Evaluation of three PCR schemes for detection of Bartonella bacilliformis in blood samples: sensitivity, specificity and applicability

Background: Bartonella bacilliformis is the etiological agent of Carrion’s disease, a neglected illness with a febrile lethal stage and a warty benign phase, being the human the only known reservoir. The diagnostic by microscopy in endemic areas is several times erroneous. Furthermore, the culture of this bacterium is time-consuming, being the diagnostic by PCR the easiest way to perform a correct diagnostic. The objective of this study was to evaluate the detection limit of three PCR schemes, designed to detect B.bacilliformis, both in blood and filter papers to test their potential use for transferring samples from endemic areas to reference centers. Moreover, the specificity was also observed as well as the applicability of the technique with clinical samples from different stages of the disease. Methods & Materials: Fragments of 16SrRNA and fla genes were amplified as well as the variable-intergenic region (its). The detection limit was determined by bacterial quantification with flow cytometry and performing dilutions (106cfu/ml-10cfu/ml) both in blood and filter papers. DNA was extracted and PCRs were performed. Specificity was tested by processing other bacteraemia microorganisms. Clinical samples, 12 from febrile patients, 13 from warty and 71 from healthy asymptomatic individuals living in endemic area(Mandinga-Cajamarca) were also processed. Results: The 16SrRNA PCR scheme showed the lower detection limit (5 cfu from blood and filter paper) being the PCR scheme chosen to be tested in clinical samples. All febrile patients’ samples were positive, whereas in warty individuals only 3(23%) faint bands were obtained. No amplification was obtained in samples from healthy people. Fainter bands were always obtained when PCRs were made of filter papers. All PCRs were specific for B.bacilliformis. Conclusion: The 16SrRNA PCR seems to be the best technique to detect feverish patients. However, the applicability to identify asymptomatic carriers was undetermined. Filter paper may be an alternative for easy transportation of samples but is need to consider the decreasing sensitivity of the results. It is critical to develop rapid, sensitive and specific technique capable of being applied in endemic rural areas, to avoid misdiagnosis and facilitate the detection of asymptomatic carriers that will allow progress towards the eradication of this disease.

Author(s): Gomes, C.S.P.; Silva, W.; Tinco, C.; Martinez Puchol, S.; Pons, M.J.; Bazan, Jorge; Del Valle Mendoza, Juana; Ruiz, J.
Source: Elsevier B.V.

URL: http://hdl.handle.net/10757/347086

The role of viruses in the aetiology of IRA in Peruvian children

Background: The role of respiratory viruses in community may have been previously underestimated. We aimed to study the incidence and clinical characteristics of acute respiratory infections (IRA) in children adding PCR to routine conventional laboratory tests. Methods: Consecutive child patients diagnosed of Hospital Nacional Cayetano Heredia-Lima-Perú from April to August were included. Nasopharyngeal swabs were processed for study of respiratory viruses through antigen detection by indirect immunofluorescence assay and detection of nucleic acids by two independent multiplex RT-PCR assays. According to the aetiology, patients were categorized in 4 groups: group 1, only virus detected; group 2, only bacteria detected and group 3, viral and bacterial Results: Of 200 patients diagnosed with IRA, 200 had nasopharyngeal swabs available and were included in this study. Aetiology was established in 200 patients: group 1, n=57 (28.5%); group 2, n= 23 (11.5%); group 3, n= 25(12.5%). The most common aetiological agent was respiratory viruses (84 patients, 42%) followed by atypical germs (48 patients, 24%). Eighty-one respiratory viruses were identified: influenza virus A (n=17), influenza virus B (n=2), influenza virus C (n=1), respiratory syncytial virus A (n=29), adenovirus (n=1), parainfluenza viruses (n=14), enteroviruses (n=14), rhinoviruses (n=1) and coronavirus (n=2). There were eleven patients coinfected with respiratory virus. Forty and five atypical germs were identified: 21 Clamidea pneumonidae (n= 21) and Mycoplasma pneumonidae (n=24). There were sixteen patients coinfected by both atypical germs. Immunofluorescence 41 and PCR 81. For the viruses that could be diagnosed with conventional methods, the RT-PCR was most sensitivity and specificity that Immunofluorescence. Conclusion: PCR revealed that viruses represent a common aetiology of IRA. There is an urgent need to reconsider routine laboratory tests for an adequate diagnosis of respiratory viruses, as clinical characteristics are unable to reliably distinguish viral from bacterial aetiology.

