Preplanned Studies: Pilot Surveillance of Babesia, Borrelia burgdorferi, and Anaplasma phagocytophilum Among Chinese Blood Donors — Xinjiang, Inner Mongolia, and Heilongjiang PLADs, China, 2022–2023

Tick-borne diseases are a growing global concern because of their potential to cause severe illness and geographical spread, which is facilitated by factors such as climate change, human mobility, and ecological disturbances. Tick-borne pathogens like Babesia, Colorado tick fever virus, Anaplasma phagocytophilum (A. phagocytophilum) and others can be transmitted through blood, necessitating vigilant monitoring (1). Transfusion-transmitted babesiosis has been reported in the US and Canada (2). From May 2020 onwards, a 13-month Babesia nucleic acid pooling testing (16-in-1) program in 14 US states and Washington, D.C. identified 36 positive blood donors with a few subsequent reports of transfusion-transmitted Babesia infection (3-4). China, with its vast territory and diverse climate, hosts over 120 tick species, particularly in provincial-level administratives (PLADs) like Xinjiang, Inner Mongolia, and Heilongjiang (5). An examination of blood donors in Mudanjiang City, Heilongjiang Province, in 2016 revealed that 1.3% of individuals tested positive for Babesia antibodies (6). Additionally, seroprevalence of B. burgdorferi in northern Xinjiang PLAD ranges from 9.05% to 16.14% (7), highlighting the urgency for targeted surveillance and intervention strategies.

Therefore, the National Health Commission of the People’s Republic of China has been conducting nucleic acid testing (NAT) for Babesia, B. burgdorferi, and A. phagocytophilum at border blood centers in Xinjiang, Heilongjiang, and Inner Mongolia PLADs since 2022 to assess the prevalence of tick-borne pathogens in areas with high tick distribution, which provides valuable insights into potential infection risks. Simultaneously, the National Center for Clinical Laboratory (NCCL) has conducted performance evaluations for all screening reagents used. Furthermore, external quality assessments (EQAs) were performed on the blood centers to ensure screening quality.

DNA fragments for detecting Babesia, B. burgdorferi, and A. phagocytophilum in a single assay were synthesized and individually cloned into the pDC316 vector (Shenggong, Shanghai, China) to generate pDC316-Babesia, pDC316-B. burgdorferi, and pDC316-A. phagocytophilum constructs. All three correctly sequenced plasmids were prepared using the thin-film hydration technique to form liposomes (Figure 1A) and were quantified by digital PCR (QIAcuity One 5plex, QIAGEN, Germany). Liposome-based quality control materials were serially diluted in human whole blood to evaluate the limit of detection (LoD) of five domestic blood screening reagents (A, B, C, D, and E), each using its own specific method for blood lysis and nucleic acid extraction. Details on blood volumes for pooled and individual testing are detailed in Supplementary Table S1. Each dilution series was tested with approximately seven replicates per lot, totaling around 21 replicates per concentration. Probit analysis was used to determine the LoD of each kit. Within-run and within-laboratory precision were assessed by testing different concentrations (approximately 1–5 LoD and above) five times a day for five consecutive days. EQA was conducted annually at 18 blood centers, involving 10 samples, which covered different concentrations of positive and negative samples. Each participating blood center’s results were scored, with a maximum score of 100 points for all correct results. Qualitative results reported by the blood centers are considered acceptable if they score 80 or above.

Figure 1. 

Flowchart for the preparation of liposomes and the screening and confirmation of blood donors. (A) Preparation of liposomes by thin-film hydration method. (B) Screening and confirmation algorithm of Babesia, B. burgdorferi, and A. phagocytophilum infection among blood donors.

Abbreviation: DOPC=dioleoyl phosphatidylcholine; DOTAP=1,2-dioleoyl-3-trimethylammonium-propane; NCCL=National Center for Clinical Laboratory ; NAT=nucleic acid testing; B. burgdorferi=Borrelia burgdorferi.

During the June–July monitoring period for the Inner Mongolia and Heilongjiang PLADs blood centers and the July–August period for Xinjiang PLAD, all blood donors from 18 blood centers were screened for Babesia, B. burgdorferi, and A. phagocytophilum (Figure 1B). Samples with initial reactive results were confirmed at the NCCL using the five Triplex-NAT kits. An initially reactive sample was considered positive when at least two assays yielded positive results.

Five domestic blood screening reagents were used to serially dilute the liposome-based quality control materials to assess the LoDs. Table 1 shows that Reagents A and B had lower LoDs. Reagent A demonstrated the lowest LoDs for both Babesia and B. burgdorferi in the individual donor (ID)-NAT test, at 33.29 copies/mL and 15.83 copies/mL, respectively. Reagent B had the lowest LoD for A. phagocytophilum, at 14.79 copies/mL. Conversely, Reagent B had the lowest LoDs for all three pathogens in the minipool (MP)-NAT test: 178.45, 99.38, and 69.41 copies/mL for Babesia, B. burgdorferi, and A. phagocytophilum, respectively. Reagent D demonstrated the highest LoD for detecting all three pathogens in both test types.

Five screening reagents were used to evaluate the liposome-based quality control materials at different concentrations to determine their precision. All reagents demonstrated good precision, as shown in Table 2, with -run and within-laboratory coefficients of variation (CVs) of <5%.

From 2022 to 2023, 92,700 blood donors were screened for Babesia, B. burgdorferi, and A. phagocytophilum infections, including 47,172 individuals in 2022 and 45,528 individuals in 2023. In Hegang City, Heilongjiang Province, 4,629 blood donors were screened using reagent A, revealing one B. burgdorferi-reactive sample (Ct value of 40.31). This sample was confirmed positive by reagents C and D, with Ct values of 37.6 and 35.29, respectively. The prevalence of B. burgdorferi among blood donors in Hegang City was 0.22‰. In 2022, the Kashi and Yili regions of Xinjiang Uygur Autonomous Region each identified one reactive sample during Babesia screening; however, both were later confirmed negative. By 2023, no reactive samples were detected in the screened areas.