Development and preliminary evaluation of a fluorescence RPA assay for the rapid detection of Necator americanus / 中国热带医学

@#Abstract: Objective To establish a rapid detection assay based on fluorescence recombinase polymerase amplification (RPA) targeting Necator americanus eggs, and to evaluate its efficacy, providing technical support for rapid detection of Necator americanus in fecal samples. Methods The fluorescence RPA primers and probe were designed based on the cox1 gene of Necator americanus and then screened the optimal combination to develop the assay. The genomic DNA of Necator americanus eggs was diluted to 7 concentration gradients including 100 pg/µL, 10 pg/µL, 1 pg/µL, 100 fg/µL, 10 fg/µL, 1 fg/µL, 0.1 fg/µL, to determine the detection limit of the assay. The specificity of the assay was demonstrated by detected genomic DNA from Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis and Fasciola hepatica. A total of 44 fecal samples were collected and DNA extraction was performed, and the modified Kato-Katz method, semi-nest PCR method, and fluorescent RPA method were simultaneously used for detection to evaluate the sensitivity and specificity. Results The established fluorescence RPA assay can specifically amplify a fragment of 194 bp of the Necator americanus cox1 gene within 20 min, with a detection limit of 10 fg/µL. There was no cross-reactivity with Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis, Fasciola hepatica after specificity validation. In 44 fecal samples, 27 positive samples were detected by the fluorescence RPA assay, and 26 positive samples were detected by both the Kato-Katz and the semi-nested PCR. The fluorescence curve of sample number 1 was slightly higher than the negative control in the later stage of the reaction, but did not show a similar trend to the positive control, and was therefore judged to be a suspected negative sample. Compared with the Kato-Katz method and the semi-nest PCR method, The sensitivity of the fluorescent RPA method were 100.00% and the specificity were 94.44%, and the consistency of the detection results was good (Kappa=0.953>0.75). Conclusions The assay based on the fluorescence RPA is an efficient, sensitive and specific technique for detecting Necator americanus and it can be applied for surveillance and early warning of hookworm infection.

Human hookworms from Argentina: Differential diagnosis of Necator americanus and Ancylostoma duodenale in endemic populations from Buenos Aires and Misiones / Ancylostomideos humanos de Argentina: Diagnóstico diferencial de Necatoramericanus y Ancylostoma duodenale en poblaciones endémicas de Buenos Aires yMisiones

Abstract Hookworm infection is endemic in many countries throughout the world; however,the information about the prevalence of each species, Necator americanus and Ancylostomaduodenale, is inaccurate in many South American countries. We aimed to determine the preva-lence of human hookworm species by combining the results of both microscopy and PCR amongendemic populations in Argentina, represented by natives and immigrants. A total of 140 serialfecal specimens were obtained from natives in the province of Misiones and an immigrantcommunity living in the province of Buenos Aires. Samples were examined using the formalin-ethyl acetate concentration technique (FECT) and one flotation technique (screening tests)and specific PCRs for N. americanus and A. duodenale. We characterized samples containingN. americanus by sequencing a fragment of the cytochrome b gene. The observed hookwormprevalence as assessed by the screening tests and PCR were 24.3% and 32.8%, respectively. PCRpositive samples were identified as N. americanus. PCR had 100% sensitivity compared with73.9% of screening tests. A total of 12 samples from individuals with hookworm-infected house-hold members were positive only by PCR. N. americanus sequences showed 90.5% identity, beingmore similar to each other than to any of the sequences obtained from GenBank. This is thefirst study that provides molecular data and characterization of N. americanus in Argentina.The complementary use of FECT and one flotation technique to screen hookworm infections,followed by PCR to differentiate the species contribute to produce better prevalence estimates.

