Project Lead(s): Giovanna Mendoza-Mujica
Carrion's disease is an endemic disease in Andean countries, such as Peru, Ecuador and Colombia. In Peru, it is a major public health concern in endemic communities living in extreme poverty and with poor sanitation.
The etiologic agent is Bartonella bacilliformis, a microorganism that invades human red blood cells, causing an acute phase characterized by a fever than can lead to anemia of varying severity, with a mortality rate of 44% to 88% in patients who do not receive appropriate antibiotic therapy.
Diagnosis during the acute phase can be made by obtaining a peripheral blood smear with Giemsa stain, but this has low sensitivity and requires laboratory testing. Serological diagnostic methods, such as Western Blot (WB), could be alternatives for diagnosis, due to their fast action, high sensitivity and specificity, allowing the early detection of cases.
This project aimed to develop a diagnostic test of Carrion's disease using the Western Blot technique, which would be sensitive, inexpensive, easy to apply and could be used at the primary care level.
Bartonella bacilliformis strains from different Peruvian endemic locations were reactivated, then subjected to ultrasonic vibration to improve antigen procurement procedures. Protein quantification was performed through the Bradford method, and antigenic band patterns of acute and eruptive strains were identified via electrophoresis.
Antigens were then purified and transferred to nitrocellulose paper, and these antigenic strips were evaluated with sera from acute and eruptive patients.
Validation of the Western Blot method was conducted by evaluating statistical parameters of diagnostic sensitivity, analytical and diagnostic specificity, repeatability, reproducibility and the correlation with the screening method (ELISA).
The Western Blot method is a reliable tool for the serological diagnosis for patients with Carrion’s disease. The team identified and determined that, for the WB confirmatory in-house test, four reactive bands (131.47, 122.60, 61.37 and 57.05 KDa) were present in positive control serum, and absent in positive or negative control samples of other etiologies.
The method was determined to be 100% repeatable and 100% reproducible with qualitative measurements, with a 95.5% sensitivity and 97% specificity, with predictive values within acceptable parameters.
Following the procedures and protocols established with the in-house Western Blot method, 600 antigenic strips were produced, which were used to confirm serological diagnosis of patients with clinical signs and symptoms of Carrion’s disease from endemic regions.
A group of eight health professionals from five regional laboratories and three intermediate laboratories with the highest number of cases of Carrion’s disease patients were trained in implementing the diagnostic method.
The team intends to scale the production of two commercial diagnostic test kits for Carrion’s disease (ELISA for screening and WB for confirmation), which would be developed at the National Center for Biological Products in the National Institute of Health, in coordination with the Peruvian Ministry of Health.