Evidencia serológica de ehrlichiosis humana en Ancash, Perú / Serological evidence of human ehrlichiosis in Ancash, Perú

Se desarrolló y estandarizó una prueba de inmunofluorescencia indirecta para detección de IgG e IgTotal para el diagnóstico de Ehrlichiosis humana. Se utilizó como antígeno, a células DH82 infectadas con Ehrlichia chaffeensis cepa Sapulpa, se consideró una dilución de 1/64 como positivo. Se evaluó 130 sueros de pacientes febriles negativos para Rickettsiosis y enfermedad de Carrión procedentes de Ancash, que ingresaron al Instituto Nacional de Salud entre los años 2004 a 2006. Se encontró que 12 (9,2%) sueros fueron positivos a Ehrlichiosis. Teniendo en cuenta que es una enfermedad emergente y con el desarrollo de esta prueba, es recomendable iniciar estudios epidemiológicos y de vigilancia de la Ehrlichiosis en el Perú.

We developed and standardized an indirect immunofluorescence test for detection of IgG and IgTotal for diagnosing human Ehrlichiosis. It was used as antigen to DH82 infected cells with Ehrlichia chaffeensis Sapulpa strain; it was considered a dilution of 1/64 as positive. We evaluated sera from 130 febrile patients negative for Rickettsiosis and Carrion disease from Ancash, who entered at Instituto Nacional de Salud (Lima, Peru) between 2004 and 2006. We found that 12 (9.2%) sera were positive for Ehrlichiosis. Given that Ehrlichiosis is an emerging disease and with the development of this test, it should start monitoring and epidemiological studies of Ehrlichiosis in Peru.

Evaluación de pruebas de Elisa e inmunofluorescencia indirecta para la detección de anticuerpos IgM contra Rickettsiosis / Elisa and indirect immunofluorescent assays evaluation for IgM antibodies detection against Rickettsiosis

Se evaluó dos pruebas modificadas de ELISA e inmunofluorescencia indirecta (IFI) para la detección de IgM basadas en el lisado total de Rickettsia akari para el diagnóstico de Rickettsiosis. Se usaron 55 sueros negativos, 100 sueros positivos confirmados por IFI IgG Total y15 sueros con diagnóstico confirmado para otras enfermedades bacterianas procedentes de la seroteca del Instituto Nacional de Salud. La prueba de ELISA IgM tuvo una sensibilidad de 78,0 por ciento, especificidad de 80,0 por ciento, valor predictivo positivo de 87,6 por ciento y el valor predictivo negativo de 66,7 por ciento, con una reacción cruzada con otras etiologías bacterianas de 20 por ciento (3/15). La prueba de IFI IgM tuvo una sensibilidad de 82,0 por ciento, especificidad de 91,7 por ciento, valor predictivo positivo de 94,3 por ciento y valor predictivo negativo de 75,3 por ciento, con una reacción cruzada de 13 por ciento (2/15). El método ELISA IgM no es una prueba útil, a diferencia de la prueba IFI IgM que podría ser aplicada en el diagnóstico de Rickettsiosis para estudios epidemiológicos y de vigilancia en el Perú.

We evaluated two modified tests of ELISA and indirect immunofluorescence (IFI) for detecting IgM based on the total lysate of Rickettsia Akari for Rickettsiosis diagnosing. 55 negative sera were used, 100 positive sera confirmed by IFI IgGTotal and 15 sera with a confirmed diagnosis for other bacterial diseases from the serum of the Instituto Nacional de Salud (Lima, Perú). IgM ELISA test had a sensitivity of78.0 per cent, 80.0 per cent specificity, positive predictive value of 87.6 per cent and negative predictive value of 66.7 per cent, and have a 20 per cent (3/15) cross-reaction with other bacterial aetiologies. The IFI IgM test had a sensitivity of 82.0 per cent, specificity of 91.7 per cent, positive predictive value of 94.3 per cent and negative predictive value of 75.3 per cent, with a 13 per cent (2/15) cross-reaction. The IgM ELISA test is not useful, unlike the IFI IgM test that could be applied in diagnosing Rickettsiosis for surveillance and epidemiological studies in Peru.

