Clinical, analytical, imaging and ECG differences in adult patients infected by Borrelia burgdorferi and doubly-infected by Babesia divergens-Borrelia burgdorferi

doi:10.21203/rs.3.rs-3133405/v1

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Clinical, analytical, imaging and ECG differences in adult patients infected by Borrelia burgdorferi and doubly-infected by Babesia divergens-Borrelia burgdorferi

https://doi.org/10.21203/rs.3.rs-3133405/v1

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Differences between double infection by Borrelia burgdorferi-Babesia divergens and Borrelia burgdorferi monoinfection in adult patients have not been reported so far. Both zoonosis, endemic in Northwestern Spain, are transmitted by Ixodes tick bites.

Clinical, analytical and other tests (imaging, ECG) characteristics of 120 adult patients with B.burgdorferi infection, of which 47 (39.2%) had B.burgdorferi-B.divergens double infection, diagnosed between 2014–2017, were retrospectively compared.

Cardiorespiratory symptoms were reported in 9/47 (19.2%) patients with B.burgdorferi-B.divergens double infection compared to 4/73 (5.5%) patients with B.burgdorferi monoinfection (P = 0.02). Dyspnea was recorded in 4/47 (8.5%) doubly infected compared to 1/73 (1.4%) monoinfected patients (P = 0.07). In addition ECG atrioventricular (AV) block was detected in 5/47 (15.6%) doubly infected compared to 1/73 (2.6%) monoinfected individuals (P = 0.09). No other clinical, laboratory or other tests differences were observed between doubly infected and monoinfected patients.

We conclude that doubly infected had more frequently cardiorespiratory symptoms, mostly dyspnea, compared to monoinfected individuals. These symptoms were unrelated to anemia. ECG AV block perhaps induced by summative myocardial damage due to both infections might play some role in the cardiorespiratory dysfunction.

Biological sciences/Microbiology

Health sciences/Diseases

Health sciences/Medical research

Asturias (Northwestern Spain) has a mild humid weather and it is covered by 210 kha of natural forest, extending over 61% of its land area. In these forested areas, ticks and wild roe and fallow deer abound, especially in its most mountainous east and southwest parts. In these areas Ixodes ricinus carriers of Borrelia burgdorferi have been frequently observed ^1,2. These circumstances explain the high seroprevalence of Lyme disease in Asturias, higher than in other regions of Spain, reaching to 5.1% in healthy blood donors of this region ³.

Although it affects primarily cattle in Europe, Babesia divergens, a parasite also transmitted by I. ricinus, can infect humans as well. B. divergens-like DNA sequences have also been reported in red deer (Cervus elaphus), roe deer (Capreolus capreolus), reindeer (Rangifer tarandus) and fallow deer ^4,5. However, there is no reported evidence that Babesia isolates from deer are infective to humans or cattle so far ⁶

Coinfection with B.burgdorferi and Babesia microti is relatively common in northwestern and upper midwestewern United States. Approximately 10% of patients with Lyme disease in southern New England are co-infected with babesiosis in locations where both diseases are zoonotic ⁷. In Europe B. microti and B. divergens positive antibodies were observed in 16.3% of Swedish patients seropositive for Borrelia ⁸ .Since 2011 two cases of severe infection by B.divergens have been reported in Asturias, probably the iceberg of an undetected B.divergens-infected population ^9,10. We detected a B.burgdorferi-B.divergens coinfection rate of 39.2% in adult patients with confirmed borreliosis from Asturias in a retrospective study (2015-17) ¹¹. The number of symptoms and duration of illness in American patients with concurrent Lyme disease and babesiosis due to B. microti were higher than in patients with either infection alone ^{7, 12,13} .

Illness characteristics of patients infected with both B. burgdorferi and B. divergens pathogens have not been reported so far. Diagnostic tools on when to suspect and how to manage babesiosis in patients with borreliosis are lacking. In order to answer these questions we planned a retrospective study comparing clinical, analytical and other studies (imaging, ECG) characteristics of 120 Asturias adult patients with confirmed borreliosis of which 47 were also infected with B. divergens.

