[Frequency of cervical cytological alterations and human papilloma virus in a sample of university students in Temuco, Chile]. / Frecuencia de alteraciones citológicas cervicales y virus papiloma humano en una muestra de estudiantes universitarias en Temuco, Chile.

BACKGROUND: The early onset of sexual activity can promote the development of cervical alterations and sexually transmitted infections, especially the human papillomavirus (HPV) very common in adolescents and young people. AIM: The condition of the cervix, HPV and sexual behavior in young women under 25 years of age were analyzed. METHODS: 182 university students, healthy, sexually active, 18-24 years old, without vaccine for HPV participated. Papanicolaou (Pap) test and classification of high and low risk HPV (HR and LR) were performed by real time polymerase chain reaction. The sexual behaviors were consulted in private. RESULTS: The 46.9% of Pap presented cytological alterations, non-specific inflammation/hemorrhagic (29.4%) and atypical smear (10.2%) being de most frequent. The overall frequency of HPV-HR was 24.3%, of these 67.4% presented an altered Pap. There was an association between cytological alterations and HPV (p < 0.0001) and years of sexual activity and atypical smear or cervical intraepithelial neoplasia grade I (CIN I) (p = 0.009). 11.9% of young women (21/177) presented atypical smear or CIN I, with 66.7% of cases HPV-HR. CONCLUSIONS: These findings alert the vulnerability of these young women who would have a potential risk of viral persistence, CIN and eventually cancer. It is important to emphasize counseling and prevention prior to the regular age of admission to the screening program for cervical cancer. This study was financed by the Universidad de La Frontera through Projects DI15-0047 and DI17-0123.

Frecuencia de alteraciones citológicas cervicales y virus papiloma humano en una muestra de estudiantes universitarias en Temuco, Chile / Frequency of cervical cytological alterations and human papilloma virus in a sample of university students in Temuco, Chile

Resumen Introducción: El inicio precoz de actividad sexual puede favorecer el desarrollo de alteraciones cervicales y de infecciones de transmisión sexual, en especial del virus papiloma humano (VPH) muy frecuente en adolescentes y jóvenes. Objetivo: Analizar el estado del cuello uterino, presencia del VPH y conductas sexuales en mujeres menores de 25 años. Material y Métodos: Participaron 182 estudiantes universitarias de 18-24 años, sanas, sexualmente activas y no vacunadas para VPH. Se realizó Papanicolaou (Pap) y clasificación del VPH en alto y bajo riesgo (AR y BR) mediante reacción de polimerasa en cadena en tiempo real. Las conductas sexuales fueron consultadas privadamente. Resultados: El 46,9% de los Pap presentaron alteraciones citológicas (inflamación inespecífica/hemorrágico: 29,4% y frotis atípicos (FA):10,2%). La frecuencia de los VPH-AR fue 24,3%; de éstos, 67,4% presentó un Pap alterado. Hubo asociación entre alteraciones citológicas y presencia de VPH (p < 0,0001) y años de actividad sexual y FA o neoplasia intraepitelial grado I (NIE I) (p = 0,009). El 11,9% de las jóvenes estudiadas (21/177) presentó FA o NIE I, con 66,7% de casos VPH-AR. Conclusiones: Estos hallazgos alertan la vulnerabilidad de estas jóvenes que tendrían un riesgo potencial de persistencia viral, NIE y eventualmente cáncer. Es importante enfatizar consejería y prevención previo a la edad normada de ingreso al programa de cribado para cáncer cérvico uterino en Chile.

Background: The early onset of sexual activity can promote the development of cervical alterations and sexually transmitted infections, especially the human papillomavirus (HPV) very common in adolescents and young people. Aim: The condition of the cervix, HPV and sexual behavior in young women under 25 years of age were analyzed. Methods: 182 university students, healthy, sexually active, 18-24 years old, without vaccine for HPV participated. Papanicolaou (Pap) test and classification of high and low risk HPV (HR and LR) were performed by real time polymerase chain reaction. The sexual behaviors were consulted in private. Results: The 46.9% of Pap presented cytological alterations, non-specific inflammation/hemorrhagic (29.4%) and atypical smear (10.2%) being de most frequent. The overall frequency of HPV-HR was 24.3%, of these 67.4% presented an altered Pap. There was an association between cytological alterations and HPV (p < 0.0001) and years of sexual activity and atypical smear or cervical intraepithelial neoplasia grade I (CIN I) (p = 0.009). 11.9% of young women (21/177) presented atypical smear or CIN I, with 66.7% of cases HPV-HR. Conclusions: These findings alert the vulnerability of these young women who would have a potential risk of viral persistence, CIN and eventually cancer. It is important to emphasize counseling and prevention prior to the regular age of admission to the screening program for cervical cancer. This study was financed by the Universidad de La Frontera through Projects DI15-0047 and DI17-0123.

Syringeable Self-Organizing Gels that Trigger Gold Nanoparticle Formation for Localized Thermal Ablation.

