Diagnosis of Plasmodium vivax by Loop-Mediated Isothermal Amplification in Febrile Patient Samples from Loreto, Perú.

Plasmodium vivax is co-endemic with Plasmodium falciparum in Peru, and optimum management requires distinguishing these two species in the blood of patients. For the differential identification of P. vivax and other Plasmodium spp., the LoopampTM Malaria Pan Detection Kit in combination with the Loopamp Malaria Pv Detection Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) was used to evaluate 559 whole blood samples collected in 2017 from febrile patients with suspected malaria attending different health facilities in the Loreto region. The Loopamp Malaria Pan Detection Kit showed a sensitivity of 87.7% (95% CI: 83.5-91.9) and a specificity of 94.4% (95% CI: 91.9-96.9) and good agreement with PCR (Cohen's kappa 0.8266, 95% CI: 0.7792-0.874). By comparison, the Loopamp Malaria Pv Detection Kit showed a similar sensitivity (84.4%, 95% CI: 79.0-89.7) and specificity (92.4%, 95% CI: 89.7-95.0) and substantial agreement with PCR (Cohen's kappa: 0.7661, 95% CI: 0.7088-0.8234).

Evaluation of Plasmodium falciparum MSP10 and its development as a serological tool for the Peruvian Amazon region.

BACKGROUND: Different antigens are needed to characterize Plasmodium falciparum infection in terms of seroreactivity and targets for invasion inhibition, in order to guide and identify the proper use of such proteins as tools for the development of serological markers and/or as vaccine candidates. METHODS: IgG responses in 84 serum samples from individuals with P. falciparum infection [classified as symptomatic (Sym) or asymptomatic (Asym)], or acute Plasmodium vivax infection, from the Peruvian Amazon region, were evaluated by enzyme-linked immunosorbent assays specific for a baculovirus-produced recombinant protein P. falciparum Merozoite Surface Protein 10 (rMSP10) and for non-EGF region selected peptides of PfMSP10 selected by a bioinformatics tool (PfMSP10-1, PfMSP10-2 and PfMSP10-3). Monoclonal antibodies against the selected peptides were evaluated by western blotting, confocal microscopy and inhibition invasion assays. RESULTS: Seroreactivity analysis of the P. falciparum Sym- and Asym-infected individuals against rMSP10 showed a higher response as compared to the individuals with P. vivax acute infection. IgG responses against peptide PfMSP10-1 were weak. Interestingly high IgG response was found against peptide PfMSP10-2 and the combination of peptides PfMSP10-1 + PfMSP10-2. Monoclonal antibodies were capable of detecting native PfMSP10 on purified schizonts by western blot and confocal microscopy. A low percentage of inhibition of merozoite invasion of erythrocytes in vitro was observed when the monoclonal antibodies were compared with the control antibody against AMA-1 antigen. Further studies are needed to evaluate the role of PfMSP10 in the merozoite invasion. CONCLUSIONS: The rMSP10 and the PfMSP10-2 peptide synthesized for this study may be useful antigens for evaluation of P. falciparum malaria exposure in Sym and Asym individuals from the Peruvian Amazon region. Moreover, these antigens can be used for further investigation of the role of this protein in other malaria-endemic areas.

BALB/c Mice resist infection with Bartonella bacilliformis.

BACKGROUND: Bartonellosis due to Bartonella bacilliformis is a highly lethal endemic and sometimes epidemic infectious disease in South America, and a serious public health concern in Perú. There is limited information on the immunologic response to B. bacilliformis infection. The objective of this research was to produce experimental infection of BALB/c mice to B. bacilliformis inoculation. FINDINGS: BALB/c mice were inoculated with 1.5, 3.0 or 4.5 x 108 live B. bacilliformis using different routes: intraperitoneal, intradermal, intranasal, and subcutaneous. Cultures of spleen, liver, and lymph nodes from one to 145 days yielded no cultivable organisms. No organs showed lesions at any time. Previously inoculated mice showed no changes in the reinoculation site. CONCLUSION: Parenteral inoculation of live B. bacilliformis via different infection routes produced no macroscopic or microscopic organ lesions in BALB/c mice. It was not possible to isolate B. bacilliformis using Columbia blood agar from 1 to 15 days after inoculation.

Acquired sulphonamide resistance genes in faecal Escherichia coli from healthy children in Bolivia and Peru.