Patients and Study Design

This retrospective study included all the patients ≥ 18 years old residing in Asturias (Northwestern Spain) with an IgG positive serology for borreliosis from 2015 through 2017 assessed at the Microbiology Service of the Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain. Detection of B. burgdorferi s.l. antibodies was done using an automated qualitative test (Vidas, BioMerieux, Madrid, Spain) ¹¹ and by immunoblot (Borrelia IgG IgM EcoLine, Sekisui Diagnostics, Russelsheim, Germany) ^3,11. Patients’ serum samples positive for Lyme disease were stored at -80ºC at the Microbiology Service of the HUCA for further studies. These patients had been followed at the Infectious Diseases, Neurology, Rheumatology and/or Dermatology Services of the HUCA or other regional affiliated hospitals because B. burgdorferi serology assessment is centralized at the HUCA at regional level. Patients’ electronic medical records were searched and their demographic, epidemiological, including borreliosis-predisposing factors, clinical, analytical, imaging, electrocardiographic and other data were collected. Patients with confirmed neurologic Lyme disease had compatible clinical symptoms and/or signs and positive Borrelia IgG serology in cerebrospinal fluid (CSF). In a subsequent study, stored serum samples seropositive for B. burgdorferi s.l. were used again to detect IgG antibodies against B. divergens by an in-house indirect fluorescent assay (IFA) and by Western Blot at the Parasitology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain ¹¹.

Ethics statement

All experimental protocols were approved by the Research Ethics Committee of the Instituto de Salud Carlos III, Madrid, Spain, reference CEI PI 59_2022.

The Research Ethics Committee of the Instituto de Salud Carlos III, Spain, reference CEI PI 59_2022 granted a formal waiver of requiring the informed consent from individual patients.

All research was performed in accordance with relevant guidelines and regulations.

Statistical analysis

The continuous variables did not follow a Gaussian distribution, according to the Kolmogorov-Smirnov test and, therefore, non-parametric tests were used for analysis. The values are reported as median (IQ range) or as percentage for continuous or categorical variables, respectively. The mono- and doubly infected groups were compared with the Mann-Whitney U test for continuous and the chi-square test and the Fisher’s exact text, when appropriate, for categorical variables. A stepwise logistic regression analysis model was constructed to identify the factors independently associated with the diagnosis of B.burgdorferi-B.divergens double infection. A P value < 0.05 for a two-tailed test was considered statistically significant. Calculations were carried out with the statistical software SPSS v. 25 (IBM Corp., Armonk, New York, USA).

A total of 120 patients, 73 monoinfected with B.burgdorferi and 47 doubly-infected with B. burgdorferi and B. divergens were included in the study¹¹. The median age of the series was 57.5 years (IQ range 43.8–72.5) and 80 patients (66.7%) were men.

Table 1 shows the demographic, epidemiologic and borreliosis predisposing factors of monoinfected and doubly-infected patients. It can be appreciated that 65.8% developed tick-bites risky outdoor activities, mostly as a hobby, 28.3% had non-professional animal contact and only 10.8% were farmers or cattle breeders. Interestingly only 38.3% of the borreliosis patients recalled a recent tick bite. There were no patients with asplenia and only 4 (0.03%) had a known immunodepression. No differences between mono and doubly- infected patients regarding demographic, epidemiologic and borreliosis predisposing factors were observed.

Table 1

Demography, epidemiology, and predisposing factors
		Monoinfected (n = 73)	Coinfected (n = 47)	P value
Demography & epidemiology
- Gender	Male	47 (64.4%)	34 (72.3%)	0.4
- Gender	Female	26 (35.6%)	13 (27.7%)
- Age years (n = 119)		58.0 (42.8–73.6)	56.4 (45.5–71.8)	0.8
- Occupation	Farmer	4 (8.5%)	4 (12.1%)	0.2
	Breeder	3 (6.4%)	2 (6.1%)
	Open air activity	0 (0%)	3 (9.1%)
	Other	40 (85.1%)	24 (72.7)
- Tick bite identified	Yes	17 (24.6%)	9 (20.9%)	0.7
- Tick bite identified	No	52 (75.4%)	34 (79.1%)
- Tick removal	Yes	12 (17.4%)	8 (18.6%)	0.9
- Tick removal	No	57 (82.6%)	35 (81.4%)
Predisposing factors	Yes	66 (90.4%)	42 (93.3%)	0.7
	No	7 (9.6%)	3 (6.7%)
- Transfusion	Yes	0 (0%)	0 (0%)	-
- Transfusion	No	69 (100%)	45 (100%)
- Asplenia	Yes	0 (0%)	0 (0%)	-
- Asplenia	No	69 (100%)	45 (100%)
- Immunosuppression	Yes	2 (2.9%)	2 (4.4%)	0.6
- Immunosuppression	No	68 (97.1%)	43 (95.6%)
- Age > 50 years	Yes	41 (57.7%)	27 (60.0%)	0.9
- Age > 50 years	No	30 (42.3%)	18 (40.0%)
- Outdoor hobbies	Yes	45 (72.6%)	34 (82.9%)	0.2
- Outdoor hobbies	No	17 (27.4%)	7 (17.1%)
- Non-professional animal contact	Yes	18 (29.0%)	16 (39.0%)	0.3
	No	44 (71.0%)	25 (61.0%)
Values are expressed as median (IQ range) or %.