Block copolymer dispersions that form gels at body temperature and that additionally are able to reduce a gold salt to nanoparticles (AuNPs) directly in the final formulation under mild conditions were designed as hybrid depots for photothermal therapy. The in situ gelling systems may retain AuNPs in the application zone for a long time so that localized elevations of temperature can be achieved each time the zone is irradiated. To carry out the work, dispersions were prepared covering a wide range of poloxamine Tetronic 1307:gold salt molar ratios in NaCl media (also varying from pure water to hypertonic solution). Even at copolymer concentrations well above the critical micelle concentration, the reducing power of the copolymer was maintained, and AuNPs were formed in few hours without extra additives. Varying the copolymer and NaCl concentrations allowed a fine tuning of nanoparticles' shape from spherical to triangular nanoplates, which determined that the surface plasmon resonance showed a maximum intensity at 540 nm or at 1000 nm, respectively. The information gathered on the effects of (i) the poloxamine concentration on AuNPs' size and shape under isotonic conditions, (ii) the AuNPs on the temperature-induced gelling transition, and (iii) the gel properties on the photothermal responsiveness of the AuNPs during successive irradiation cycles may help the rational design of one-pot gels with built-in temperature and light responsiveness.

[Human papilloma virus and Chlamydia trachomatis by number of sexual partners and time of sexual activity on university students in the Region of La Araucanía, Chile]. / Virus papiloma humano y Chlamydia trachomatis según número de parejas sexuales y tiempo de actividad sexual en estudiantes universitarias en la Región de La Araucanía, Chile.

BACKGROUND: Human papilloma virus (HPV) and Chlamydia trachomatis are the most prevalent sexually transmitted infections (STIs), among teenagers and young people, with risk factors: active sex life and multiple partners. Chlamydia trachomatis infection may favor HPV infection and this, the development of cervical cancer. Both infections can lead to consequences on sexual and reproductive health. OBJECTIVE: To determine frequency of HPV and C. trachomatis in asymptomatic university women less than 25 years, associating them with number of sexual partners (n°SxP) and time of sexual activity (TSxA). Material andMethods: 151 cervical samples for HPV and C. trachomatis, were processed by conventional and in real time reaction polymerase chain. RESULTS: HPV 21, 8%, C. trachomatis 11, 2% and co-infection (HPV/C.trachomatis), 4.6%. Aimong HPV +, 80, 6% showed high risk HPV. The n°SxP was strongly associated with HPV. Aimong young coinfected HPV/C. trachomatis, 71.4% had 3 or more PSx. Chlamydia trachomatis was more frequent (64,7%) that HPV within range of 3-5 years according to the TSxA, Discussion: A high prevalence of HPV and C. trachomatis was observed. Young women with coinfection HPV/C. trachomatis could be a high-risk group need to monitor their infections. It suggests the implementation of university programs in education, counseling and prevention in sexual health.

Virus papiloma humano y Chlamydia trachomatis según número de parejas sexuales y tiempo de actividad sexual en estudiantes universitarias en la Región de La Araucanía, Chile / Human papilloma virus and Chlamydia trachomatis by number of sexual partners and time of sexual activity on university students in the Region of La Araucanía, Chile

Introducción: El virus papiloma humano (VPH) y Chlamydia trachomatis son las infecciones de transmisión sexual (ITS) más frecuentes, en adolescentes y jóvenes, con factores de riesgo: vida sexual activa y múltiples parejas. Chlamydia trachomatis puede favorecer el ingreso de VPH y éste el desarrollo del cáncer cérvico uterino. Ambas infecciones pueden dejar secuelas en la salud sexual y reproductiva. Objetivo: Determinar frecuencias VPH y C trachomatis en estudiantes universitarias asinto-máticas bajo 25 años, asociándolas con número de parejas sexuales (n°PSx) y tiempo de actividad sexual (TASx). Material y Método: Se procesaron 151 muestras/exo y endo cervicales para VPH y C. trachomatis, mediante reacción de polimerasa en cadena convencional y en tiempo real. Resultados: La frecuencia fue: VPH 21,8%, C. trachomatis 11,2% y co-infección 4,6%. De las jóvenes con infección por VPH, 80,6% presentó VPH alto riesgo oncogénico. El n°PSx se asoció fuertemente a VPH. Entre las jóvenes con co-infección VPH/C. trachomatis, 71,4% tenían tres o más PSx. Según TASx, C. trachomatis fue más frecuente (64,7%) entre 3-5 años que VPH. Conclusión: Se observó alta prevalencia de VPH y C trachomatis. Mujeres jóvenes con co-infección VPH/C. trachomatis podrían ser un grupo de alto riesgo con necesidad de mo-nitorear sus infecciones. Es sugerida la implementación de programas universitarios en educación, orientación y prevención en ITS.

