Antimicrobial resistance and new insights in the diagnosis of Carrión's Disease

Abstract

Carrión's disease is an overlooked disease restricted to the poorest areas of the Andes, with only a few research groups working in this field around the world. Until recently, it was thought that Carrion's disease met the most relevant criteria for being eradicated, but before that happens a number of issues need to be addressed. One of the problems is the lack of well-defined effective treatment program. Both in vitro antimicrobial resistance studies and clinical trials are needed to determine the best treatment approaches. On the other hand, and perhaps the most urgent need, it is necessity to have an easy way to make the correct diagnosis. New immunological studies will help to identify new molecules with diagnostic potential.

The results of this thesis have been separated into 2 different aspects of Carrión's disease; On one hand, antimicrobial resistance (Chapter I) and, on the other hand, the diagnosis and characterization of clinical samples (Chapter II). Chapter I describes a study on resistance mechanisms developed in the presence of the 4 most common antibiotics used in the treatment of Carrion's disease. The ability of Bartonella bacilliformis to become resistant to the main antibiotics used to treat Carrión's disease is evidenced both by the development of antibiotic target alterations and by the overexpression of expulsion pumps. However, total or partial reversion of acquired resistance suggests the existence of a high biological cost derived from the selection of antimicrobial resistance. These instability of the acquired antibiotic resistance may underlie the lack of antibiotic-resistant clinical isolates and the high frequency of microbiological failures during antibiotic treatments. Although studies of mutants obtained in vitro can not be transferred directly to the community, chloramphenicol appears to be the best treatment option in vitro.

Chapter 2 analyzes several aspects of the diagnosis of Carrion's disease that are addressed in 3 studies. In the first study, samples obtained from patients of an suspected Oroya fever outbreak were molecularly characterized. However, we propose that this outbreak was erroneously attributed to B. bacilliformis and our results suggest that the causative agent was Sphingomonas faeni. As far as we know, this was the first reported outbreak caused by Sphingomonas spp. acquired in the community, making clear the need to develop diagnostic techniques that can be implemented in endemic areas. In a second study we evaluated the limit of detection of several PCR approaches to detect B. bacilliformis from blood. It seems that the sensitivity of these techniques could allow the diagnosis of acute cases of Carrion's disease, but its

applicability to detect healthy carriers or acute cases with low bacteraemia remains unclear. In this article we propose the concomitant use of the 3 PCR approaches in combination with the clinical information. Finally, in the third study of this chapter the objective was the identification and characterization of immunogenic candidates of B. bacilliformis that may in the future be used in a rapid diagnostic tool. Four immunogenic B. bacilliformis candidates were selected: 2 proteins were identified with an anti-human IgM secondary antibody (Pap31 and SCS-α) and another 2 with anti- human IgG secondary antibody (GroEL and SCS-β).

The design of treatment schemes that minimize resistance selection along with the development of diagnostic techniques that can be implemented in rural and isolated areas is essential for the control, elimination and eradication of Carrion's disease.

La enfermedad de Carrión es una enfermedad desatendida restringida a las zonas más pobres de la región Andina. Uno de los problemas es la falta de programas de tratamiento eficaces bien definidos. Por otro lado, y quizás sea la necesidad más imperiosa, es preciso tener una manera fácil de realizar el diagnóstico. Nuevos estudios inmunológicos ayudarán a identificar nuevas moléculas con potencial diagnóstico.

Los resultados de esta tesis han sido separados en 2 aspectos diferentes de la enfermedad de Carrión; la resistencia a los antimicrobianos (Capítulo I) y el diagnóstico y caracterización de muestras clínicas (Capítulo II). El capítulo I describe un estudio sobre los mecanismos de resistencia desarrollados en presencia de los 4 antibióticos más comunmente utilizados en el tratamiento de la enfermedad de Carrión, evidenciándose la capacidad de Bartonella bacilliformis de volverse resistente a estos antibióticos, tanto por el desarrollo de alteraciones en sus dianas, como por la sobreexpresión de bombas de expulsión.

El capítulo 2 analiza aspectos del diagnostico de la enfermedad de Carrión que se abordan en 3 estudios. En el primero se caracterizan molecularmente muestras obtenidas de pacientes de un supuesto brote de fiebre de Oroya. Sin embargo, proponemos que este brote se atribuyó erróneamente a B. bacilliformis sugieriéndose que el agente causante fue Sphingomonas faeni. En el segundo hemos evaluado el límite de detección de varios esquemas de PCR para detectar B. bacilliformis. Parece que la sensibilidad de estas técnicas podría permitir el diagnóstico de casos agudos de la enfermedad de Carrión, pero su aplicabilidad para detectar a los portadores sanos o con una baja bacteriemia sigue sin estar clara. Por fin, en el tercer estudio de este capítulo el objetivo fue la identificación y caracterización de candidatos inmunogénicos de B. bacilliformis que puedan en un futuro ser utilizados en una herramienta de diagnostico rápida. Se identificaron 4 proteínas inmunogénicas: Pap31, GroEL, SCS-α y SCS-β.

El diseño de esquemas de tratamiento que minimicen la selección de resistencia junto con el desarrollo de técnicas de diagnóstico que puedan ser implementadas en zonas rurales es esencial para el control y eliminación de la enfermedad de Carrión.