Dynamics of Spirochetemia and Early PCR Detection of Borrelia miyamotoi

We investigated whether Borrelia miyamotoi disease can be detected in its early stage by using PCR for borrelial 16S rRNA, which molecule (DNA or RNA) is the best choice for this test, and whether spirochetes are present in blood during the acute phase of B. miyamotoi disease. A total of 473 patients with a suspected tickborne infection in Yekaterinburg, Russia, in 2009, 2010, and 2015 were enrolled in this study. Blood samples were analyzed by using quantitative PCR or ELISA, and a diagnosis of borreliosis was confirmed for 310 patients. For patients with erythema migrans, 5 (3%) of 167 were positive for B. miyamotoi by PCR; for patients without erythema migrans, 65 (45%) of 143 were positive for B. miyamotoi by PCR. The median concentration for RNA was 3.8 times that for DNA. Median time for detection of B. miyamotoi in blood was 4 days.

The diagnosis of Lyme disease (borreliosis) is based primarily on identification of an erythema migrans rash and positive results for serologic laboratory tests (11). However, PCR amplification of B. burgdorferi sensu lato DNA is not sensitive enough for routine diagnosis (11,12).
B. miyamotoi infection usually manifests as an influenza-like disease causing high fever, headache, and myalgia but, in most instances, without erythema migrans (3). B. miyamotoi produces glycerophosphodiester phosphodiesterase (glpQ), which is absent in B. burgdorferi sensu lato and is therefore useful for serologic discrimination between relapsing fever and Lyme borreliosis. ELISAs and confirmatory Western blot assays of human serum samples have been used to detect antibodies against B. miyamotoi in the Netherlands (13) and the United States (14). Wagemakers et al. (15) detected antibodies against variable small protein 1 and variable large proteins (Vlp15, Vlp16, and Vlp18) in blood samples of patients with B. miyamotoi disease. PCR and thin and thick blood smears of peripheral blood stained with Wright stain or Giemsa were used to detect infection in patients suspected of having relapsing fever (16).
Unlike DNA, which is usually present as a single copy per cell, or mRNAs, which constitute the minor population of total cellular RNA, each bacterium contains hundreds to thousands of 16S rRNA molecules during the growth phase. Therefore, we hypothesized that an assay based on PCR amplification of cDNA molecules, representing highly and consistently transcribed Borrelia genes such as 16S rRNA, could improve the sensitivity of detection of Borrelia spp. Backstedt et al. (17) reported that leptospiral RNA-based quantitative PCRs (qPCRs) for human blood were >100-fold more sensitive than a DNA-based approach. Furthermore, detection of 16S rRNA (e.g., by nucleic acid sequence-based amplification) can distinguish viable from nonviable bacterial cells (18).
Hematogenous dissemination is a major pathogenetic event in Lyme borreliosis. Investigations of level and duration of spirochetemia caused by B. miyamotoi are needed for understanding the pathogenesis of the infection, as well as development of new diagnostic tools.
Our study had 3 goals. First, we sought to determine whether B. miyamotoi disease can be detected in its early stage by using PCR for borrelial 16S rRNA. Second, we assessed which molecule (DNA or RNA) is the best choice for this test. Third, we investigated the dynamics of B. miyamotoi spirochetemia during the acute phase of the disease.

Study Design
Patients suspected of having tickborne diseases in Russia are hospitalized if they have been bitten by a tick and show development of signs and symptoms of acute infection (fever, chills, headache, fatigue, muscle aches) within a few weeks. A diagnosis of acute Lyme borreliosis was determined by the presence of erythema migrans, detection of borrelial IgM and IgG seroconversion, and detection of pathogen DNA or RNA. In this study, the case definition for B. miyamotoi disease was based only on PCR results. We have previously reported detailed information about case definitions for B. miyamotoi disease (3).
A total of 459 patients admitted to Municipal Clinical Hospital No. 33 (Medical Association Novaya Bolnitsa) in Yekaterinburg, Russia, during May-August 2009 and June-July 2010 for suspected tickborne infection were enrolled in this study. We obtained blood samples from all patients during the first 3 days of hospitalization for analysis by qPCR and ELISA. We performed PCR for detection of 16S rRNA of B. miyamotoi, B. burgdorferi sensu lato, Ehrlichia chaffeensis, and E. muris; the 5′-untranslated region gene of tickborne encephalitis virus (TBEV); and the major outer membrane protein 2 gene of Anaplasma phagocytophillum as described (3).
We tested serum samples for borrelial IgM and IgG and TBEV IgM by ELISA (EUROIMMUN AG, Lubeck, Germany). We estimated the number of copies of B. miyamotoi and B. burgdorferi sensu lato DNA and RNA in PCR-positive patient blood samples by using a qPCR for 16S rRNA.
For determining duration of spirochetemia, we collected repeat blood samples from 9 PCR-positive B. miyamotoi disease patients in 2010 and 14 B. miyamotoi disease patients admitted to Municipal Clinical Hospital No. 33 in June-July 2015. We collected blood samples from these patients during the first 3 days after admission and then repeatedly for 4 days after initial detection of B. miyamotoi in the blood of these patients.