Background: Human papilloma virus (HPV) and Chlamydia trachomatis are the most prevalent sexually transmitted infections (STIs), among teenagers and young people, with risk factors: active sex life and multiple partners. Chlamydia trachomatis infection may favor HPV infection and this, the development of cervical cancer. Both infections can lead to consequences on sexual and reproductive health. Objective: To determine frequency of HPV and C. trachomatis in asymptomatic university women less than 25 years, associating them with number of sexual partners (n°SxP) and time of sexual activity (TSxA). Material andMethods: 151 cervical samples for HPV and C. trachomatis, were processed by conventional and in real time reaction polymerase chain. Results: HPV 21, 8%, C. trachomatis 11, 2% and co-infection (HPV/C.trachomatis), 4.6%. Aimong HPV +, 80, 6% showed high risk HPV. The n°SxP was strongly associated with HPV. Aimong young coinfected HPV/C. trachomatis, 71.4% had 3 or more PSx. Chlamydia trachomatis was more frequent (64,7%) that HPV within range of 3-5 years according to the TSxA, Discussion: A high prevalence of HPV and C. trachomatis was observed. Young women with coinfection HPV/C. trachomatis could be a high-risk group need to monitor their infections. It suggests the implementation of university programs in education, counseling and prevention in sexual health.

Diagnóstico diferencial de tuberculosis en base a PCR en lesiones granulomatosas clasificadas histopatológicamente / PCR-based differential diagnosis of tuberculosis in histopathologically classified granulomatous lesions

El diagnóstico diferencial de tuberculosis en tejidos fijados en formalina e incluidos en parafina es necesario porque la morfología de la lesión tuberculosa es variada, hay diversos granulomas clasificados en necrobióticos, tuberculoideos, supurativos, sarcoideo, a cuerpo extraño/crónico inespecífico. Las lesiones granulomatosas ocurren en tuberculosis y también en otras infecciones (hongos, parásitos, brucelosis, lepra) en condiciones tóxicas, alérgicas, autoinmunes, tumores y otras. El diagnóstico histológico no es confirmatorio de tuberculosis y en ausencia de una baciloscopia positiva, se hace necesaria la confirmación molecular para el diagnóstico diferencial. Evaluamos la eficacia de la técnica de PCR para la detección de tuberculosis en tejidos fijados y comparamos esos resultados con la histología del granuloma y la baciloscopia. Analizamos 444 biopsias de diferentes tejidos (ganglios, piel, pleura, pulmón, intestino, tejido óseo, mama y otros) de 5 tipos de granulomas: G1.tuberculoideo con necrosis caseosa; G2.tuberculoideo sin necrosis caseosa; G3. supurativo; G4. sarcoideo l; G5. a cuerpo extraño/inespecífico. Utilizamos dos PCR-IS6110 nested para detección del complejo Mycobacterium tuberculosis y un pan PCR-hsp65 nested para detección de Mycobacterium spp. Los resultados obtenidos muestran que la detección de tuberculosis mediante PCR fue significativamente superior que mediante baciloscopia. G1: PCR 69,6%, baciloscopia 31,3%; G2: PCR 26,8%, baciloscopia 6,1%; G3: PCR 16,7%, baciloscopia 6,7%; G4: PCR 7%, baciloscopia 4%; G5: PCR 6,7%, baciloscopia 0%. Concluimos que el diagnóstico molecular de tuberculosis mediante un PCR robusto adaptado a tejidos fijados es eficaz, rápido, sensible y contribuye a la precisión del diagnóstico diferencial en diferentes tipos de granulomas (AU)

The differential diagnosis of tuberculosis in fixed paraffin embedded-tissues is necessary due to both the diverse morphology of tuberculous lesions and the varying histological types of granulomas (necrobiotic, tuberculoid, suppurative, sarcoidal and foreign body/inespecific). Granulomatous lesions occur in tuberculosis, in other infections (fungal, parasitic, brucelosis, lepra), in toxic, allergic and autoimmune, tumours and in conditions of unknown etiology. Diagnosis of tuberculosis cannot be confirmed by histopathology alone and in absence of a positive acid-fast bacilli (AFB) stain, molecular confirmation of tuberculosis is necessary for a correct differential diagnosis. The aim of our study was to assess PCR efficacy for mycobacterial infection detection in fixed tissues and to correlate those findings with granuloma histology and with AFB staining. We analyzed 444 biopsies from various tissues (lymph nodes, skin, pleura, lung, intestine, bone tissue, breast and others) with 5 granuloma types: G1: with caseous necrosis; G2: without caseous necrosis; G3: suppurative; G4: sarcoidal; G5: chronic/nonspecific. For molecular detection, we used nested PCR-IS6110 for Mycobacterium tuberculosis complex and a nested pan PCR-hsp65 for Mycobacterium sp.. The results obtained demonstrated that PCR was significantly better than AFB stain for tuberculosis detection. G1: PCR 69.6%, AFB staining 31.3%. G2: PCR 26.8%, AFB staining 6.1%; G3: PCR 16.7%, AFB staining 6.7%; G4: PCR 7%, AFB staining 4%. G5: PCR 6.7%, AFB staining 0%. We conclude that molecular diagnosis of tuberculosis using robust PCR-based testing adapted to fixed tissues is a fast, efficient and sensitive method that increases the accuracy of the differential diagnosis of granulomatous lesions (AU)

NaCl-triggered self-assembly of hydrophilic poloxamine block copolymers.

