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

Abstract

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.

Keywords: Babesia microti; Babesiosis detection; Blood-borne disease; Nucleic acid amplification test; Parasitic disease; Quantitative PCR; Tick-borne infection.

Fig. 1

B. microti infection relationship with the age of patients. Whole blood samples obtained from 192 patients from Morris, Ocean and Monmouth Counties of New Jersey examined by FISH or microscopic examination of Giemsa-stained thin smears indicates a higher babesiosis incidence in patients older than 40 years of age

Fig. 2

Determine the sensitivity of detection of B. microti using in vitro grown parasites by qPCR. a Amplification plots of Bmtpk gene in monoplex qPCR assay starting with 10⁶ gene copies (8ng DNA). Five-fold dilutions of genomic DNA of B. microti purified from in vitro grown culture using Bmtpk primers and molecular beacon probe were used to determine quantities of B. microti. Dotted line indicates ‘no template’ control. b A high coefficient of correlation (r ² = 0.9822) between the amplification cycle number (Ct values) and Bmtpk copy number representing the parasite numbers obtained from the standard curve indicates that qPCR can be used effectively to evaluate even low level of parasitemia in patients

Fig. 3

Detection of B. microti presence in the qPCR positive patient samples by IFA (Top Panels) Two representative samples (J22 and J67) from Ocean and Monmouth Counties not tested for Babesia by microscopy at JSUMC show green fluorescence due to reactivity of antibodies in patient plasma with the parasites followed by detection with Alexa 488 conjugated secondary antibodies, when observed by using FITC filter indicating positive IFA results (marked by arrows). (Bottom Panels) Blue fluorescence due to DAPI staining shows the parasites present (marked by arrows) in each field of view of the Nikon 80i fluorescence microscope at × 1000 magnification when Apo-Plan TIRF objective was used. Scales shown represent respective panels of Giemsa-stained microscopy and IFA