Resumen La infección por Ancylostomideos es endémica en muchos países del mundo, pero la información sobre la prevalencia de las especies que la causan, Necator americanus y Ancylostoma duodenale, es inexacta en América del Sur. Nuestro objetivo fue determinar la prevalencia de especies de Ancylostomideos humanos en poblaciones de Argentina nativas o provenientes de áreas endémicas, combinando los resultados de microscopía y PCR. Un total de 140 muestras fecales seriadas fueron obtenidas de individuos nacidos en la provincia de Misiones con residencia en esta y de miembros de una comunidad oriunda del Paraguay establecida en la provincia de Buenos Aires. Las muestras fueron examinadas por la técnica de formol-acetato de etilo (FAE) y una técnica de flotación como pruebas de cribado, y se efectuaron PCR específicas para N. americanus y A. duodenale. Caracterizamos muestras que contienen N. americanus secuenciando un fragmento del gen del citocromo b. La prevalencia de Ancylostomideos según las pruebas de cribado y el método PCR fueron del 24,3 y 32,8%, respectivamente. Las muestras positivas por PCR se identificaron como N. americanus. La PCR tuvo una sensibilidad del 100,0% en comparación con el 73,9% de las pruebas de detección. Hubo 12 muestras de individuos con miembros de la familia infectados con anquilostomas que solo por PCR fueron positivas. Las secuencias de N. americanus mostraron un 90,5% de identidad y fueron más similares entre sí que a cualquiera de las secuencias obtenidas de GenBank. Este es el primer estudio que proporciona datos moleculares y la caracterización de N. americanus en Argentina. El uso complementario de FAE y una técnica de flotación para detectar infecciones por anquilostomas, seguido de PCR para diferenciar las especies, contribuye a producir mejores estimaciones de prevalencia.

Diferencias morfológicas relevantes para la identificación específica de larvas de uncinarias y Strongyloides stercoralis / Relevant morphological differences for the specific identification of larvae of hookworms and Strongyloides stercoralis

Objetivos: Describir las características morfológicas diferenciales más relevantes de los estadios larvarios de uncinarias (Ancylostoma duodenale y Necator americanus), y S. stercoralis, para su identificación específica en laboratorio clínico convencional. Material y métodos: Estudio observacional durante julio del 2015 a agosto del 2016. Se realizaron cultivos en placa de agar de muestras fecales positivas por microscopía a larvas de nematodos o huevos de uncinaria. Las larvas desarrolladas en cultivo fueron descritas morfológicamente mediante claves, se identificaron las características morfológicas diferenciales más relevantes y se ilustraron mediante microfotografías. Resultados: Las características morfológicas más relevantes para la diferenciación específica fueron la longitud de la cavidad bucal y la prominencia del poro genital en el estadio rabditiforme; y la forma del cuerpo, el extremo terminal de la cola y el diámetro del extremo anterior del intestino en relación al esófago en el estadio filariforme. Conclusiones: Las uncinarias A. duodenale y N. americanus, y S. stercoralis pueden identificarse específicamente por microscopía óptica mediante el análisis morfológico de sus estadíos larvarios desarrollados en cultivo. Debido al requerimiento de equipos e insumos de uso común en laboratorio de microbiología, se recomienda esta metodología para uso en laboratorios clínicos convencionales. (AU)

Objectives: To determine the most relevant morphologic differences of larval stages of hookworms (Ancylostoma duodenale y Necator americanus) and S. stercoralis for their specific identification in a conventional laboratory. Methods: An observational study was carried-out between July and August 2016. Positive fecal samples at microscopy to nematode larvae or to eggs of hookworms were cultured in agar plate. Larvae that developed in culture were described morphologically using passwords. We identified the most relevant differential morphologic features and illustrated them using microphotography. Results: The most relevant morphologic features that allow differentiating these nematodes were the longitude of the oral cavity and the prominence of the genital primordium for the rabditiform larva; the shape of the body, the terminal end of the tail and the diameter of the anterior extreme of the intestine in relation to the esophagus for the filariform larvae. Conclusions: Hookworms and S. stercoralis can be specifically identified by optic microscopic examination of their larval stages developed in culture. We recommend this methodology for conventional microbiology laboratories due to the requirement of simple equipment. (AU)

Differentiating the morphology of infective larvae between two human hook-worms / 中国血吸虫病防治杂志

The morphological differentiation of the infective larvae between human Ancylostoma duodenale and Necator ameri-canus is of great significance for the epidemiological survey of hookworm diseases and human parasitology teaching.Understand-ing of features of the oral spear and transverse lines on the tunica vaginalis is able to accurately differentiate the infective larvae between these two human hookworms.

