cajamarca low burden cases (<400 epg), nor in Huacullani high burden cases (≥ 400 epg), although there was in Huacullani low burden cases (<400 epg). Six cases of egg emission appeared negative by MM3-COPRO, including one with a high egg count (1248 epg). CONCLUSIONS: The coproantigen-detection test allows for high sensitivity and specificity, fast large mass screening capacity, detection in the chronic phase, early detection of treatment failure or reinfection in post-treated subjects, and usefulness in surveillance programs. However, this technique falls short when evaluating the fluke burden on its own.

Fasciola hepatica phenotypic characterization in Andean human endemic areas: valley versus altiplanic patterns analysed in liver flukes from sheep from Cajamarca and Mantaro, Peru.

Fascioliasis is a zoonotic parasitic disease caused by Fasciola hepatica and Fasciola gigantica. Of both species, F. hepatica is the only one described in the Americas, mainly transmitted by lymnaeid snail vectors of the Galba/Fossaria group. Human fascioliasis endemic areas are mainly located in high altitude areas of Andean countries. Given the necessity to characterize F. hepatica populations involved, the phenotypic features of fasciolid adults infecting sheep present in human fascioliasis endemic areas were analysed in the Cajamarca Valley and Mantaro Valley (valley transmission patterns) and the northern Bolivian Altiplano (altiplanic transmission pattern). A computer image analysis system (CIAS) was applied on the basis of standardized measurements. The aforementioned highland populations were compared to standard lowland natural and experimental populations of European origin. Liver fluke size was studied by multivariate analyses. Two phenotypic patterns could be distinguished in F. hepatica adult size: the valley pattern (Cajamarca and Mantaro, Peru) and the altiplanic pattern (northern Altiplano, Bolivia). Results showed that the Andean valley population and European standard populations presented a phenotypic homogeneity. The Altiplano population showed a large size range with a pronouncedly lower minimum size indicating that uterus gravidity is reached at a smaller size than in valley populations. The results of this study demonstrate that there is no apparent relationship between the shape of fasciolid adults with regard to altitudinal difference or geographical origin and that allometry-free shape appears as a more stable trait than size in fasciolid species. Results are analysed in terms of intensity/crowding effect aspects and permanent/seasonal transmission characteristics.

Fasciolosis humana y animal en el Perú: impacto en la economía de las zonas endémicas / Human and animal fascioliasis in Peru: impact in the economy of endemic zones

Fasciola hepatica es el agente etiológico de la fasciolosis en el Perú. La fasciolosis es un problema de salud pública por la alta prevalencia de la infección humana, especialmente niños y un problema veterinario de importancia por las altas tasas de infección del ganado en la mayoría de regiones del País. La fasciolosis es endémica en la sierra y la costa, y esporádica en la región amazónica. La infección humana se reporta en 18 regiones y la animal en 21 de las 24 regiones del Perú. La transmisión humana ocurre en poblaciones andinas rurales dedicadas a la agricultura, pero hay un creciente número de casos en ciudades. Diferentes situaciones epidemiológicas de la infección humana se presentan: i) Regiones con casos no autóctonos, donde la infección es por consumo de vegetales contaminados traídos de zonas endémicas; ii) Regiones con poblados hipo- y mesoendémicos, transmisión local y prevalencia < 10 por ciento; y iii) Regiones con poblados hiperendémicos, transmisión local y prevalencia ≥ 10 por ciento. El parásito infecta a vacunos, ovinos, equinos, caprinos, porcinos, camélidos, cobayos y conejos. La pérdida ganadera anual por la fasciolosis es no menor de US$ 50 millones, estimada por la prevalencia de la infección y los decomisos de hígados de vacunos en mataderos. Es difícil estimar el impacto económico de la fasciolosis humana por su estatus de enfermedad desatendida, lo cierto es que la fasciolosis es endémica, y en algunos casos hiperendémica, en las regiones más pobres de nuestro país, donde la situación debe ser reconocida como una emergencia de salud pública.

