ORIGINAL RESEARCH ARTICLE
Targeting Multicopy Prophage Genes for the Increased Detection of Borrelia burgdorferi Sensu Lato (s.l.), the Causative Agents of Lyme Disease, in Blood
- 1Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom
- 2PhelixRD Charity 230 Rue du Faubourg St Honoré, Paris, France
The successful treatment of Lyme disease (LD) is contingent on accurate diagnosis. However, current laboratory detection assays lack sensitivity in the early stages of the disease. Because delayed diagnosis of LD incurs high healthcare costs and great suffering, new highly sensitive tests are in need. To overcome these challenges, we developed an internally controlled quantitative PCR (Ter-qPCR) that targets the multicopy terminase large subunit (terL) gene encoded by prophages that are only found in LD-causing bacteria. The terL protein helps phages pack their DNA. Strikingly, the detection limit of the Ter-qPCR was analytically estimated to be 22 copies and one bacterial cell in bacteria spiked blood. Furthermore, significant quantitative differences was observed in terms of the amount of terL detected in healthy individuals and patients with either early or late disease. Together, the data suggests that the prophage-targeting PCR has significant power to improve success detection for LD. After rigorous clinical validation, this new test could deliver a step-change in the detection of LD. Prophage encoded markers are prevalent in many other pathogenic bacteria rendering this approach highly applicable to bacterial identification in general.
Lyme disease (LD) is the most common tick-born disease with approximately 476,000 patients in the United States annually during 2010–2018 (Kugeler et al., 2021). LD is caused by a group of bacteria classified together as the Borrelia burgdorferi sensu lato (s.l.) complex, that comprises a clade of more than 20 species including B. burgdorferisensu stricto (s.s.) which dominates in United States, and B. garinii and B. afzelii which are prevalent in Europe and Asia. The LD-causing bacteria are generally transmitted to humans after they are bitten by ticks of the Ixodes family infected with LD causing Borrelia. However, recent reports have raised concerns over Borrelia transmission through blood transfusion based on observations that Borrelia can survive and circulate in the human bloodstream (Pavia and Plummer, 2018).
Currently, LD diagnosis is based on the overt clinical manifestation of disease in the form of erythema migrans (EM) skin lesions, commonly known as a ‘bull’s-eye’ rash and a history of tick exposure. Although EM lesions occur in 70 to 80% of infected individuals, only a third of these patients develop the classic ‘bull’s-eye’ rash, and many other types of skin lesions can occur which are easily confused with EM (Chaaya et al., 2016). In addition to the EM uncertainty, other common symptoms of LD such as fatigue, muscle pain, headache, and perceived cognitive dysfunction largely overlap with an array of other diseases, including other tick-borne diseases. One such example is Relapsing Fever (RF), which is caused by close relatives of the LD-causing bacteria, such as Borrelia miyamotoi(Wormser et al., 2019). The two Borrelia ‘groups’ responsible for LD and RF have caused great concern and clinical confusion, as they are morphologically similar and present with almost indistinguishable clinical symptoms (Bergström and Normark, 2018). Despite this, they respond to different antibiotics and treatment regimens (Koetsveld et al., 2017). Another example of confusion surrounding LD is the co-infection caused by Bartonella spp. This genus of bacteria is emerging as an increasingly common human infection (Anderson and Neuman, 1997). Much of the controversy surrounding LD and co-infections with Bartonella and/or B. miyamotoi is due to the lack of a reliable and sensitive diagnostic method to detect and distinguish between the three groups of bacteria, the LD and RF causing Borrelia and Bartonella (Schutzer et al., 2019). Therefore, laboratory tests to determine and distinguish between LD and co-infections play a vital role in the correct diagnosis and consequent treatment with different antibiotics.