Tetronic 1307 (T1307) is a hydrophilic poloxamine (HLB>24) with a high molecular mass owing to its long PEO and PPO blocks. In spite of good biocompatibility, its use as a component of drug delivery systems is limited by its high critical micelle concentration (CMC) and temperature (CMT). The aim of this work was to elucidate whether the addition of NaCl or the combination of salts and temperature may bring T1307 micellization and gelling features into more practically useful values. Increasing NaCl concentration in the 0.154 M (isotonic) to 2M (hypertonic) range made the copolymer more hydrophobic and more prone to self-assemble into unimodal micelles, as observed by means of π-A isotherms, (1)H NMR, dynamic light scattering (DLS), small-angle neutron scattering (SANS), and pyrene fluorescence. The decrease in CMC and CMT observed for T1307 in 0.5 M NaCl medium (tolerable hypertonic solution), compared to water, notably favored the solubility of hydrophobic drugs such as curcumin and quercetin. Moreover, phase diagram, intrinsic viscosity and sol-to-gel transition were markedly affected by NaCl concentration. Overall, the strong dependence of T1307 self-assembly features on NaCl opens interesting possibilities for tuning the performance of T1307 as a component of nanocarriers and in situ gelling systems.

A Controlled Study of Tuberculosis Diagnosis in HIV-Infected and Uninfected Children in Peru.

BACKGROUND: Diagnosing tuberculosis in children is challenging because specimens are difficult to obtain and contain low tuberculosis concentrations, especially with HIV-coinfection. Few studies included well-controls so test specificities are poorly defined. We studied tuberculosis diagnosis in 525 children with and without HIV-infection. METHODS AND FINDINGS: 'Cases' were children with suspected pulmonary tuberculosis (n = 209 HIV-negative; n = 81 HIV-positive) and asymptomatic 'well-control' children (n = 200 HIV-negative; n = 35 HIV-positive). Specimens (n = 2422) were gastric aspirates, nasopharyngeal aspirates and stools analyzed by a total of 9688 tests. All specimens were tested with an in-house hemi-nested IS6110 PCR that took <24 hours. False-positive PCR in well-controls were more frequent in HIV-infection (P≤0.01): 17% (6/35) HIV-positive well-controls versus 5.5% (11/200) HIV-negative well-controls; caused by 6.7% (7/104) versus 1.8% (11/599) of their specimens, respectively. 6.7% (116/1719) specimens from 25% (72/290) cases were PCR-positive, similar (P>0.2) for HIV-positive versus HIV-negative cases. All specimens were also tested with auramine acid-fast microscopy, microscopic-observation drug-susceptibility (MODS) liquid culture, and Lowenstein-Jensen solid culture that took ≤6 weeks and had 100% specificity (all 2112 tests on 704 specimens from 235 well-controls were negative). Microscopy-positivity was rare (0.21%, 5/2422 specimens) and all microscopy-positive specimens were culture-positive. Culture-positivity was less frequent (P≤0.01) in HIV-infection: 1.2% (1/81) HIV-positive cases versus 11% (22/209) HIV-negative cases; caused by 0.42% (2/481) versus 4.7% (58/1235) of their specimens, respectively. CONCLUSIONS: In HIV-positive children with suspected tuberculosis, diagnostic yield was so low that 1458 microscopy and culture tests were done per case confirmed and even in children with culture-proven tuberculosis most tests and specimens were false-negative; whereas PCR was so prone to false-positives that PCR-positivity was as likely in specimens from well-controls as suspected-tuberculosis cases. This demonstrates the importance of control participants in diagnostic test evaluation and that even extensive laboratory testing only rarely contributed to the care of children with suspected TB. TRIAL REGISTRATION: This study did not meet Peruvian and some other international criteria for a clinical trial but was registered with the ClinicalTrials.gov registry: ClinicalTrials.gov NCT00054769.

Eficacia del diagnóstico diferencial de tuberculosis mediante reacción de polimerasa en cadena en lesiones granulomatosas de tejidos fijados en formalina e incluidos en parafina / Efficacy of PCR for the differential diagnosis of tuberculosis in granulomatous lesions of paraffin-embedded formalin fixed tissues

Background: Granulomatous lesions occur in tuberculosis (TB), other infections, toxic, allergic, and autoimmune diseases among others. In absence of a an acid-fast bacilli (AFB) confirmation of TB is necessary. Objective: To assess the efficacy of PCR for TB detection and to correlate with granuloma histology and AFB staining. Methods: We analyzed 380 fixed paraffin-embedded tissues (PETs) of granulomas with and without caseous necrosis; suppurative; sarcoidal; or of chronic nonspecific nature. Nested PCR-IS6110 for Mycobacterium tuberculosis complex (MTB) and a nested pan-Mycobacterium for the hsp65 gene were used for Mycobacterium spp detection. Results: PCR was more sensitive than AFB staining for all five catageories of granulomas: G1: PCR 71%, AFB staining 28%. G2: PCR 37%, AFB 8%. G3: PCR 17%, AFB staining 7%. G4: PCR 8%, AFB staining 4%. G5: PCR 6%, AFB staining 0%. Conclusions: Molecular diagnosis of TB using PCR-based testing is a fast, efficacious and sensitive method that increased the accuracy of PET histological diagnosis associated with granulomatous lesions.