Patogenicidad de las amebas de vida libre aisladas de fuentes de agua en Lima / Patogenicity of free-living amoebas isolates in body waters from Lima

Objetivos. Determinar la presencia de amebas de vida libre (AVL) en fuentes de agua del departamento de Lima y evaluar su capacidad patógena en ratones normales e inmunosuprimidos. Materiales y métodos. Se recolectaron muestras de agua de ríos, lagos, piscinas y pozos de Lima. El aislamiento se realizó por cultivo en agar no nutritivo al 2 por ciento con E. coli o E. aerogenes a 37 °C. Posteriormente se instiló en ratones normales e inmunosuprimidos (betametasona 1,4 mg/mL, dosis única) una suspensión con las amebas aisladas. A los 15 díasse evaluaron histológicamente los daños en ambos grupos. Resultados. Se identificaron AVL en 40/83 muestras, 31,3 por ciento de las muestras se desarrollaron en los cultivos. Se aislaron 26 cepas de AVL de siete géneros (Hartmannella, Acanthamoeba, Mayorella, Naegleria, Vahlkampfia, Vannella y Saccamoeba), Acanthamoeba fue la más frecuente (44,2 por ciento). Se encontraron AVL en 53,8 por ciento de los ratones inmunosuprimidos y 15,4 por ciento de los normales (p menor que 0,05). Conclusiones. Existen AVL en cuerpos de agua de Lima, las cuales tienen un potencial patógeno en pacientes inmunosuprimidos, existiendo un riesgo de infección amebiana asociada con estas fuentes de agua.

Objectives. To determine the presence of free-living amoeba (FLA) in water bodies from Lima department and assess their pathogenic ability in normal mice and immunosuppressed. Material and methods. Water samples were collected from rivers, lakes, swimmingpools and wells in Lima, Peru. The isolation was performed by culture in non-nutritious agar with 2 per cent of E. coli or E. aerogenes at 37 °C. Subsequently were injected into normal mice and immunosuppressed (betamethasone 1.4 mg/mL, a single dose) with a suspension of amoeba isolated. For the 15 days were assessed histological damage in both groups. Results. AVL were identified in 40/83 samples, 31.3 per cent of the samples were developed in cultures. 26 strains were isolated from AVL of seven genera (Hartmannella, Acanthamoeba,Mayorella, Naegleria, Vahlkampfia, Vannella and Saccamoeba), Acanthamoeba was the most frequent (44.2 per cent). AVL was found in 53.8 per cent of immunosuppressed mice and 15.4 per cent of the normal (p minor that 0.05). Conclusions. AVL exist in water bodies from Lima, which have a potential pathogen in immunosuppressed patients, there is a risk of amoebic infection associated with these water sources.

Human spotted fever rickettsial infections.

Serum specimens from patients at 4 sites in Peru were tested for evidence of spotted fever group rickettsial infection. Results showed that 30 (18%) of 170 patients had spotted fever group rickettsial infections, which likely caused their illnesses. These findings document laboratory-confirmed spotted fever from diverse areas of Peru.

Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study.

Sweden is an area potentially endemic for spotted fever rickettsioses. Rickettsia helvetica has been isolated from its tick vector Ixodes ricinus, and in a handful of cases linked to human disease. This study demonstrates for the first time in Sweden the transmission of rickettsial infection after a tick bite and the attack rate in an endemic area. We present three cases of documented rickettsial infection and a prospective serological study of Swedish recruits who were trained in the area where the patients lived and showed seroconversion to spotted fever rickettsiae. All patients showed a four-fold increase in antibody titer to the spotted fever rickettsia, R. helvetica, and immunohistochemical examination revealed rickettsia-like organisms in the walls of skin capillaries and veins. Electron microscopy showed organisms resembling R. helvetica and immunogold labeling with two anti-rickettsial antibodies demonstrated specific labeling of the rickettsial organisms in the skin biopsy specimens. Eight of the thirty-five recruits showed a four-fold increase in IgG titer reflecting a high rate of exposure. The results of this study demonstrate that spotted fever rickettsioses should be taken into consideration in the diagnosis of tick-transmitted infections in Sweden.

Phylogenetic analysis of a novel molecular isolate of spotted fever group Rickettsiae from northern Peru: Candidatus Rickettsia andeanae.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

Characterization of spotted fever group rickettsiae in flea and tick specimens from northern Peru.

Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.

Inmunohistoquímica en el diagnóstico de fiebre amarilla / Immunohistochemistry in the diagnosis of yellow fever

Objetivo: Confirmar la presencia del antígeno del virus de la fiebre amarilla en muestras de hígado. Material y Métodos: Se aplicó la técnica de inmunohistoquímica en muestras de hígado de 34 pacientes procedentes de las diferentes regiones del país con diagnóstico clínico de fiebre amarilla, durante los años 1999 a 2001. A los cortes de tejido (hígado parafinizado) se aplicó esta técnica con anticuerpos monoclonales y policlonales contra FA y el complejo biotina-streptavidina. El control positivo fue el hígado de un paciente con diagnóstico serológico e histopatológico de fiebre amarilla y el control negativo una muestra de hígado obtenida de la necropsia de un paciente con patología no hepática. Resultados: La positividad se dio por una tinción marrón en el citoplasma hepático. Las muestras negativas carecen de esta tinción. Se confirmó la presencia de antígeno de fiebre amarilla durante los años 1999 al 2001. Las 34 muestras procedieron de los departamentos de San Martín, Junín, Cuzco, Huánuco, Loreto, Pasco, y Ucayali. Conclusión: La técnica de inmunohistoquímica constituye una herramienta de diagnóstico por su alta sensibilidad y especificidad (ambas 90 por ciento ), para estudios epidemiológicos en los que se puede realizar un diagnóstico retrospectivo.

Evidence of rickettsial and leptospira infections in Andean northern Peru.

Between May and October 2002, a cluster of acute febrile illnesses occurred in the subtropical Andean foothills of Peru. Serologic evidence in villages where disease had been documented showed that the prevalence of IgM antibody to Leptospira ranged from 6% to 52%, that of IgM antibody to spotted fever group (SFG) rickettsia ranged from 10% to 19%, and that of IgM antibody to Coxiella burnetii from 1% to 15%. Measurement of IgG antibodies for SFG rickettsiae suggested that this disease was endemic. In contrast, IgG antibodies against C. burnetii were largely absent. In humans, microagglutination tests identified pathogenic variants of Leptospira. The presence of an SFG rickettsial infection was confirmed in four febrile patients following polymerase chain reaction and sequencing of the conserved 17-kD common antigen gene (htrA). Collectively, these analyses indicated that Rickettsia sp., C. burnetii, and Leptospira sp. were circulating in the region during the time of disease outbreak and implicate the involvement of an as yet undetermined SFG rickettsia in northwestern Peru.

An outbreak of leptospirosis among Peruvian military recruits.

Acute undifferentiated febrile illnesses are common in tropical developing countries but are difficult to diagnose on clinical grounds alone. Leptospirosis is rarely diagnosed, despite evidence that sporadic cases and epidemics continue to occur worldwide. The purpose of this study was to diagnose an outbreak of acute undifferentiated febrile illness among Peruvian military recruits that developed after a training exercise in the high jungle rainforest of Peru. Of 193 military recruits, 78 developed an acute febrile illness with varied manifestations. Of these, 72 were found to have acute leptospirosis by a microscopic agglutination test (MAT). An enzyme-linked immunosorbent assay using Leptospira biflexa antigen was insensitive for the detection of anti-leptospiral IgM antibodies compared with the MAT (20 of 72, 28%). This outbreak of acute undifferentiated febrile illness among Peruvian military recruits was due to leptospirosis. High clinical suspicion, initiation of preventative measures, and performance of appropriate diagnostic testing is warranted in similar settings to identify, treat, and prevent leptospirosis.

Rickettsiosis of the genus Rickettsia in South America.

In South America, human cases of infection by the genus Rickettsia have been described in several countries in the last twenty years. The role of international organizations, such as the Centers for Disease Control and Prevention in Atlanta, Georgia, USA and the World Health Organization Collaborating Center for Tropical Diseases at the University of Texas Medical Branch at Galveston, Texas, USA, was very important in the last twenty years for the development of surveillance systems and for the increase in notification of rickettsial diseases by the countries of South America. We hope that the next goal will be prevention and control of rickettsial diseases in the countries of South America, as well as maintaining the programs developed during the last twenty years, so that a good health system and improved social conditions will be possible.