Preservación de huevos de Necator americanus con acetato de sodio-ácido acético-formalina (SAF). Estudio de caso / Preservation of Necator americanus eggs with sodium acetate-acetic acid-formalin(SAF). Case study

Resumen Introducción. La técnica de Kato Katz es el método recomendado para realizar el recuento de huevos en materia fecal y determinar la intensidad parasitaria por helmintos. La calidad de los resultados se ve afectada por la tardanza en el procesamiento de la muestra, por lo que se requiere de preservantes que faciliten la lectura posterior a las 24 horas de recolección de la muestra. Objetivo. Demostrar validez de la solución con acetato de sodio-ácido acético-formalina (SAF) como preservante de huevos de Necator americanus por medio del estudio de un caso clinico. Materiales y métodos. Descripción de caso clínico con diagnóstico de parásitos intestinales por coprológico, intensidad de la infección por la técnica de Kato Katz, evaluación de la presencia de huevos N. americanus por la técnica de Kato Katz durante 10 meses en muestra preservada en SAF y determinación de especie de Uncinaria sp. por qPCR. Resultados. Los huevos de N. americanus se conservan de manera adecuada en morfología durante los primeros cinco meses. Conclusiones. A fin de ejecutar la técnica de Kato Katz, la fijación con SAF demostró ser adecuada para el mantenimiento de la intensidad parasitaria y morfología de los huevos de N. americanus en un período de hasta cinco meses.

Abstract Introduction: The Kato Katz technique is the recommended method for fecal egg counts and determining the intensity of parasitic helminths. The quality of the results is affected by the delay in the analysis of the sample. Therefore, fixatives are necessary to make the reading easier 24 hours after sample collection. Objective: To prove the usefulness of sodium acetate-acetic acid-formalin (SAF) solution as a fixative for Necator americanus eggs by means of a clinical case study. Materials and methods: Clinical case description and intestinal parasites diagnosis by direct stool microscopic analysis and parasite infection intensity by means of the Kato Katz technique. N. americanus eggs were detected by Kato Katz method for 10 months on a SAF-fixed stool sample. Identification of Uncinaria sp. by qPCR. Results: N. americanus eggs are adequately preserved in SAF during the first five months. Conclusions: SAF fixative is suitable for Kato Katz method and proved to be capable of maintaining parasite intensity and N. americanus egg morphology over a period of up to five months.

Hookworm Infection: A Neglected Cause of Overt Obscure Gastrointestinal Bleeding

Hookworm infections are widely prevalent in tropical and subtropical areas, especially in low income regions. In the body, hookworms parasitize the proximal small intestine, leading to chronic intestinal hemorrhage and iron deficiency anemia. Occasionally, hookworms can cause overt gastrointestinal bleeding, but this is often ignored in heavily burdened individuals from endemic infectious areas. A total of 424 patients with overt obscure gastrointestinal bleeding were diagnosed by numerous blood tests or stool examinations as well as esophagogastroduodenoscopy, colonoscopy, capsule endoscopy or double-balloon enteroscopy. All of the patients lived in hookworm endemic areas and were not screened for hookworm infection using sensitive tests before the final diagnosis. The patients recovered after albendazole treatment, blood transfusion, and iron replacement, and none of the patients experienced recurrent bleeding in the follow-up. All the 31 patients were diagnosed with hookworm infections without other concomitant bleeding lesions, a rate of 7.3% (31/424). Seventeen out of 227 patients were diagnosed with hookworm infections in the capsule endoscopy (CE), and 14 out of 197 patients were diagnosed with hookworm infections in the double balloon enteroscopy (DBE). Hookworm infections can cause overt gastrointestinal bleeding and should be screened in patients with overt obscure gastrointestinal bleeding (OGIB) in endemic infectious areas with sensitive methods. Specifically, the examination of stool specimens is clinically warranted for most patients, and the proper examination for stool eggs relies on staff's communication.