Introducción: Lesiones granulomatosas ocurren en tuberculosis (TBC), otras infecciones, condiciones tóxicas, alérgicas y autoinmunes, entre otras. Con baciloscopia negativa, es necesario confirmar el diagnóstico de TBC. Objetivo: Evaluar la eficacia de la RPC para detectar TBC comparado con baciloscopia en relación a la histología del granuloma. Métodos: Analisis de 380 tejidos fijados en formalina e incluidos en parafina (TFFP) con diferentes tipos de granulomas: con necrosis caseosa; sin necrosis caseosa; supurativo; sarcoidal; a cuerpo extraño/inespecífico. Utilizamos RPC anidada-IS6110 para detección del complejo Mycobacterium tuberculosis (MTB) y una pan-RPC anidada-hsp65 para Mycobacterium spp. Resultados: La detección de TBC mediante RPC fue significativamente superior a baciloscopia en los cinco tipos de granuloma: G1: RPC 71%, baciloscopia 28%; G2: RPC 37%, baciloscopia 8%; G3: RPC 17%, baciloscopia 7%; G4: RPC 8%, baciloscopia 4%; G5: RPC 6%, baciloscopia 0%. Conclusión: El diagnóstico de TBC por RPC es un método rápido, eficaz y de gran sensibilidad, que aumenta la precisión del diagnóstico diferencial de lesiones granulomatosas de TFFP procesados rutinariamente en histopatología.

[Norovirus detection in children with community-acquired or nosocomial diarrhea at Guillermo Grant Benavente Hospital in Concepción, Chile]. / Detección de norovirus en niños con diarrea adquirida en la comunidad o nosocomial en el Hospital Guillermo Grant Benavente de Concepción, Chile.

INTRODUCTION: Norovirus (NoV) are RNA viruses highly contagious, stable in the environment, genetically variable, and the most common cause of viral sporadic acute gastroenteritis worldwide. This is the first study carried out in Concepcion, Chile, to investigate the presence of NoV as an etiologic agent of viral diarrheas in hospitalized children. Objective. To detect the presence and genogroup of NoV in children with diarrhea and to compare it with rotavirus (RV) and adenovirus (AdV). MATERIAL AND METHODS: A one year descriptive, prospective study in children 0-14 years old. A single diarrheic stool sample per patient was analyzed for the presence of NoV, RV and AdV. Clinical data were unknown at the moment of sampling. Real time RT-PCR with Taqman™ probes for NoV and the immunocromatography VIKIA™ kit for RoV /AV detection were used. RESULTS: Infection for NoV (25.5%) was significantly higher than for RV (15.9%) and AdV (6.2%). It was even greater in infants younger than 2yr. old (n: 103): NoV 34%, RV 17.5%, AdV 7.8%. Children 2-4 yr. old had 11.8% infection of NoV and RV. Children older than 4, only had 12% RV and 4% AdV. Children hospitalized for diarrhea (n: 92) had: 21.7% of both NoV and RV, and 7.6% AdV; whereas children hospitalized for other causes (n: 53) had 32.1% NoV,5.7% RV and 3.8% AV. The proportion of infection due to NoV was significantly higher in males (31.5%) than in females (19.4%). The average frequency during the year was higher for NoV (30.3%) than for RV (14.7%) except in summer. CONCLUSION: The presence of NoV was higher than RV in children with diarrhea. NoV infection showed defined characteristics regarding age, gender, seasonal occurrence and nosocomial transmission that are important epidemiological features.

Detección de norovirus en niños con diarrea adquirida en la comunidad o nosocomial en el Hospital Guillermo Grant Benavente de Concepción, Chile / Norovirus detection in children with community-acquired or nosocomial diarrhea at Guillermo Grant Benavente Hospital in Concepción, Chile

Introduction: Norovirus (NoV) are RNA viruses highly contagious, stable in the environment, genetically variable, and the most common cause of viral sporadic acute gastroenteritis worldwide. This is the first study carried out in Concepcion, Chile, to investigate the presence of NoV as an etiologic agent of viral diarrheas in hospitalized children. Objective. To detect the presence and genogroup of NoV in children with diarrhea and to compare it with rotavirus (RV) and adenovirus (AdV). Material and Methods: A one year descriptive, prospective study in children 0-14 years old. A single diarrheic stool sample per patient was analyzed for the presence of NoV, RV and AdV. Clinical data were unknown at the moment of sampling. Real time RT-PCR with Taqman™ probes for NoV and the immunocromatography VIKIA™ kit for RoV /AV detection were used. Results: Infection for NoV (25.5%) was significantly higher than for RV (15.9%) and AdV (6.2%). It was even greater in infants younger than 2yr. old (n: 103): NoV 34%, RV 17.5%, AdV 7.8%. Children 2-4 yr. old had 11.8% infection of NoV and RV. Children older than 4, only had 12% RV and 4% AdV. Children hospitalized for diarrhea (n: 92) had: 21.7% of both NoV and RV, and 7.6% AdV; whereas children hospitalized for other causes (n: 53) had 32.1% NoV,5.7% RV and 3.8% AV. The proportion of infection due to NoV was significantly higher in males (31.5%) than in females (19.4%). The average frequency during the year was higher for NoV (30.3%) than for RV (14.7%) except in summer. Conclusion: The presence of NoV was higher than RV in children with diarrhea. NoV infection showed defined characteristics regarding age, gender, seasonal occurrence and nosocomial transmission that are important epidemiological features.