The clinical features and complications of the patients are reported in Table 2. Overall, 48.3% had neurological symptoms, raising to 53.2% in doubly-infected patients, 14/120 (11.7%) had unilateral or bilateral facial paralysis and 25.8% cutaneous symptoms with erythema migrans reported in 22/120 (18.3%). On the other hand 32.5% had osteomuscular symptoms, mostly arthralgias (27.5%), and 20.8% had constitutional symptoms, mostly fever (11.7%). There were no differences between mono and doubly- infected patients regarding these clinical manifestations. On the other hand 13/120 (10.8%) had cardiorespiratory symptoms, with dyspnea as the most frequent (5/120, 4.12%). Of note, doubly-infected patients had more frequently cardiorespiratory symptoms compared to monoinfected (9/47 [19.1%] vs. 4/73 [5.65%], P = 0.02) especially dyspnea (4/47 [8.5%] vs. 1/73 [1.4%], P = 0.07 ). No differences in the physical exam between mono and doubly-infected individuals were detected No differences regarding type of borreliosis, its complications or presence of an alternative diagnosis between mono and doubly- infected patients were observed.

Table 2

Clinical features and complications
		Monoinfected (n = 73)	Coinfected (n = 47)	P value
SYMPTOMS
- Duration of symptoms	days (n = 92)	7.0 (1.0–45.0)	15.00 (6.00-105.0)	0.11
Constitutional symptoms	Yes	15 (21.1%)	10 (22.2%)	0.9
Constitutional symptoms	No	56 (78.9%)	35 (77.8%)
- Fever	Yes	9 (12.7)	5 (11.1%)	0.8
- Fever	No	62 (87.3%)	40 (88.9%)
- Asthenia	Yes	8 (11.3%)	6 (13.3%)	0.7
- Asthenia	No	63 (88.7%)	39 (86.7%)
- Anorexia	Yes	2 (2.8%)	0 (0%)	0.5
- Anorexia	No	69 (97.2%)	45 (100%)
- Weight loss	Yes	0 (0%)	0 (0%)	-
- Weight loss	No	71 (100%)	45 (100%)
Osteomuscular symptoms	Yes	22 (31.0%)	17 (37.8%)	0.5
Osteomuscular symptoms	No	49 (69.0%)	28 (62.2%)
- Arthralgias	Yes	19 (26.8%)	14 (31.1%)	0.6
- Arthralgias	No	52 (73.2%)	31 (68.9%)
- Arthritis	Yes	12 (16.9%)	5 (11.1%)	0.4
- Arthritis	No	59 (83.1%)	40 (88.9%)
- Myalgias	Yes	7 (9.9%)	9 (20.0%)	0.12
- Myalgias	No	64 (90.1%)	36 (80.0%)
Digestive symptoms	Yes	3 (4.2%)	0 (0%)	0.3
Digestive symptoms	No	68 (95.8%)	45 (100%)
- Abdominal pain	Yes	2 (2.8%)	0 (0%)	0.5
- Abdominal pain	No	69 (97.2%)	45 (100%)
- Nausea	Yes	0 (0%)	0 (0%)	-
- Nausea	No	71 (100%)	45 (100%)
- Vomiting	Yes	1 (1.4%)	0 (0%)	1
- Vomiting	No	70 (98.6%)	45 (100%)
- Diarrhea	Yes	0 (0%)	0 (0%)	-
- Diarrhea	No	71 (100%)	45 (100%)
Cardiorespiratory symptoms	Yes	4 (5.6%)	9 (20.0%)	0.02