Antimicrobial resistance and sulphonamide resistance determinants were studied in 20 co-trimoxazole resistant Escherichia coli in faecal samples from healthy children in Bolivia and Peru. Methods used were disc diffusion susceptibility tests, PCR, sequence analysis and plasmid conjugation assays. All isolates but one were resistant to at least two different classes of antimicrobials; 19 isolates also carried at least one sul-gene. The most frequent gene was sul2 followed by sul1 and sul3, which was detected in one isolate. This is the first observation of sul3 on the American continent. In conclusion, the high prevalence of sul-genes in this material of faecal commensal E. coli isolates points to a potential role of the faecal flora in the emergence and spread of antimicrobial resistance.

Report of an unusual case of persistent bacteremia by Bartonella bacilliformis in a splenectomized patient.

We report a case of a 56-year-old man with a history of splenectomy for idiopathic thrombocytopenic purpura who developed persistent bacteremia in the acute phase of human bartonellosis. This patient did not develop hemolytic anemia. Only after several courses of antibiotic treatment was the infection eradicated. This is an unusual case of overwhelming post-splenectomy infection by Bartonella bacilliformis, which provides clinical evidence that the spleen is a critical effector organ of clearance of this infection as well as the effector organ of bartonellosis-associated hemolytic anemia.

Identificación de Bartonella sp mediante reacción en cadena de la polimerasa y métodos microbiológicos / Identification of Bartonella sp by polymerase chain reaction and microbiological methods

En el Perú la Angiomatosis bacillar (AB) y la enfermedad de Arañazo de Gato (EAG) han sido reportados confirmando el diagnóstico en laboratorios de investigación o del extranjero, no contándose con métodos de rápido diagnóstico. Se captaron 23 pacientes y se estudiaron 8 muestras de tejidos con sospecha de EAG,AB o Verruga Peruana (VP). Se realizaron cultivos en condiciones requeridas. Extracción de DNA de sangre, cultivos y tejidos mediante buffers de digestión fueron estandarizados para cada caso. Se amplificó el DNA con primers 16S-ITRFf y 23S-ITRr ó CS-ITRf y CSITRr; en gel de agarosa 1 por ciento, y cuantificados con DNA -Hind III. Cultivos controles mostraron banda en B. bacilliformis de 948 pb, B. quintana 1310 pb, B henselae 1427 pb. M. tuberculosus 563 pb. Un paciente con diagnóstico confirmado de EAG por B. henselae, mostró banda concordante con control de B. henselae utilizando los primers 16S y 23S. Una muestra de VP mostró banda concordante con control de B. bacilliformis. una muestra confirmada por patología de AB mostró bandas concordantes a B. henselae utilizando el primer CS. Se confirmaron casos de EAG, AB y VP en base a muestras de aspirados de ganglio o biopsia mediante PCR. El PCR permitió el diagnóstico diferencial de adenomagalias por EAG y Micobacterias, y de AB en la Verruga Peruana.

A prospective study of cat-scratch disease in Lima-Peru

Cat-Scratch Disease (CSD) is a benign lymphadenitis that may progress to severe or recurrent forms, and it is occasionally associated with morbidity. Between January of 1998 and March of 1999, forty-three suspected CSD patients were assessed in the Hospital Cayetano Heredia and the Instituto de Salud del Niño, in Lima, Peru. Twelve patients had a confirmed diagnosis, 8 of whom were women, and the mean age was 10 years old. The majority (53 percent) of the cases were encountered in the summer. All patients reported having had contact with cats. Fever, malaise, lymphadenopathy and skin lesions were the most frequent clinical features. Twelve patients had indirect immunofluorescence antibody test titers of between 1/50 and 1/800 for Bartonella henselae and Bartonella clarridgeiae. Two lymph node biopsies were histologically compatible with CSD. No positive blood cultures could be obtained. This is the first Peruvian prospective study able to identify B. henselae and B. clarridgeiae in pediatric patients

Identificación de Bartonella bacilliformis por métodos moleculares / Bartonella bacilliformis diagnosis by molecular methods

Introducción: La técnica de PCR amplifica una secuencia de ADN con la enzima polimerasa; es muy sensible y especifica. Objetivo: Estandarizar una técnica de PCR para identificar Bartonella bacilliformis en sangre total de pacientes con bartonelosis aguda. Material y métodos: Se usó muestras de sangre total de seis pacientes con diagnóstico clínico y microbiológico de bartonelosis aguda. Se extrajo el ADN de sangre total usando el detergente guanidina DNAZOL BD. Se amplificó el ADN usando los cebadores de extensión "primers" 16S y 23S del espaciador de trascripción interna (ITS). Se hizo electroforesis de los productos de amplificación en gel de agarosa. Se compararon los pesos moleculares de las bandas observadas en la electroforesis con un marcador de 100 pares de bases. Resultados: Se determinó que la concentración de ADN extraído por DNAZOL BD corresponde alrededor de 6 ng de ADN. El producto amplificado de muestras de sangre total fue alrededor de 1000 pares de bases, idéntico al extraído de los hemocultivos de B. bacilliformis y claramente diferente del de otras especies. Las diluciones de las extracciones mejor detectadas por PCR fueron 1/5 y 1/10. Conclusiones: El ADN de B. bacilliformis extraído con DNAZOL BD de sangre total de pacientes con bartonelosis aguda es amplificado por PCR utilizando los primers 16S y 23S; es posible usar esta técnica para el diagnóstico etiológico rápido.