Introducción: Los norovirus (NoV) son virus ARN altamente contagiosos, resistentes, variables genéticamente y una de las etiologías más frecuente de gastroenteritis viral esporádica mundial. Este es el primer trabajo en Concepción, Chile, de búsqueda de NoV como etiología viral de diarreas en niños hospitalizados. Objetivo: Determinar la presencia y genogrupo de NoV en niños con diarrea y compararla con la frecuencia de rotavirus (RV) y adenovirus (AdV). Material y Método: Estudio descriptivo, prospectivo de un año, en niños de 0-14 años ingresados por diarrea aguda o que la adquirieron dentro del hospital. La muestra de deposiciones diarreica se tomó una sola vez por paciente. Las fichas clínicas se analizaron al finalizar el estudio etiológico. Para la detección de NoV se utilizó RPC-TR a en tiempo real con sondas Taqman® y para detección de RV/AdV, el kit VIKIA® de inmunocromatografia. Resultados: La infección por NoV (25,5%) fue significativamente más frecuente que por RV (15,9%) y AdV (6,2%). La mayor presencia de infección fue en pacientes bajo2 años de edad (n: 103): NoV 34,0%, RV 17,5%, AdV 7,8%. La detección en niños hospitalizados por diarrea fue: NoV y RV 21,7% cada uno; AdV 7,6%. En niños con diarrea nosocomial hospitalizados por otras causas se detectó NoV en 32,1%, RV en 5,7% y AdV en 3,8%. La presencia de NoV fue significativamente mayor en varones (31,5%) que en niñas (19,4%). El promedio de diarreas durante el año fue mayor para NoV (30,3%) que para RV(14,7%), excepto en verano. Discusión y Conclusión: La presencia de NoV fue mayor que la de RoV en niños con diarrea y con una tendencia nosocomial que podría deberse a las características del virus que favorece infecciones de ambiente confinado, como hospitales, asilos y cruceros. La infección por NoV presentó características definidas, en edad, género, ocurrencia estacional y relevancia nosocomial, que aportan datos epidemiológicos importantes.

[Efficacy of PCR for the differential diagnosis of tuberculosis in granulomatous lesions of paraffin-embedded formalin fixed tissues]. / Eficacia del diagnóstico diferencial de tuberculosis mediante reacción de polimerasa en cadena en lesiones granulomatosas de tejidos fijados en formalina e incluidos en parafina.

BACKGROUND: Granulomatous lesions occur in tuberculosis (TB), other infections, toxic, allergic, and autoimmune diseases among others. In absence of a an acid-fast bacilli (AFB) confirmation of TB is necessary. OBJECTIVE: To assess the efficacy of PCR for TB detection and to correlate with granuloma histology and AFB staining. METHODS: We analyzed 380 fixed paraffin-embedded tissues (PETs) of granulomas with and without caseous necrosis; suppurative; sarcoidal; or of chronic nonspecific nature. Nested PCR-IS6110 for Mycobacterium tuberculosis complex (MTB) and a nested pan-Mycobacterium for the hsp65 gene were used for Mycobacterium spp detection. RESULTS: PCR was more sensitive than AFB staining for all five catagories of granulomas: G1: PCR 71%, AFB staining 28%. G2: PCR 37%, AFB 8%. G3: PCR 17%, AFB staining 7%. G4: PCR 8%, AFB staining 4%. G5: PCR 6%, AFB staining 0%. CONCLUSIONS: Molecular diagnosis of TB using PCR-based testing is a fast, efficacious and sensitive method that increased the accuracy of PET histological diagnosis associated with granulomatous lesions.

Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate. For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and PCR: a prospective case-control study.

BACKGROUND: The diagnosis of pulmonary tuberculosis presents challenges in children because symptoms are non-specific, specimens are difficult to obtain, and cultures and smears of Mycobacterium tuberculosis are often negative. We assessed new diagnostic approaches for tuberculosis in children in a resource-poor country. METHODS: Children with symptoms suggestive of pulmonary tuberculosis (cases) were enrolled from August, 2002, to January, 2007, at two hospitals in Lima, Peru. Age-matched and sex-matched healthy controls were enrolled from a low-income shanty town community in south Lima. Cases were grouped into moderate-risk and high-risk categories by Stegen-Toledo score. Two specimens of each type (gastric-aspirate, nasopharyngeal-aspirate, and stool specimens) taken from each case were examined for M tuberculosis by auramine smear microscopy, broth culture by microscopic-observation drug-susceptibility (MODS) technique, standard culture on Lowenstein-Jensen medium, and heminested IS6110 PCR. Specimens from controls consisted of one nasopharyngeal-aspirate and two stool samples, examined with the same techniques. This study is registered with ClinicalTrials.gov, number NCT00054769. FINDINGS: 218 cases and 238 controls were enrolled. 22 (10%) cases had at least one positive M tuberculosis culture (from gastric aspirate in 22 cases, nasopharyngeal aspirate in 12 cases, and stool in four cases). Laboratory confirmation of tuberculosis was more frequent in cases at high risk for tuberculosis (21 [14.1%] of 149 cases with complete specimen collection were culture positive) than in cases at moderate risk for tuberculosis (one [1.6%] of 61). MODS was more sensitive than Lowenstein-Jensen culture, diagnosing 20 (90.9%) of 22 patients compared with 13 (59.1%) of 22 patients (p=0.015), and M tuberculosis isolation by MODS was faster than by Lowenstein-Jensen culture (mean 10 days, IQR 8-11, vs 25 days, 20-30; p=0.0001). All 22 culture-confirmed cases had at least one culture-positive gastric-aspirate specimen. M tuberculosis was isolated from the first gastric-aspirate specimen obtained in 16 (72.7%) of 22 cases, whereas in six (27.3%), only the second gastric-aspirate specimen was culture positive (37% greater yield by adding a second specimen). In cases at high risk for tuberculosis, positive results from one or both gastric-aspirate PCRs identified a subgroup with a 50% chance of having a positive culture (13 of 26 cases). INTERPRETATION: Collection of duplicate gastric-aspirate specimens from high-risk children for MODS culture was the best available diagnostic test for pulmonary tuberculosis. PCR was insufficiently sensitive or specific for routine diagnostic use, but in high-risk children, duplicate gastric-aspirate PCR provided same-day identification of half of all culture-positive cases.