Preparation of Blood Samples
We used differential centrifugation to separate spirochetes from erythrocytes. Blood samples were centrifuged at 160 × g for 10 min to pellet erythrocytes. We then transferred 500 μL of leukocytes and platelet-rich plasma into 1.5-mL tubes for centrifugation at 10,000 × g for 10 min to collect leukocytes and spirochetes. We extracted DNA and RNA from the pellet and 100 μL of the supernatant by using an AmpliSens Riboprep Kit (Central Research Institute of Epidemiology, Moscow, Russia). After cell lysis, we divided samples into 2 equal aliquots for separate isolation of B. miyamotoi RNA and DNA targets of 16S rRNA. We extracted RNA by using a universal internal RNA recombinant control having a known number of RNA copies per milliliter and DNA by using a universal internal DNA recombinant control having a known number of DNA copies per milliliter. RNA samples were not treated with DNase; therefore, RNA concentrations in this article are RNA/ DNA concentrations.
The RNA template was subjected to reverse transcription (Thermo Fisher Scientific, Waltham, MA, USA). In 2015, a commercial PCR kit (AmpliSens B. miyamotoi-FL; Central Research Institute of Epidemiology) was used according to the manufacturer's instructions for B. miyamotoi glpQ gene screening of patients with suspected tickborne diseases.
We tested serum samples collected at the time of hospitalization and 1-2 weeks later for borrelial IgM and IgG. We obtained serologic evidence of exposure to Borrelia spp. by using 2 ELISAs (EI 2132-9601 M and EI 2132-9601-2 G; EUROIMMUN AG) and detected TBEV IgM by using a semiquantitative ELISA (EI 2661-9601 M; EUROIMMUN AG).

Data Analysis
We used the Wilcoxon signed-rank test for comparing copy numbers of B. miyamotoi RNA and DNA in blood samples and the Kaplan-Meier estimator to assess persistence of DNA or RNA of pathogens in blood. We performed time-to-event analysis by using the following assumptions: 1) starting time was the first day of illness; 2) observations for no antimicrobial drug treatment were complete data; 3) observations for late antimicrobial drug treatment (when DNA or RNA were eliminated from blood before start of treatment) were complete data; and 4) observations for antimicrobial drug treatment were incomplete data (right censored). The Kaplan-Meier estimator assumes that at any time, patients who are censored have the same survival prospects as those who continue to be followed up (19).
We used the Clopper-Pearson interval for calculating CIs for proportions. Descriptive statistics are given as mean and SD or median and interquartile range (IQR). We analyzed data by using SPSS software (IBM, Armonk, NY, USA) or R software (https://www.r-project.org/).

RNA and DNA Concentrations
Because onset of B. miyamotoi disease is acute, patients are usually admitted to a hospital during the first 3 days of the disease in Russia. In our study, 79% of patients were admitted to the hospital during the first 3 days, which indicates that concentrations of B. miyamotoi RNA or DNA are accurate for this period.
The maximum pathogen RNA concentration was observed on day 1 of the disease (Figure 1, panel A). On day 2, RNA copy number varied widely (median 3,700-45,360 copies/mL); it remained in this range on subsequent days. The concentration of B. miyamotoi DNA varied in a similar way (Figure 1, panel B); the highest value of 9,085 copies/mL was found on the first day, after which the value decreased to 797.5 copies/mL. Because there were only 2 observations on day 6, observed ranges of DNA (and RNA) concentrations are speculative. The Wilcoxon signed-rank test showed that the copy number of RNA in blood was significantly higher than the copy number of DNA (p<0.001). The RNA:DNA ratio also showed a wide range (median ratio 3.8, IQR 2.1-7.5).
Early Lyme disease is usually manifested only by erythema migrans; thus, patients are often hospitalized late in their illness. The median time gap between onset of  Figure 2, panel A). However, despite such a wide range, 90% of all observations were in the first 8 days of the disease. We showed in a previous study (20) that 7 (9.1%) positive samples, which were obtained from patients given a diagnosis of B. miyamotoi disease during the second week of disease (or later), were assumed to be caused by a relapse of fever. However, this assumption could not be confirmed because of lack of availability of clinical materials. Pathogen DNA or RNA were detected in blood samples from patients with Lyme borreliosis obtained up to day 24 of disease ( Figure 2,  panel B).
The study showed a high number of false-negative PCR results in patients with and without EM ( Table 1). The time gap between onset of disease and blood sampling can affect results of blood tests. Therefore, all blood samples from the group of patients without EM were obtained based on the time after onset of symptoms. We found that blood samples from B. miyamotoi PCR-positive patients were obtained significantly earlier (z = -3.29, p<0.001, by Mann-Whitney U-test). The median time of blood sampling was 2.0 days (IQR 2.0-4.0 days) for PCR-positive patients without EM and 4.0 days (IQR 2.3-6.0 days) for PCR-negative patients without EM after initial symptoms. Therefore, PCR is applicable for diagnosis of B. miyamotoi disease only during the first few days of the disease.