Cardiorespiratory symptoms	No	67 (94.4%)	36 (80.0%)
- Syncope	Yes	1 (1.4%)	2 (4.4%)	0.6
- Syncope	No	70 (98.6%)	43 (95.6%)
- Chest pain	Yes	2 (2.8%)	3 (6.7%)	0.4
- Chest pain	No	69 (97.2%)	42 (93.3%)
- Dyspnea	Yes	1 (1.4%)	4 (8.9%)	0.07
- Dyspnea	No	70 (98.6%)	41 (91.1%)
- Palpitations	Yes	0 (0%)	0 (0%)	-
- Palpitations	No	71 (100%)	45 (100%)
Neurologic symptoms	Yes	33 (46.5%)	25 (55.6%)	0.3
Neurologic symptoms	No	38 (53.5%)	20 (44.4%)
- Loss of strength	Yes	11 (15.5%)	9 (20.0%)	0.5
- Loss of strength	No	60 (84.5%)	36 (80.0%)
- Gait disturbance	Yes	8 (11.3%)	5 (11.1%)	1
- Gait disturbance	No	63 (88.7%)	40 (88.9%)
- Cranial nerve involvement	Yes	7 (9.9%)	3 (6.7%)	0.7
- Cranial nerve involvement	No	64 (90.1%)	42 (93.3%)
- Paresthesia	Yes	8 (11.3%)	7 (15.6%)	0.5
- Paresthesia	No	63 (88.7%)	38 (84.4%)
- Dizziness	Yes	3 (4.3%)	2 (4.4%)	1
- Dizziness	No	67 (95.7%)	43 (95.6%)
- Headache	Yes	13 (18.3%)	6 (13.3%)	0.5
- Headache	No	58 (81.7%)	39 (86.7%)
- Hyperesthesia	Yes	0 (0%)	1 (2.2%)	0.4
- Hyperesthesia	No	71 (100%)	44 (97.8%)
- Other symptoms	Yes	9 (12.7%)	4 (8.9%)	0.5
- Other symptoms	No	62 (87.3%)	41 (91.1%)
Ophthalmologic symptoms	Yes	3 (4.2%)	1 (2.2%)	1
Ophthalmologic symptoms	No	68 (95.8%)	44 (97.8%)
- Photophobia	Yes	2 (2.8%)	1 (2.2%)	1
- Photophobia	No	69 (97.2%)	44 (97.8%)
Cutaneous symptoms	Yes	18 (25.4%)	13 (28.9%)	0.7
Cutaneous symptoms	No	53 (74.6%)	32 (71.1%)
- Erythema migrans	Yes	14 (19.7%)	8 (18.2%)	0.8
- Erythema migrans	No	57 (80.3%)	36 (81.8%)
- Rash	Yes	5 (7.0%)	2 (4.4%)	0.7
- Rash	No	66 (93.0%)	43 (95.6%)
- Other cutaneous involvement	Yes	3 (4.2%)	5 (11.1%)	0.3
- Other cutaneous involvement	No	68 (95.8%)	40 (88.9%)
Other symptoms	Yes	9 (12.7%)	1 (2.2%)	0.09
Other symptoms	No	62 (87.3%)	44 (97.8%)
PHYSICAL EXAM
General	Yes	2 (2.8%)	2 (4.4%)	0.6
General	No	69 (97.2%)	43 (95.6%)
- Jaundice	Yes	0 (0%)	0 (0%)	-
- Jaundice	No	71 (100%)	45 (100%)
- Pharyngeal erythema	Yes	0 (0%)	1 (2.2%)	0.4
- Pharyngeal erythema	No	71 (100%)	44 (97.8%)
- Lymphadenopathy	Yes	2 (2.8%)	2 (4.4%)	0.6
- Lymphadenopathy	No	69 (97.2%)	43 (95.6%)
- Hepatomegaly	Yes	0 (0.0%)	1 (2.2%)	0.4
- Hepatomegaly	No	71 (100%)	44 (97.8%)
- Splenomegaly	Yes	0 (0.0%)	1 (2.2%)	0.4
- Splenomegaly	No	71 (100%)	44 (97.8%)
Neurologic	Yes	17 (23.9%)	11 (24.4%)	0.9
Neurologic	No	54 (76.1%)	34 (75.6%)
- Meningeal signs	Yes	2 (2.8%)	0 (0%)	0.5
- Meningeal signs	No	69 (97.2%)	45 (100%)
- Nystagmus	Yes	2 (2.8%)	0 (0%)	0.5