Diagnosis of pediatric pulmonary tuberculosis by stool PCR.

Pediatric pulmonary tuberculosis diagnosis is difficult because young children are unable to expectorate sputum samples. Testing stool for tuberculosis DNA from swallowed sputum may diagnose pulmonary tuberculosis. Hospitalized children with suspected tuberculosis had stool, nasopharyngeal, and gastric aspirates cultured that confirmed pulmonary tuberculosis in 16/236 patients. Twenty-eight stored stools from these 16 children were used to evaluate stool polymerase chain reaction (PCR) for tuberculosis diagnosis compared with 28 stool samples from 23 healthy control children. Two DNA extraction techniques were used: fast-DNA mechanical homogenization and Chelex-resin chemical extraction. DNA was tested for tuberculosis DNA with a hemi-nested IS6110 PCR. PCR after Fast-DNA processing was positive for 6/16 culture-proven tuberculosis patients versus 5/16 after Chelex extraction (sensitivity 38% and 31%, respectively). All controls were negative (specificity 100%). If sensitivity can be increased, stool PCR would be a rapid, non-invasive, and relatively bio-secure initial test for children with suspected pulmonary tuberculosis.

[Microsatellite instability and human papilloma virus genotypes in preneoplastic and neoplastic uterine cervix lesions]. / Inestabilidad microsatelital en lesiones preneoplásicas y neoplásicas del cuello uterino: Correlación con el genotipo del virus papiloma humano.

BACKGROUND: The association between some specific human papilloma virus (HPV) types and cervix cancer is well known. However, the genetic conditions that favor the development of cervical cancer are less well known. AIM: To determine the presence of satellite instability (MSI) in preneoplastic and neoplastic lesions of the cervix and correlate these findings with HPV genotypes. MATERIAL AND METHODS: Biopsy samples of cervical lesions were studied. Sixteen had low grade lesions, 22 had high grade lesions and 28 had an epidermoid cancer. Viral types were identified with polymerase chain reaction, dot-blot hybridization and restriction fragment length polymorphism. MSI was determined using a panel of eight highly informative microsatellites. RESULTS: Microsatellite instability in at least one locus was observed in 91, 56 and 69% of low grade lesions, high grade lesions and epidermoid carcinomas, respectively. MSI-High grade, MSI-Low grade instability and microsatellite stability were observed in 5, 60 and 46% of samples, respectively. Two of three samples with high grade instability had HPV 52 genotype. Other viral subtypes had frequencies that ranged from 78% to 100%, with the exception of HPV16 that was present in only 53% of samples with low grade instability. CONCLUSIONS: Two thirds of biopsy samples from cervical lesions had MSI, mechanism that can be involved in the first stages of cervical carcinogenesis. The low frequency of high grade instability, its association with HPV52 and the low frequency of HPV16 in samples with low grade instability, suggest different coadjutant mechanisms in cervical carcinogenesis.

Inestabilidad microsatelital en lesiones preneoplásicas y neoplásicas del cuello uterino: Correlación con el genotipo del virus papiloma humano / Microsatellite instability and human papilloma virus genotypes in preneoplastic and neoplastic uterine cervix lesions