Hookworm Infection Caused Acute Intestinal Bleeding Diagnosed by Capsule: A Case Report and Literature Review

Hookworm infections are rare causes of acute gastrointestinal bleeding. We report a middle aged man with primary nephrotic syndrome and pulmonary embolism. During the treatment with steroids and anticoagulants, the patient presented acute massive hemorrhage of the gastrointestinal tract. The results of gastroscopy showed red worms in the duodenum. Colonoscopy and CT angiogram of abdomen were unremarkable. Capsule endoscopy revealed fresh blood and multiple hookworms in the jejunum and ileum. Hookworms caused the acute intestinal bleeding. The patient responded well to albendazole. Hematochezia was markedly ameliorated after eliminating the parasites. Hence, hookworm infection should be considered in the differential diagnosis of a patient with obscure gastrointestinal bleeding. Capsule endoscopy may offer a better means of diagnosis for intestinal hookworm infections.

Biological control of the grapevine diseases ‘grey mold’ and ‘powdery mildew’ by Bacillus B27 and B29 strains.

Uncinula necator and Botrytis cinerea are the most destructive pathogens of the grapevine in Tunisia and elsewhere. We used two strains of Bacillus subtilis group, B27 and B29 to control powdery mildew and the grey mold disease of the grapevine. Green house experiments showed that B29 and B27 strains of the bacteria efficiently reduced the severity of powdery mildew up to 50% and 60%, respectively. Further, they decreased Botrytis cinerea development on grape leaf by 77% and 99%, respectively. The mode of action has been shown to be chitinolytic. These two bacteria showed significant production of total proteins discharged into the culture medium. Determination of some chitinolytic enzymes revealed the involvement of N-acetyl glucosaminidase (Nagase), the chitin-1,4-chitobiosidase (Biase) and endochitinase in degrading the mycelium of B. cinerea.

Biosynthesis of P(3HB-co-3HV-co-3HHp) terpolymer by Cupriavidus necator PHB4 transformant harboring the highly active PHA synthase gene of Chromobacterium sp. USM2

Aims: This study evaluates potentials of Cupriavidus necator PHB4 transformant harboring the highly active polyhydroxyalkanoate synthase gene (phaC) of a locally isolated Chromobacterium sp. USM2 for its ability to incorporate 3-hydroxyheptanoate (3HHp) monomer. Methodology and results: A mixture of fructose and sodium heptanoate fed to the culture gave rise to poly(3- hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyheptanoate), [P(3HB-co-3HV-co-3HHp)] terpolymer synthesis, with traces of 3HHp monomers confirmed through gas chromatography (GC), proton (1H) and carbon (13C) NMR spectra. Conclusion, significance and impact of study: This study has revealed that the PHA synthase of Chromobacterium sp. USM2 has a broad range of substrate specificity. The synthase is able to polymerize 3-hydroxyalkanoate monomers having 4–7 carbon atoms.

Waste cooking oil as substrate for biosynthesis of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): Turning waste into a value-added product

Aims: Improper disposal of domestic wastes, such as waste cooking oil (WCO), contributes to the deterioration of the environment and may lead to health problems. In this study, we evaluated the potential of plant-based WCO as a carbon source for the commercial biosynthesis of the bio-plastics, poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). The consumption of WCO for this purpose would mitigate their pollution of the environment at the same time. Methodology and Results: WCO collected from several cafeterias in USM was tested as the carbon source for polyhydroxyalkanoates (PHA) production. A selection of suitable nitrogen source was first conducted in order to obtain an acceptable number of dry cell weight (DCW) and PHA content. Urea was found to be a suitable nitrogen source for the two bacterial strains used in our study, Cupriavidus necator H16 and its transformed mutant, C. necator PHB¯4 harboring the PHA synthase gene of Aeromonas caviae (PHB¯4/pBBREE32d13). With WCO as the sole carbon source, C. necator H16 yielded a relatively good dry cell weight (DCW=25.4 g/L), with 71 wt% poly(3-hydroxybutyrate) P(3HB) content. In comparison, the DCW obtained with fresh cooking oil (FCO) was 24.8 g/L. The production of poly(3 hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from WCO by the transformant C. necator PHB¯4 was comparable, yielding a DCW of 22.3 g/L and P(3HB-co-3HHx) content of 85 wt%. Lipase activities for both bacterial strains reached a maximum after 72 h of cultivation when time profile was conducted. Conclusion, significance and impact of study: The use of WCO as a carbon source in the biosynthesis of the bioplastic, PHA, turns a polluting domestic waste into a value-added biodegradable product. This renewable source material can thus be exploited for the low cost production of PHA.