Duration of Spirochetemia
In 2010 and 2015, we obtained blood samples repeatedly during the first 6 days of disease from 23 patients with confirmed B. miyamotoi disease ( Table 2) and used data for these samples to estimate the duration of spirochetemia. We excluded samples from patients who had started antimicrobial drug therapy before or at the same time as the blood tests and those from patients who had a time gap between blood samples >2 days. We performed time-toevent analysis for 17 patients. Pathogen DNA or RNA was detected in blood up to day 5 of illness (Table 2), and all samples showed negative results on day 6 of illness. Median time for detection of pathogen DNA or RNA in blood was 4.0 days (95% CI 3.1-4.9 days) (Figure 3).

Discussion
Borreliosis is the most prevalent tickborne disease in Russia (21,22). Our findings showed that, in Yekaterinburg, ≈23% cases of borreliosis (confirmed clinically, serologically, or by PCR) were caused B. miyamotoi. Findings that indicated that B. miyamotoi disease might not be a rare infection have been previously suggested for the United States (8,14,23) and the Netherlands (13).
We found that erythema migrans was not apparent in 70 (93%) patients with B. miyamotoi disease, and only in 7% of case-patients was erythema migrans manifested. We obtained data for the 5 PCR-confirmed case-patients with B. miyamotoi disease and erythema migrans and the case-patient co-infected with B. burgdorferi sensu lato and B. miyamotoi. We believe that these 5 patients were also co-infected with Borrelia spp. and that the B. burgdorferi sensu lato DNA was not detected because of the low sensitivity of the PCR. Additional research is needed to test this assumption. Recent studies reported that in the United States, 14% of patients with B. miyamotoi disease were coinfected with B. burgdorferi (7,23), including a patient with erythema migrans (23).
Our study showed poor sensitivity (≈11%) of qPCR detection for 16S rRNA for B. burgdorferi sensu lato.

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 24, No. 5, May 2018 In comparison, the sensitivity of PCR for B. burgdorferi sensu lato varied widely (7.5%-78.1%) (12,24,25). Several studies have reported low sensitivity of PCR for B. burgdorferi sensu lato DNA for routine diagnostic purposes because of low numbers of spirochetes circulating in the bloodstream during acute infection (8,12,(26)(27)(28)(29). For relapsing fever caused by Borrelia spp., the number of glpQ gene copies for B. duttonii and B. reccurentis ranged from 10 2 to 10 5 copies/mL for 7 (0.3%) of 2,057 healthy participants and from 10 3 to 10 8 copies/mL for 15 (3.9%) of 382 patients with fever who were surveyed in Tanzania (16). The number of copies of the B. miyamotoi gene in serum samples of 2 patients in Japan was 7.2 × 10 3 and 2.8 × 10 4 copies/mL by 16S rRNA qPCR (30). A mean copy number of 7,787 copies/mL was reported for B. miyamotoi disease patients in the northeastern United States (23). The number of copies of the B. miyamotoi gene in serum samples from 2 patients in Japan was 7.2 × 10 3 and 2.8 × 10 4 copies/mL by 16S rRNA qPCR (30). A mean copy number of 7,787 copies/mL was reported for B. miyamotoi disease patients in the northeastern United States (23). Our study identified the bacterial load in blood samples of patients with B. miyamotoi disease (<9,085 copies/ mL) and showed that an early diagnosis of this disease is possible if a PCR for the 16S rRNA gene is used. The concentration of B. miyamotoi RNA in blood is higher than that for DNA. We found a low median RNA:DNA ratio (≈4), which indicates that use of RNA as the target molecule is inappropriate.
We obtained data on the duration of spirochetemia, which contributes to the early diagnosis of B. miyamotoi disease. It is possible to detect borrelial DNA by PCR during the first 3 days of the disease. However, bacterial DNA is then no longer detectable in the blood, so PCR detection is ineffective after 4 days. We detected 73 Borrelia spp. ELISA-positive patients without erythema migrans who seroconverted within the observation period and were PCR negative for both Borrelia spp. On average, these samples were obtained later than B. miyamotoi PCR-positive samples, suggesting that they might have been collected after the end of the period of spirochetemia and caused the PCRnegative results.
We characterized the duration of B. miyamotoi spirochetemia during acute illness. Although there was no strong clinical confirmation, our data showed that B. miyamotoi DNA and RNA might be detected in the circulation within 30 days after onset of disease. This extended parasitemia is probably related to disease relapse (20). Lee et al. (8) provided indirect results for B. miyamotoi spirochetemia and detected B. miyamotoi DNA in blood samples of 4 patients during the period with little tick exposure in the northeastern United States. These authors suggested that this off-season spirochetemia with a low bacterial density was most likely the result of bacteria being dislodged periodically from persistent deep-tissue lesions (8).
Thus, a long-term study of pathogen persistence is required. The duration of B. miyamotoi spirochetemia is relatively short. Thus, the true number of ambulatory and hospitalized patients infected with B. miyamotoi will not be known until a sensitive, reliable, diagnostic laboratory test (i.e., serologic test) is available to detect causative agents in patients with acute infections in disease-endemic areas.