Background: The association between some specific human papilloma virus (HPV) types and cervix cancer is well known. However, the genetic conditions that favor the development of cervical cancer are less well known. Aim: To determine the presence of satellite instability (MSI) in preneoplastic and neoplastic lesions of the cervix and correlate these findings with HPV genotypes. Material and methods: Biopsy samples of cervical lesions were studied. Sixteen had low grade lesions, 22 had high grade lesions and 28 had an epidermoid cancer. Viral types were identified with polymerase chain reaction, dot-blot hybridization and restriction fragment length polymorphism. MSI was determined using a panel of eight highly informative microsatellites. Results: Microsatellite instability in at least one locus was observed in 91, 56 and 69 percent of low grade lesions, high grade lesions and epidermoid carcinomas, respectively. MSI-High grade, MSI-Low grade instability and microsatellite stability were observed in 5, 60 and 46 percent of samples, respectively. Two of three samples with high grade instability had HPV 52 genotype. Other viral subtypes had frequencies that ranged from 78 percent to 100 percent, with the exception of HPV16 that was present in only 53 percent of samples with low grade instability. Conclusions: Two thirds of biopsy samples from cervical lesions had MSI, mechanism that can be involved in the first stages of cervical carcinogenesis. The low frequency of high grade instability, its association with HPV52 and the low frequency of HPV16 in samples with low grade instability, suggest different coadjutant mechanisms in cervical carcinogenesis

[Genotypying of clinical isolates of Helicobacter pylori by cagA, vacA and babA2 virulence associated genes. First detection of a babA2 positive strain in Chilean patients]. / Genotipificación de aislados clínicos de Helicobacter pylori en base a genes asociados a virulencia cagA, vacA y babA2. Primer aislamiento de una cepa babA2 positiva en pacientes chilenos.

BACKGROUND: Helicobacter pylori-associated gastroduodenal diseases depends on host characteristics, environmental conditions and bacterial virulence factors, such as cagA, vacA y babA2 gene products. Moreover, peptic ulcer disease has been related with cagA+, vacAs1m1 strains, while metaplasia and gastric cancer has been associated to cagA+, vacAs1 and babA2+ H pylori strains. Gene babA2 has not yet been described in clinical isolates from Chilean patients. AIM: To investigate the presence of cagA, vacA (s and m) and babA2 genes in clinical isolates of H pylori from Chilean patients. MATERIAL AND METHODS: Sixty six isolates from 41 patients were genotyped by PCR, using primers for s1a, s1b, s2, m1, m2, cagA and babA2 genes as previously described. RESULTS: cagA gene was detected in 16 isolates (24.2%) while vacAs1a, vacAs1b, vacAs2, vacAm1 and vacAm2 were detected in 28 (42.4%), 14 (21.2%), 17 (25.8%), 21 (31.8%) and 29 isolates (43.9%), respectively. One isolate (1.5%) was babA2 positive, being the first isolate with this genotype described in Chile. Besides the babA2+ genotype this clinical isolate also presented cagA+ and vacAs1a which has been related with metaplasia or gastric cancer. Five isolates showed an ulcerogenic profile cagA+, vacAs1m1. CONCLUSIONS: The results presented indicate the prevalence of vacAs1m1 genotype among the clinical isolates analyzed, and a low frequency of babA2 genotype.

[Detection of human papilloma virus in cytologic samples or biopsies of the cervix]. / Estudio comparativo de detección del virus papiloma humano (VPH) en muestras citológicas y biopsias de cuello uterino.

BACKGROUND: The DNA quality for the detection and typification of Human Papilloma Virus (HPV) varies according to the type of sample in which it is studied. This may affect the sensitivity and specificity of the method employed. AIM: To study the yield and specificity of HPV detection and typification in uterine cervical samples obtained by cervical brushing fresh frozen and formalin fixed tissue. MATERIAL AND METHODS: Cytological, fresh frozen and fixed tissue samples from 44 patients (nine with low grade lesions and 35 with high grade lesions) were studied. Nested polymerase chain reaction for genes E6/E7 was used to typify HPV groups as low risk or high risk. RESULTS: Of all the cytological samples obtained by brushing 84% of fixed samples and 43% of fresh frozen samples were positive for HPV. The yields were significantly different when comparing brushing with fixed tissue or fresh frozen tissue and fixed tissue with fresh frozen tissue (p <0.05). The frequency of high risk HPV fluctuated from 41% in fresh frozen tissue to 98% in cytological samples. Low risk HPV was detected in 16% of fresh frozen tissue and 68% of cytological samples. A mixed infection was detected in 66%, 41% and 14% of cytological, fresh frozen and fixed tissue samples respectively. CONCLUSIONS: Cytological samples obtained by brushing had the highest yield for the detection of cervical infection with HPV.

Alterations in the profile of blood cell types during malaria in previously unexposed primigravid monkeys.

Malaria in nonimmune, primigravid women threatens both mother and fetus. We used the Plasmodium coatneyi/rhesus monkey model to examine factors associated with this. Clinical and immunologic responses during the blood stage of chronic malaria (4 months) were evaluated in 8 malaria-naive primigravid (PMI) and 8 age-matched nulligravid (NMI) infected monkeys, compared with those in 8 primigravid, noninfected control monkeys. Although parasitemia levels were similar, recrudescence was more frequent and prolonged, and anemia was more severe in PMI than in NMI monkeys. During infection, CD2+, CD4+, and CD8+ lymphocyte levels were higher in NMI than in PMI monkeys. Monocyte and neutrophil levels were lower in PMI than in NMI monkeys. During chronic, untreated malaria, NMI monkeys had a B lymphocyte count 23 times greater than that of PMI monkeys. Pregnancy-induced immunomodulation, defined as a lack of appropriate cellular responses to malaria, was indiscernible until the immune system was challenged by a pathogen.