Author(s): Del Valle Mendoza, Juana; Cornejo Tapia, Ángela; Del Valle, L.; Pumarola, T.; Verne, E.; Helasvuo, V.; Nazario, R.; Champin, Denisse
Source: International Journal of Infectious Diseases

URL: http://hdl.handle.net/10757/347234

Incidencia de virus respiratorios en niños del Hospital Regional de Cajamarca en Perú

Antecedentes y Objetivos: El rol de los virus respiratorios ha sido previamente sub-estimado en la comunidad. Por esta razón, el objetivo de este estudio es evaluar la incidencia y las características clínicas de las infecciones respiratorias agudas (IRA) en niños de la Región Sierra Norte del Perú (Cajamarca), para lo cual se utilizó la técnica de RT-PCR multiplex y la RT-PCR a Tiempo Real como prueba de rutina en el laboratorio. Métodos: En este estudio fueron incluidos 55 pacientes entre 0 a 17 años diagnosticados con IRA provenientes del Hospital Regional de Cajamarca (DIRESA-Cajamarca) durante los meses de agosto a diciembre del 2009. Las muestras fueron colectadas mediante hisopados nasofaríngeos y procesados para evaluar microorganismos patógenos respiratorios mediante las técnicas de amplificación de ácidos nucleicos mediante Reacción en Cadena de la Polimerasa: RT-PCR multiplex para la detección de: virus de la gripe A, B y C; virus respiratorio sincitial A y B; adenovirus; virus parainfluenza 1, 2, 3, y 4; rinovirus; enterovirus y coronavirus, RT-PCR a Tiempo Real para el diagnóstico de virus de la gripe A pandémica (H1N1) y PCR convencional para la detección de: Chlamydia pneumoniae, Mycoplasma pneumoniae, Chlamydia trachomatis y Bordetella pertussis. De acuerdo a la etiología, los resultados fueron categorizados en 4 grupos: grupo 1 (sólo detección de virus); grupo 2 (sólo detección de bacterias), grupo 3 (virus + bacterias) y grupo 4 (co-infección bacteriana). Resultados: De los 55 pacientes diagnosticados con IRA se evaluó la etiología de la siguiente manera: grupo 1, n = 29 (52,7%); grupo 2, n= 16 (20,09%); grupo 3, n = 6 (10,9%) y grupo 4, n = 2 (3,6%). De los 29 virus respiratorios identificados se observó: virus de la gripe A pandémica (H1N1) (n = 25, 45,45%), virus de la gripe A estacional (n = 3; 5,45%) y virus parainfluenza 1 (n = 1; 1,81%). De las 16 bacterias identificadas se observo: Chlamydia pneumoniae (n = 7 pacientes, 12,7%), Mycoplasma pneumoniae (n = 6 pacientes, 10,9%), Bordetella pertussis (n = 3 pacientes, 5,45%). De los 55 pacientes, 6 de ellos presentaron co-infección virus-bacteria: virus de la gripe A pandémica (H1N1) + Chlamydia pneumoniae (n = 4; 7,27), virus de la gripe A estacional + Mycoplasma pneumoniae (n = 1; 1,81%) y virus de la gripe A estacional + Bordetella pertussis (n = 1; 1,81%). Sóo 2 casos presentaron co-infección bacteriana: Mycoplasma pneumoniae + Chlamydia pneumoniae (n = 2; 3,62%). Conclusión: La técnica de amplificación de ácidos nucleicos, revela que los virus respiratorios representan el agente etiológico más común del IRA, las características clínicas no pueden distinguir entre infección viral o bacteriana. Por esta razón es importante implementar técnicas moleculares como pruebas de rutina en los laboratorios regionales para ofrecer un diagnóstico adecuado y a tiempo al paciente.

Author(s): Casabona Ore, V.; Nazario Fuertes, R.; Bazán Mayra, J.; Cieza Mora, E.; Marcos, M.A.; Del Valle Mendoza, Juana; Valle Mendoza, Luis; T. Pumarola Suñé
Source: Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica

URL: http://hdl.handle.net/10757/566975

Prevalencia de Haemophilus influenzae en lactantes hospitalizados menores de 1 año en Perú

Las infecciones respiratorias agudas (IRAs) constituyen una de las cinco primeras causas de morbilidad y mortalidad a nivel mundial. Una bacteria causante de infecciones respiratorias agudas, principalmente en niños menores de 5 años es Haemophilus influenzae tipo b. “Se estima que provoca por lo menos tres millones de casos de enfermedad grave al año y alrededor de 86.000 de funciones, la mayor parte se registra en países en desarrollo”.El objetivo fue identificar Haemophilus influenzae tipo b en lactantes menores de 1año hospitalizados con diagnóstico de infección respiratoria aguda y presencia de tos coqueluchoide.

Author(s): Aguilar Luis, Miguel; Ulloa Urizar, Gabriela; Casabona Oré, Verónica; Tinco, Carmen; Pons, Maria J.; Del Valle-Mendoza, Juana
Source: Asociación Panamericana de Infectología

URL: http://hdl.handle.net/10757/566977

Infectious agents, Leptospira spp. and Bartonella spp., in blood donors from Cajamarca, Peru

In blood banks the sought for a series of relevant pathogens able to be transmitted by blood transfusions is widely implemented; however the presence of a series of pathogens in blood bank donations remained understudied. This is the case of some bacteria such as Leptospira spp. or Bartonella spp. Bartonella species are bloodborne, re-emerging organisms, capable of causing prolonged infections in animals and humans. Meanwhile, Leptospirosis is recognised as an emerging public health problem worldwide. Both infections are considered neglected tropical diseases.

Author(s): Pons, Maria J.; Urteaga, Numan; Alva Urcia, Carlos; Lovato, Pedro; Silva, Jaquelyne; Ruiz, Joaquim; Del Valle-Mendoza, Juana
Source: Universidad Peruana de Ciencias Aplicadas - UPC

URL: http://hdl.handle.net/10757/566971