Fasciola hepatica is the causative agent of fasciolosis in Peru; the disease is an important public health problem by the high prevalence of the human infection affecting mainly children and a major veterinary problem by the high rates of infected livestock. The human disease is endemic in the Sierra and the Coast but sporadic in the Amazonia, and reported in 18 Departments, while the animal infection in 21 of 24 Departments of Peru. Transmission occurs in Andean rural populations engaged in agriculture, but recently an increasing number of people became infected in the cities. The epidemiological situation in Peru includes i) Departments with non-autochtonous cases, where infection occurs by consumption of contaminated vegetables brought from endemic areas or infection is acquired by visit to endemic areas; ii) Departments with hypoendemic and mesoendemic villages, where transmission occurs by ingest of contaminated vegetables and prevalence ≤10 percent; and iii) Departments with hyper-endemic villages with human prevalence >10 percent, with an intense transmission by consumption of contaminated vegetables. The disease affects bovine, sheep, goat, swine, equine, South American camelids, rabbits and guinea pigs. The negative impact of fasciolosis in the livestock economy is not lesser than US$ 50 million per year, estimation based on the prevalence and the number of condemned livers in the abattoirs. It is difficult to estimate the economic impact of this infection in the human health due to its status of neglected disease, but fasciolosis is hyper-endemic in the poorest Andean areas of Peru where the situation has to be recognized as a public health emergency.

[Human and animal fascioliasis in Peru: impact in the economy of endemic zones]. / Fasciolosis humana y animal en el Perú: impacto en la economía de las zonas endémicas.

Fasciola hepatica is the causative agent of fasciolosis in Peru; the disease is an important public health problem by the high prevalence of the human infection affecting mainly children and a major veterinary problem by the high rates of infected livestock. The human disease is endemic in the Sierra and the Coast but sporadic in the Amazonia, and reported in 18 Departments, while the animal infection in 21 of 24 Departments of Peru. Transmission occurs in Andean rural populations engaged in agriculture, but recently an increasing number of people became infected in the cities. The epidemiological situation in Peru includes i) Departments with non-autochtonous cases, where infection occurs by consumption of contaminated vegetables brought from endemic areas or infection is acquired by visit to endemic areas; ii) Departments with hypoendemic and mesoendemic villages, where transmission occurs by ingest of contaminated vegetables and prevalence ≤10%; and iii) Departments with hyper-endemic villages with human prevalence >10 %, with an intense transmission by consumption of contaminated vegetables. The disease affects bovine, sheep, goat, swine, equine, South American camelids, rabbits and guinea pigs. The negative impact of fasciolosis in the livestock economy is not lesser than US$ 50 million per year, estimation based on the prevalence and the number of condemned livers in the abattoirs. It is difficult to estimate the economic impact of this infection in the human health due to its status of neglected disease, but fasciolosis is hyper-endemic in the poorest Andean areas of Peru where the situation has to be recognized as a public health emergency.

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.

Risk factors for Fasciola hepatica infection in children: a case-control study.

We conducted a case-control study to clarify the risk factors for human fascioliasis in the Northern Peruvian Altiplano. Cases (n=61) were individuals who were diagnosed as having chronic fascioliasis by faecal and/or serologic (Fas2-ELISA) studies. Controls (n=61) had negative serologic and coprological results for Fasciola hepatica. We identified an association between fascioliasis and four variables (40 analysed): the habit of drinking alfalfa juice (OR=4.5; 95% CI 1.8-11.1; P<0.001); familiarity with aquatic plants (OR=4.3; 95% CI 1.8-10.6; P<0.001); dog ownership (OR=5; 95% CI 1.7-15.1; P=0.002); and raising more than five sheep (OR=0.3; 95% CI 0.1-0.8; P=0.01). According to clinical presentation and laboratory studies, dizzy spells (P=0.01), history of jaundice (P=0.01), peripheral eosinophilia (P=0.005) and Ascaris lumbricoides in stools (P=0.001) were associated with fascioliasis. The principal exposure factor for F. hepatica infection was drinking alfalfa juice. In conclusion, we suggest that human fascioliasis in Peru should be suspected in patients from livestock-rearing areas, who present with recurrent episodes of jaundice and who have a history of consumption of alfalfa juice or aquatic plants, or who have eosinophilia.