- Nystagmus	No	69 (97.2%)	45 (100%)
- Unilateral facial paralysis	Yes	8 (11.3%)	3 (6.7%)	0.5
- Unilateral facial paralysis	No	63 (88.7%)	42 (93.3%)
- Bilateral facial paralysis	Yes	2 (2.8%)	1 (2.2%)	1
- Bilateral facial paralysis	No	69 (97.2%)	44 (97.8%)
- Other cranial nerve involvement	Yes	2 (2.8%)	0 (0%)	0.5
- Other cranial nerve involvement	No	69 (97.2%)	45 (100%)
- Romberg sign	Yes	1 (1.4%)	0 (0%)	1
- Romberg sign	No	70 (98.6%)	45 (100%)
- Babinski sign	Yes	2 (2.8%)	1 (2.2%)	1
- Babinski sign	No	69 (97.2%)	44 (97.8%)
- Muscle weakness	Yes	12 (16.9%)	7 (15.6%)	0.8
- Muscle weakness	No	59 (83.1%)	38 (84.4%)
Ophthalmologic	Yes	1 (1.4%)	1 (2.2%)	1
Ophthalmologic	No	70 (98.6%)	44 (97.8%)
- Retinal infarction	Yes	0 (0%)	0 (0%)	-
- Retinal infarction	No	71 (100%)	45 (100%)
- Retinal hemorrhage	Yes	0 (0%)	0 (0%)	-
- Retinal hemorrhage	No	71 (100%)	45 (100%)
- Conjunctival hyperemia	Yes	1 (1.4%)	1 (2.2%)	1
- Conjunctival hyperemia	No	70 (98.6%)	44 (97.8%)
TYPE OF BORRELIOSIS
- Early localized borreliosis	Yes	22 (31.9%)	14 (31.1%)	0.9
	No	47 (68.1%)	31 (68.9%)
- Early disseminated borreliosis	Yes	12 (17.4%)	7 (15.6%)	0.8
	No	57 (82.6%)	38 (84.4%)
- Late borreliosis	Yes	3 (4.3%)	3 (6.7%)	0.7
	No	66 (95.7%)	42 (93.3%)
- Past Borrelia infection	Yes	21 (30.4%)	16 (35.6%)	0.6
	No	48 (69.6%)	29 (64.4%)
- Neurologic Lyme disease ¹	Yes	12 (17.4%)	7 (15.6%)	0.8
	No	57 (82.6%)	38 (84.4%)
COMPLICATIONS	Yes	0 (0.0%)	1 (2.2%)	0.4
COMPLICATIONS	No	71 (100%)	44 (97.8%)
- Respiratory distress	Yes	0 (0%)	0 (0%)	-
- Respiratory distress	No	71 (100%)	45 (100%)
- Heart failure	Yes	0 (0.0%)	1 (2.2%)	0.4
- Heart failure	No	71 (100%)	44 (97.8%)
- Disseminated intravascular coagulation	Yes	0 (0%)	0 (0%)	-
- Disseminated intravascular coagulation	No	71 (100%)	45 (100%)
- Splenic infarct	Yes	0 (0%)	0 (0%)	-
- Splenic infarct	No	71 (100%)	45 (100%)
- Splenic rupture	Yes	0 (0%)	0 (0%)	-
- Splenic rupture	No	71 (100%)	45 (100%)
- Other complications	Yes	0 (0%)	0 (0%)	-
- Other complications	No	71 (100%)	45 (100%)
ALTERNATIVE DIAGNOSIS ²	Yes	21 (29.6%)	15 (34.1%)	0.6
ALTERNATIVE DIAGNOSIS ²	No	50 (70.4%)	29 (65.9%)
Values are expressed as median (IQ range) or %.
¹ Patients with neurologic Lyme disease had compatible symptoms and/or signs and positive Borrelia IgG antibodies in cerebrospinal fluid (CSF)
² Alternative diagnosis represents patients with a primary diagnosis with clinical symptoms and/or signs different from borreliosis

