Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR.

BACKGROUND: Tick-borne infections have been increasing steadily over the years, with co-infections with Borrelia burgdorferi and Babesia microti/divergens emerging as a serious health problem. B. burgdorferi is a spirochetal bacterium that causes Lyme disease while protozoan pathogens belonging to Babesia species are responsible for babesiosis. Currently used serological tests do not always detect acute Lyme disease or babesiosis, and fail to differentiate cured patients from those who get re-infected. This is a major problem for proper diagnosis particularly in regions endemic for tick-borne diseases. Microscopy based evaluation of babesiosis is confirmatory but is labor intensive and insensitive such that many asymptomatic patients remain undetected and donate blood resulting in transfusion transmitted babesiosis. RESULTS: We conducted multiplex qPCR for simultaneous diagnosis of active Lyme disease and babesiosis in 192 blood samples collected from a region endemic for both diseases. We document qPCR results obtained from testing of each sample three times to detect infection with each pathogen separately or together. Results for Lyme disease by qPCR were also compared with serological tests currently used for Lyme disease when available. Considering at least two out of three test results for consistency, 18.2% of patients tested positive for Lyme disease, 18.7% for co-infection with B. burgdorferi and B. microti and 6.3% showed only babesiosis. CONCLUSIONS: With an 80% sensitivity for detection of Lyme disease, and ability to detect co-infection with B. microti, multiplex qPCR can be employed for diagnosis of these diseases to start appropriate treatment in a timely manner.

A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests.

BACKGROUND: Ticks transmit Babesia microti, the causative agents of babesiosis in North America and Europe. Babesiosis is now endemic in Northeastern USA and affects people of all ages. Babesia species infect erythrocytes and can be transmitted through blood transfusion. Whole blood and blood products, which are not tested for Babesia, can cause transfusion-transmitted babesiosis (TTB) resulting in severe consequences in the immuno-compromised patients. The purpose of this study was epidemiological evaluation of babesiosis in a tick-infested state. RESULTS: We examined blood samples from 192 patients who visited clinics during the active tick-borne diseases season, using a newly developed qPCR assay that uses the specific molecular beacon probe. Due to the absence of clear symptomology, clinical laboratories did not test 131 samples by IFA, FISH or microscopic examination of Giemsa-stained blood smears. Babesia infection was detected in all age groups by FISH and microscopy; notably patients >40 years of age represented 64% of tested samples and 13% were younger patients. We tested all samples using qPCR and found that 38% were positive for Babesia. Of 28 samples that were positive by FISH, 27 (96%) were also positive by qPCR indicating high congruency between nucleic acid based tests. Interestingly, of 78 asymptomatic samples not tested by FISH, 22 were positive by our qPCR. Direct detection of Babesia relies upon microscopic examination of patient blood smears, which is labor intensive, difficult to scale up, requires specific expertise and is hence, often not performed. In fact, a clinical laboratory examined only 23 of 86 blood samples obtained from two different counties by microscopy. By considering individuals positive for Babesia infection when results from currently available microscopy, FISH or serological tests were positive, we found that our qPCR is highly sensitive (96.2%) and showed a specificity of 70.5% for Babesia. CONCLUSION: Robust qPCR using specific probes can be highly useful for efficient and appropriate diagnosis of babesiosis in patients in conjunction with conventional diagnostics, or as a stand-alone test, especially for donated blood screening. The use of a nucleic acid amplification test based screening of blood and blood products could prevent TTB.