Fascioliasis in relatives of patients with Fasciola hepatica infection in Peru.

High prevalence rates of human fascioliasis have been described in several regions of Peru. We surveyed 20 families in an endemic area of Peru in order to determine the proportion of infection with F. hepatica in relatives of diagnosed subjects and in order to identify associated risk factors. The study included feces and blood samples of 93 subjects. Ages ranged from one to 53 (mean = 18.6; SD = 14.2). The overall prevalence of fascioliasis by fecal examinations was 33.3% (n = 83) and by serology, 51.9% (n = 86). The prevalence in age group I (< or = 19 years old) by coprological and serological tests was 61.4% and 75.9%, respectively; in group II (> 19 years old) 15.4% and 37.5%. The main associated risk factor with fascioliasis was eating salads (OR = 3.29, CI = 1.2 - 9.0, p = 0.02). In conclusion, human fascioliasis is highly prevalent in the relatives of index cases and the most significant risk factor of acquiring fascioliasis in the family is eating salads in endemic areas.

Fascioliasis in relatives of patients with Fasciola hepatica infection in Peru

Altas tasas de fasciolosis humana han sido descritas en varias regiones del Perú. Estudiamos 20 familias en una área endémica del Perú para determinar la proporción de infección con F. hepatica en los familiares de los sujetos diagnosticados y para identificar factores de riesgo asociados. El estudio incluyó un total de 93 sujetos, quienes contribuyeron con muestras de heces y sangre. Las edades comprendieron desde 1 a 53 años (media = 18.6; DS = 14.2). La prevalencia general de fasciolosis por exámenes de heces fue 33.3% (n = 83) y por serología, 51.9% (n = 86). La prevalencia en el grupo de edad I (< 19 años de edad) por pruebas coprológicas y serológicas fueron 61.4% y 75.9%, respectivamente; en el grupo II (> 19 años de edad) 15.4% y 37.5%. El principal factor de riesgo asociado con fasciolosis fue comer ensaladas (OR = 3.29, IC = 1.2-9.0, p = 0.02). En conclusión, la fasciolosis humana es altamente prevalente en familiares de los casos índices y el factor de riesgo más significante para adquirirla en la familia es comer ensaladas en las áreas endémicas.

[Hyperendemicity of human fasciolosis in the Mantaro Valley, Peru: factors for infection with Fasciole hepatica]. / Hiperendemicidad de fasciolosis humana en el Valle de Mantaro, Perú: factores de riesgo de la infección por Fasciola hepática.

A sample of 206 children (between 1 and 16 years-old) in Huertas (n = 110) and Julcán (n = 96) districts of the Mantaro Valley, Peru, were studied with a clinico-epidemiological interview, serology and fecal parasitologic tests to describe the prevalence and analyze risk factors of infection by Fasciola hepatica (Fh). Fh prevalence by fecal examinations in Huertas (H) was 28.3% and Julcán (J), 12.6%. Using serological tests, the prevalences were: for H, 36.3% and for J, 22.7%. Univariate analysis showed a significant association between Fh infection and 3 variables in H: living near small streams (OR= 4.8; P < 0.05) or farms (OR= 2.88; P < 0.05) and the habit of drinking hot herbal drinks "emolientes" (OR= 2.92; P < 0.05); and in other 3 variables in J: having a single room per house (OR= 21.0; P < 0.05), defecating in the fields (OR= 4.77; P < 0.05), history of taking antiparasitic medication in the last six months (OR= 4.61; P < 0.05). Multivariate analysis, applied to the whole set of data revealed as possible risk factors for infection with Fh: the habit of drinking "emolientes" (OR= 5.16; P < 0.05) and living near small streams (OR= 17.22; P < 0.05) or in farms (OR= 8.13; P < 0.05). According to these results one of the most important and less recognized risk factors is the habit of drinking "emolientes". Due to the clinical importance of human fascioliasis, we conclude that its presence in the Mantaro Valley is an important public health problem, effective early diagnosis would allow rapid treatment and avoidance of the serious impairment brought by chronic infection.

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.

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.