Molecular Detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.

Relación entre geohelmintiasis intestinales y variables químicas, hematológicas e IgE, en una comunidad yukpa del estado Zulia, Venezuela / Relationship between intestinal geohelminthiases and chemical, hematologic, and IgE variables at a Yukpa community of Zulia State, Venezuela

Para relacionar helmintiasis intestinales y parámetros hematológicos, bioquímicos y serológicos, se estudiaron 37 individuos yukpas del municipio Perijá, estado Zulia, durante el año 2009. Las muestras de heces se evaluaron mediante el concentrado con formol-éter, Kato-Katz y cultivo de Harada-Mori. Los parámetros hematológicos, las proteínas totales, albúminas, globulinas y relación A/G, fueron determinados por autoanalizadores hematológicos y bioquímicos. La IgE fue determinada por ELISA. 70,3% de los indígenas presentaron una o más especies de helmintos. Ascaris lumbricoides y Trichuris trichiura presentaron 35,5% de prevalencia c/u; les siguieron los Ancylostomideos con 27,4%. Según el recuento de huevos, los casos de ancylostomiasis fueron considerados leves, mientras que en ascariasis el mayor porcentaje como moderados y en trichuriasis, leves. De los cultivos de Harada-Mori realizados, 10 fueron positivos a larvas filariformes de Necator americanus. La mayoría de los individuos estudiados presentó eosinofilia (x=3.384 eosinófilos/mL) y valores de IgE por encima de lo normal (>150UI/mL). Los valores de proteínas totales, albúmina, globulinas, relación A/G, hemoglobina, hematocrito y contaje de plaquetas fueron normales en la mayoría de los indígenas. No se encontró relación entre las geohelmintiasis y variables químicas o hematológicas estudiadas, excepto que los parasitados con geohelmintos presentaron eosinofília intensa y elevada IgE.

During 2009, 37 Yukpa individuals from the Perija municipality, Zulia State, were studied to determine the relationships between intestinal helminthiasis and hematologic, biochemical and serologic parameters. Feces samples were evaluated through formol-ether concentrate, Kato-Katz, and Harada-Mori culture. Hematologic parameters, total proteins, albumins, globulins, and A/G relationship, were determined by hematologic and biochemical auto-analyzers. IgE was determined by ELISA. 70.3% of the population presented one or more helminth species. Ascaris lumbricoides and Trichuris trichiura showed a 35.5% prevalence each, followed by Ancylostomideos with 27.4%. According to egg counts, ancylostomiasis cases were considered mild, while in ascariasis, the greater percentage were moderate and in trichuriasis, mild. Of the Harada-Mori cultures done, 10 were positive for Necator americanus filariform larvae. Most of the individuals studied presented eosinophilia (x=3,384 eosinophils/mL) and IgE values above normal (>150 IU/mL). Total protein, albumin, globulin, A/G relationship, hemoglobin, and platelet counts were normal in most individuals. No relationship between geohelminthiases and the chemical and hematologic variables studied was found, except that parasitized individuals presented an intense eosinophilia and high IgE values.

A Case of Severe Anemia by Necator americanus Infection in Korea

This report describes clinical and parasitological findings of an 82-yr-old female patient who lived in a local rural village and suffered from severe chronic anemia for several years. She was transferred to the National Police Hospital in Seoul for management of severe dyspnea and dizziness. At admission, she showed symptoms or signs of severe anemia. Gastroduodenoscopy observed hyperemic mucosa of the duodenum and discovered numerous moving roundworms on the mucosa. Endoscopy isolated seven of them, which were identified as Necator americanus by characteristic morphology of cutting plates in the buccal cavity. The patient was treated with albendazole and supportive measures for anemia, and her physical condition much improved. This case suggests the possibility that hookworm N. americanus is still transmitted in a remote local mountainous area in Korea.