Background: The PCR methodology amplifies a sequence of DNA with the Polimerase enzyme; the sensibility and specificity is very high. Objective: To develop a PCR methodology designed to work on extracts from whole blood samples rather than from isolated, cultured organisms. Methods. The whole blood of six patients with clinical and microbiologic diagnosis of acute bartonelosis by B. bacilliformis was used. The DNA was extracted with DNAZOL® BD (lysis solution with guanidine detergent), and the extract subjected to PCR using primers 16S and 23S for the Intergenic Transcribed Spacer (ITS). The amplified products were subjected to electrophoresis on 1 per cent agarose gels. Results: The concentration of the extracted product with DNAZOL® BD was around 6 ng. The amplified single sized product of 1000 base pairs was identical to that from authentic cultures of B. bacilliformis and clearly different from that of other species. The dilutions detected by PCR were 1/5 and 1/10. Conclusion: The amplification of the DNA of B. bacilliformis extracted with DNAZOL® BD from whole blood of patients with acute bartonelosis using the primers 16S and 23S is possible using this method for a fast etiologic diagnosis. ( Rev Med Hered 2002; 13: 58-63 ).

A prospective study of Cat-Scratch Disease in Lima-Peru.

Cat-Scratch Disease (CSD) is a benign lymphadenitis that may progress to severe or recurrent forms, and it is occasionally associated with morbidity. Between January of 1998 and March of 1999, forty-three suspected CSD patients were assessed in the Hospital Cayetano Heredia and the Instituto de Salud del Niño, in Lima, Peru. Twelve patients had a confirmed diagnosis, 8 of whom were women, and the mean age was 10 years old. The majority (53%) of the cases were encountered in the summer. All patients reported having had contact with cats. Fever, malaise, lymphadenopathy and skin lesions were the most frequent clinical features. Twelve patients had indirect immunofluorescence antibody test titers of between 1/50 and 1/800 for Bartonella henselae and Bartonella clarridgeiae. Two lymph node biopsies were histologically compatible with CSD. No positive blood cultures could be obtained. This is the first Peruvian prospective study able to identify B. henselae and B. clarridgeiae in pediatric patients.

Linfadenomegalia cervical por Mycobacterium kansasii en un paciente inmunocompetente / Cervical lymphademegaly by Mycobacterium kansasii in one inmunocompetent patient

Se describe un paciente de 1 año 3 meses, procedente de Lima, sin antecedente de viajes ni enfermedades previas de importancia, quien desarrolló en forma brusca malestar general y adenomegalias submaxilar, preauricular y cervical izquierdos, siendo el resto del examen no contributorio. Sus exámenes serológicos, a toxoplasmosis y CMV, y los hemocultivos fueron negativos; y, el PPD de o mm. El diagnóstico de Mycobacterium spp. Se realizó en base a un cultivo del aspirado ganglionar y PCR y se identificó M. kansasii mediante la prueba para Micobacterias INNO-LiPA. Se discute la presentación inusual de M. kansasii en un paciente pediátrico inmunocompetente, un análisis por PCR probado en la muestra del paciente y que permitiría la identificación rápida del agente etiológico como Mycobacterium spp. en casos similares, y el diagnóstico diferencial con Enfermedad por Arañazo de Gato.

Reporte de un caso inusual de bacteriema persistente por Bartonella bacilliformis en un paciente esplenectomizado y estudios de PCR y Western blot / Report of persistent an unusual case of bacteriemia by Bartonella bacilliformis in a splenectomized patient and studies of PCR and Western blot

Reportamos el caso de un varón de 56 años esplenectomizado que desarrolló bacteriemias persistentes por Bartonella bacilliformis adquirida durante un viaje a una zona endémica. Se confirmó el diagnóstico por métodos microbiológicos y moleculares. Discutimos a través de una revisión de la literatura las circunstancias de persistencia de bacteriemia en esta enfermedad.