Table 3 depicts the laboratory, radiologic imaging and electrocardiographic studies of the patients. Anemia, or increased levels of lactate dehydrogenase (LDH) or bilirrubin, parameters associated with hemolytic anemia, were not observed neither in patients with mono nor doubly-infected. Normal levels of C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR) were observed in patients with mono or double infection. Atrioventricular (AV) block was detected in 5/47 (15.6%) doubly compared to 1/73 (2.7%) monoinfected individuals (P = 0.09). No other imaging, electrocardiographic or laboratory studies differences were observed between doubly and monoinfected patients.

Table 3

Imaging, electrocardiographic and laboratory studies
		Monoinfected (n = 73)	Coinfected (n = 47)	P value
Chest X-ray	Normal	38 (97.4%)	26 (92.9%)	0.6
Chest X-ray	Abnormal	1 (2.6%)	2 (7.1%)
Electrocardiogram	Normal	36 (97.3%)	27 (84.4%)	0.09
Electrocardiogram	AV block	1 (2.7%)	5 (15.6%)
Laboratory blood determinations
Total leukocyte counts	cells/µL (n = 109)	7500.0 (6165.0-9065.0)	7240.0 (5612.5–9520.0)	0.6
Absolute neutrophil counts	cells/µL (n = 109)	4180.0 (3170.0-5765.0)	4520.0 (2885.0-6552.5)	0.8
Absolute lymphocyte counts	cells/µL (n = 107)	1940.0 (1460.0-2590.0)	1740.0 (1470.0-2307.5)	0.3
Absolute eosinophil counts	cells/µL (n = 106)	130.0 (77.5-232.5)	135.0 (72.5–245.0)	0.9
Platelets	per µL (n = 109)	232000 (183000–294000)	236000 (200250–285250)	1
Hemoglobin	gr/dL (n = 109)	14.10 (13.40–15.20)	14.40 (13.38–15.23)	0.8
C-reactive protein	mg/L (n = 89)	0.350 (0.100-0.925)	0.300 (0.100–1.400)	0.7
Erythrocyte sedimentation rate	mm/h (n = 66)	12.0 (4.8–22.0)	11.0 (5.0–29.0)	0.6
Aspartate aminotransferase	U/L (n = 37)	24.0 (19.0-36.3)	21.0 (20.0–28.0)	0.9
Alanine aminotransferase	U/L (n = 93)	19.0 (15.0–31.0)	23.0 (17.0–28.0)	0.2
Lactate dehydrogenase	U/L (n = 39)	208.0 (182.0-237.0)	207.0 (168.8-254.5)	0.9
Total bilirubin	mg/dL (n = 73)	0.90 (0.90–0.90)	0.90 (0.90–0.90)	0.3
Alkaline phosphatase	U/L (n = 89)	73.0 (59.0–87.0)	62.0 (52.5–83.3)	0.2
Creatinine	mg/dL (n = 106)	0.85 (0.74–1.02)	0.80 (0.74–0.93)	0.4
Values are expressed as median (IQ range) or %.

The microbiological diagnostic procedures are described in Table 4. Positive antibodies against B. divergens assessed by indirect immunofluorescence assay (IFI) were identified in 47/47 (100%), and by inmunoblot in 39/47 (95.1%) of coinfected patients. In addition antibodies against B. divergens extract proteins were identified in 40 /47 (85.1%) and antibodies against the GST-rBD37 recombinant protein in 19 /47 (47.5%) of coinfected individuals ^9,14.