Parasitosis intestinal en niños de zonas palúdicas de Antioquia (Colombia) / Intestinal parasitoses in children living in malarious zones of Colombia

Introducción: malaria, parasitosis intestinales y desnutrición coexisten e interactúan en los habitantes de las zonas palúdicas; se requiere conocer la magnitud de cada una de esas enfermedades. Objetivo: identificar la prevalencia de parásitos intestinales en niños (4-10 años) de Urabá y Bajo Cauca (Antioquia, Colombia) y conocer el cambio postratamiento antiparasitario intestinal específico. Metodología: en niños con paludismo, se hicieron encuestas parasitarias intestinales antes y 8 y 30 días postratamiento (albendazol + secnidazol). Se usaron coprológicos directo y por concentración, en muestra única. Resultados: el día-1, 80% mostraron algún helminto patógeno y 39%, algún protozoo patógeno; hubo helmintos y protozoos patógenos en 35%. Las prevalencias específicas el día 1 fueron: Ascaris lumbricoides 43,5%, Trichuris trichiura 68,2%, Necator americanus 37,6%, Entamoeba histolytica 15,3%, Giardia lamblia 21,2%. La intensidad parasitaria protozoaria fue siempre baja (1-2 cruces) y por helmintos fue moderada-intensa en 84% para A. lumbricoides, 83% para T. trichiura y 50% para N. americanus. Medida el día 8, la “reducción neta de la prevalencia protozoaria”, atribuible al tratamiento, fue: E. histolytica 50%, G. lamblia 71% (p < 0,05). Medida el día 8, la “reducción neta de la intensidad de helmintos”, atribuible al tratamiento, fue significativa para A. limbricoides y N. americanus. La reducción neta por el albendazol medida al día 8 fue: A. lumbricoides 91%, N. americanus 87%, T. trichiura 27%; siempre fue significativa la reducción de la cantidad de huevos (p < 0,05). Conclusión: las parasitosis intestinales en Turbo y El Bagre, en niños maláricos de 4-10 años, tienen muy alta prevalencia y constituyen un grave problema de salud pública.

Introduction: Malaria, intestinal parasitoses and malnutrition coexist and interact in people of malarious areas; it is required to know the magnitude of these diseases. Objective: to identify the prevalence of intestinal parasites in children (aged 4-10 years) of malarious areas of Antioquia (northwestern Colombia), and to know the changes of parasites at days 8 and 30 after specific treatment. Methodology: intestinal parasites surveys were applied before treatment (albendazole + secnidazole) and at days 8 and 30 after treatment. Examination of stools (direct and concentration) were used in single samples. Results: at day 1, 80% of the children showed some pathogenic helminth, 39% had some pathogenic protozoan, and 35% had both helminths and protozoa. Specific prevalences at day 1 were as follows: Ascaris lumbricoides 43.5%, Trichuris trichiura 68.2%, Necator americanus 37.6%, Entamoeba histolytica, 15.3%, Giardia lamblia 21.2%. The intensity of infection was slight (1-2 crosses) for protozoan parasites and it was moderateintense for helminth parasites, namely: 84% for A. lumbricoides, 83% for T. trichiura and 50% for N. americanus. The “net effect on protozoan prevalence” attributable to treatment, measured at day 8, was E. histolytica 50% and G. lamblia 71% (p < 0.05). The “net effect on helminth burden” (NEHB) attributable to treatment (between days 1 and 8) was significant for A. lumbricoides and N. americanus. The NEHB of albendazole, measured at day 8, was 91% for A. lumbricoides, 27% for T. trichiura and 87% for N. americanus. Egg reduction was always significant (p < 0.05). Conclusion: Prevalence rates of intestinal parasites in Turbo and El Bagre, in children with malaria, aged 4-10 years, are still high and constitute a severe public health problem.