Scientists have faced several challenges with LD detection including patients presenting with a delayed antibody response and a low number of Borrelia cells typically found in human clinical samples (Moore et al., 2016). Although it is particularly difficult to diagnose LD early, it is critical, as it is far easier to treat the disease when it is detected at an early stage (Theel et al., 2019). Bacteria-targeting approaches, such as polymerase chain reaction (PCR) detecting the Borrelia chromosomal DNA, can potentially identify early LD but is relatively insensitive detecting only between 30-50% of positive cases, and is therefore deemed to have little clinical utility (Schutzer et al., 2019). The reasons behind the poor sensitivity of the current PCR methods in Lyme detection are twofold; first, the current PCRs target Borrelia genomic DNA regions that have only one copy in each bacterium, such as the bacterial 16S rRNA gene, RecA gene, and the 5S-23S intergenic regions (Brettschneider et al., 1998; Liveris et al., 2012; Waddell et al., 2016; Lohr et al., 2018; Schutzer et al., 2019). Second, at least some Borrelia species are ‘tissue-bound’ and are only transiently found circulating in the blood (Liang et al., 2020).
In response to these diagnostic challenges, we adopted a novel approach, taking advantage of the fact that most pathogenic bacteria carry multiple complete or partial prophages (phages associated with bacteria) (Argov et al., 2019). These prophage sequences can form the bases of a template from which quantitative PCR (qPCR) primers and probes can be designed. It is known that Borrelia carry a large number of linear and circular plasmids (comprising between 33-40% of the Borrelia genome), among which the cp26 and cp32, and the lp54 linear plasmid, are evolutionarily stable (Casjens et al., 2017). Of these paralogous plasmids, cp32 has been experimentally determined to be a Borrelia burgdorferi prophage thus it is highly likely that many of its homologs are also prophages (Eggers and Samuels, 2000).
In this paper we have demonstrated for the first time in Borrelia-related diagnostics that it is possible to overcome the sensitivity challenges associated with LD detection. We highlight the potential of our test to discriminate between healthy volunteers, early LD, and late LD patients. We present data from a systematic and comprehensive study that evaluate the use of the multicopy phage terminase large subunit (terL) gene as a molecular marker for the detection of Borrelia species. The analytical performance of the terL-targeting qPCR (referred to as Ter-qPCR) was thoroughly evaluated, and the test was shown to be able to detect one single Borrelia cell from blood samples. The diagnostic potential was evaluated using a set of blood and serum samples collected from healthy volunteers and individuals who were clinically diagnosed with LD.
In summary, we demonstrate that a quantitative phage-based PCR has the potential to change the diagnosis of LD from blood samples. This approach of detecting bacteria-specific phages may be applicable to infections other than LD such as sepsis caused by Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa etc. (Minasyan, 2019), as long as suitable phages are identifiable.
Results and Discussion
Each Borrelia species has a distinct amount of species specific variation in its prophage sequences; thus these prophages can be used as a proxy to identify the bacteria because of the tight correlation between them and the exact prophages found in each Borrelia host. As there are multiple prophages per Borrelia cell, the detectable signal is higher for prophages than bacteria. Furthermore, evidence suggests that Borrelia prophages can be released outside the Borrelia cells following encounters with stressors such as antibiotics (Eggers and Samuels, 2000). In this study, we confirmed that Borrelia prophages can escape from the bacterial host cell in a spontaneous manner. Taking advantage of the multicopy and free movement of Borrelia prophages, the approach to target prophages instead of bacteria will bypass the cryptic and tissue-bound feature that typifies human Borrelia infections (Liang et al., 2020). Thus, we have a greater chance of detecting the prophages in blood even when the bacteria may not be present or present in extremely low numbers. In this sense, prophages are somewhat analogous to Borrelia ‘footprints’.
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Borrelia Strains and Cultures
The Borrelia strains used in this study are listed in Table 1.
- Ten strains were provided by Professor Sven Bergström, Department of Molecular Biology, Umea University, Sweden. Seven strains were purchased from the Pasteur Institute and DSMZ (German Collection of Microorganisms and Cell Cultures GmbH).
- Two strains were provided by the Center for Disease Control and Prevention (CDC), United States, and
- two by Cecilia Hizo-Teufel from the German National Reference Centre for Borrelia.