Anemia severa causada por Necator americanus: reporte de un caso / Severe anemia caused by Necator americanus: case report

En climas templados, yungas (zona intermedia entre valle y trópico) y trópico, prevalecen dos parásitos hematófagos: Necator americanusy Ancylostoma duodenale, responsable de una anemia crónica, que lleva a los niños a tener niveles tan bajos de Hb, que a veces sonconsiderados incompatibles con la vida; rara vez originan sangrado agudo severo.Describimos el caso de un niño, de seis meses de edad, procedente de Coroico (Nor Yungas) con disentería inicial, misma que luego secomplica con melenas y rectorragia de sangre rutilante que origina una hipovolemia severa, rayana en el choque. Sometido a exploraciónquirúrgica por persistencia del sangrado, previo gammagrafía para descartar un divertículo de Meckel, no se halla el sitio de sangradopor lo que se realiza laparotomía exploratoria y posteriormente una endoscopía, identifi cándose en duodeno foco de sangrado originadopor parásitos, que por su morfología se asume que se trate de Necator americanus. Con este diagnóstico, recibe transfusiones para suestabilizar su hipovolemia y Mebendazol, con buena evolución, remisión del sangrado de tubo digestivo, y buena tolerancia a la alimentación.El “sangrado agudo” por uncinarias, es excepcional, hecho que justifi ca la presentación del caso y permite realizar un recordatorio breve delos mecanismos responsables de ello...

In temperate climate, yungas (intermediate zone between valley and tropical lowland), two blood sucking parasites are prevalent: Necator americanus and Ancylostoma duodenale, responsible for chronic anaemia in children that causes extremely low Hb levels sometimesconsidered as incompatible with life; only in rare cases acute bleeding occurs.We describe the case of a six months old child stemming from Coroico (Nor Yungas) initially with dysentery that becomes complicated by melaena and rectal bleeding originating severe hypovolaemia bordering on shock. After gammagraphy in order to exclude Meckel’s diverticle, the child was submitted to surgical exploration, where no source of bleeding could be found, therefore an explorative laparotomy was performed and later on an endoscopy, where the source of bleeding was identifi ed in the duodenum as caused by the parasite Necator Americanus. With this diagnose, blood transfusions were administered to the child in order to stabilize the hypovolaemia and Mebendazol was given. The evolution was good, the bleeding stopped and feeding was well tolerated.Acute bleeding caused by hook worms is exceptional; therefore we present this case as a short reminder of the responsible mechanisms.

Histopathological observation of acquired immunity in skin of hamsters elicited by Necator americanus third-stage infective larvae / 中国地方病学杂志

Objective To obtain acquired immunity evidence in hamsters elicited by third stage hookworm larvae of Necator americanas(NaL3).morphology changes of NaL3 and inflammatory responses in the skin and undedying subcutaneous tissue and muscles of hamsters were observed.Methods Hamsters were immunized subcutaneously with one dose of 150 NaL3 at 2 weeks earlier,and then challenged pereutaneously with 900 NaL3.Skins were excised from post-challenge hamsters at 6,24,72 hours and 1,2 weeks,and then examined under light microscopy.Non-immunized hamsters served as negative controls.Results In non-immunized hamsters the number of NaL3 were 15,33,11.0 and 0 at 6,24,72 hours and 1,2 weeks post-infection.No damaged or dead NaL3 section was observed.All NaL3 exhibited no structural damage and infihrating inflammatory cells were absent from the sunDunding tissues.There were no cutaneous changes.In contrast.the total number of Nak sections in the skin of immunized hamsters were 25,53,15,5 and 4 at 6,24,72 hours and 1,2 weeks post-challenge.Among these NaL3 sections,damaged and dead section number were 0,24,6,0,0 and 0,0,7,5,4.At 24 hours post-challenge the Nak exhibited cutieular swelling and damage.By 72 hours post-challenge pyknosis of the somatic cells nuclei and sparseness or loss of definition in the internal structures of NaL3 were seen.One or two weeks after chanenge,the NaL3 showed severe damage or even dead with remnants.Inflammatory responses including macrophages,epithelioid cells and eosinophils infiltrating and granulomata forming were mainly seen around the NaL3 sections in the skin of immunized hamsters.Conclusions Hamsters initially immunized with NaL3 exhibited obvious acquired immunity protection against percutaneously challenged infection as evidenced by vigorous inflammatory responses in the skin and underlying subcutaneous tissue and muscle.

Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

Revisión de Tema: Nemátodos intestinales de importancia médica en Colombia ¿un problema resuelto? / intestinal nematodes of medical importance in Colombia: a solved problem?

Las enteroparasitosis, en general, constituyen un gran problema de salud pública en el mundo. De éstas, las nematodosis figuran entre las más destacadas en Colombia. Debido a que estos parásitos pueden ingresar al organismo por vía oral o por la piel, los hábitos higiénico-sanitarios deficientes, entre ellos el fecalismo, facilitan su transmisión y conservación.Entre las nematodosis más prevalentes en nuestro país tenemos aquéllas causadas por Ascaris lumbricoides, Trichuris trichiura (tricocéfalo), uncinarias (Necator americanus y Ancylostoma duodenale) y Strongyloides stercoralis. Cuando la carga de dichos parásitos es considerablemente alta o se acompaña de alteraciones en las defensas del hospedero, se pueden producir complicaciones que comprometen seriamente la salud del paciente.En la actualidad se sabe que el control farmacológico de estas nematodosis es efectivo y seguro. No obstante, sin autocuidado y mantenimiento sostenible de buenas condiciones higiénico-sanitarias, no es posible su erradicación.Por lo anterior, esta revisión dirigida al personal de la salud, especialmente a los médicos generales, discutirá los aspectos más relevantes de estas nematodosis y de sus complicaciones, haciendo énfasis en su biología, fisiopatología, manifestaciones clínicas, diagnóstico, tratamiento y algunos datos epidemiológicos.

Abstract Intestinal parasites, in general, constitute a great worldwide, public health problem. Of them, nematodes are among the most outstanding in Colombia. Because these parasites can enter into the organism either by ingestion or through the skin, faulty hygienic-sanitary habits facilitate their transmission and conservation. Among nematodosis the more frequent in Colombia are those caused by Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm), hookworms (Necator americanus and Ancylostoma duodenale) and Strongyloides stercoralis. When the burden of these parasites is considerably high and/or is accompanied by alterations in host defenses, complications can take place that seriously affect health. Presently it is known that pharmacological control of these nematodosis is effective and safe. Nevertheless, without selfcare, and maintenance of good hygienic-sanitary conditions, their eradication will not be feasible. This review of outstanding aspects of nematodosis, with emphasis on their biology, physiopathology, clinical manifestations, diagnosis, treatment, and some epidemiologic data, intends to update physicians as a basis for adequate management of this problem.

Um século de experiência no controle da ancilostomíase / One century of experience with the control of ancylostomiasis

In the beginning of the XX century, ancylostomiasis was considered a serious public health problem and governments started the first systematic planning for the control of an endemic disease, in large scale. Thereafter, ancylostomiasis was considered a less important subject, because efficient anti-helminthic drugs were introduced, this in combination with the economic development in rich countries, reduced undernourishment and improved housing and sanitary conditions. Consequently, the residual cases of the infection became in general asymptomatic. However, in the third world the problem is still present, and must be considered with realism and managed accordingly. Various suggestions for controling the disease are presented in this paper.

A ancilostomíase despertou a atenção do mundo, no começo do século XX, como grave problema de saúde pública e deu origem aos primeiros planos sistemáticos de controle de uma endemia em larga escala. Passou depois a ser vista como questão de menor importância quando surgiram medicamentos anti-helmínticos eficazes e o desenvolvimento econômico dos países mais ricos reduziu neles a subnutrição, melhorando ao mesmo tempo as condições habitacionais e sanitárias que tornaram as infecções residuais clinicamente assintomáticas. O problema persiste; entretanto, no terceiro mundo que deve encará-lo com realismo e agir em conseqüência. Sugestões são feitas nesse sentido.