Table 4

Microbiological studies
		Monoinfected (n = 73)	Coinfected (n = 47)	P value
Borrelia burgdorferi IgM serology	Positive	43 (58.9%)	27 (57.4%)	0.9
Borrelia burgdorferi IgM serology	Negative	30 (41.1%)	20 (42.6%)
Borrelia burgdorferi IgG serology	Positive	73 (100%)	47 (100%)	-
Borrelia burgdorferi IgG serology	Negative	0 (0%)	0 (0%)
Borrelia burgdorferi immunoblot	Positive	62 (88.6%)	39 (95.1%)	0.5
	Negative	1 (1.4%)	0 (0%)
	Doubtful	7 (10.0%)	2 (4.9%)
Babesia divergens protein extract ¹	Positive	0 (0%)	40 (85.1%)	< 0.0001
Babesia divergens protein extract ¹	Negative	73 (100%)	7 (14.9%)
Babesia divergens indirect immunofluorescence assay	Positive	0 (0%)	47 (100%)	< 0.0001
Babesia divergens indirect immunofluorescence assay	Negative	73 (100%)	0 (0%)
Bd37 recombinant protein ¹	Positive	0 (0%)	19 (44.2%)	< 0.0001
	Negative	73 (100%)	24 (55.8%)
¹ B. divergens protein extracts and the purified Glutathione S-Transferase-tagged Bd37 recombinant protein (GST-rBD37) were used as target substrates for B. divergens Western-blot assays

Table 5 shows the treatment administered to the patients. The most commonly administered antibiotic was doxycycline and there were no significant differences between mono and doubly-infected patients in any of the regimens administered.

Table 5

Antibiotic treatment
		Monoinfected (n = 73)	Coinfected (n = 47)	P value
Doxycycline	Yes	21 (31.3%)	19 (44.2%)	0.17
Doxycycline	No	46 (68.7%)	24 (55.8%)
Doxycycline dosage	mg/day (n = 39)	200 (200–200)	200 (200–200)	0.3
Amoxicillin	Yes	5 (7.5%)	5 (11.4%)	0.5
Amoxicillin	No	62 (92.5%)	39 (88.6%)
Amoxicillin dosage	mg/day (n = 11)	1500 (650–2625)	1500 (938–2625)	1
Ceftriaxone	Yes	10 (14.9%)	7 (15.9%)	9
Ceftriaxone	No	57 (85.1%)	37 (84.1%)
Ceftriaxone dosage	mg/day (n = 17)	1500 (1000–2000)	2000 (1000–2000)	0.8
Cefuroxime	Yes	1 (1.5%)	0 (0%)	1
Cefuroxime	No	66 (98.5%)	44 (100%)
Azithromycin	Yes	0 (0%)	1 (2.2%)	0.4
Azithromycin	No	67 (100%)	44 (97.8%)
Penicillin G	Yes	1 (1.5%)	0 (0%)	1
Penicillin G	No	66 (98.5%)	44 (100%)
Treatment duration	weeks (n = 108)	2.00 (0.00–3.00)	2.00 (0.00–3.00)	0.7
Values are expressed as median (IQ range) or %

A forward stepwise logistic regression analysis model, including all variables with a P value ≤ 0.1 in the univariate analyses, revealed that the only significantly independent predictive factor for B. burgdorferi s.l.-B. divergens double infection was the presence of cardiorespiratory symptoms (OR 4.184, 95% CI 1.205–14.493, P = 0.024).

Babesia divergens-Borrelia burgdorferi s.l. doubly infected patients had more frequently cardiorespiratory symptoms, mostly dyspnea, compared to B. burgdorferi s.l. monoinfected individuals. These symptoms were unrelated to anemia that neither mono nor doubly infected had, and might be enhanced by ECG AV block. This cardiac arrhythmia could be caused by summative myocardial damage induced by both infections.

Babesiosis due to B. divergens is the most severe among the infections due to all Babesia species in humans with a quite high mortality, up to 40% in hospitalized patients, although nowadays it is decreasing ¹⁵. Other Babesia species such as B. microti have a lower mortality rate reaching 10–20% in hospitalized individuals ^{16, 17}. Mortality attributable to Babesia is not due exclusively to the pathogen or the host but also to the interaction between both of them. This interaction explains the severity of some cases, two of them reported from Asturias, one in a young immunocompetent patient, and the lower severity of others such as those reported in the present study ^9,10.

Previous reports showed that experimental coinfection by B. microti and B. burgdorferi s.l. induced increased arthritis severity in mice which correlated with a significant reduction of expression of the IL-10 and IL-13 cytokines ¹⁸. B. microti-B. burgdorferi coinfection in humans increased the intensity and duration of the illness and its symptoms in American patients ^7,12. Fatigue, but also headache, nausea, sweating, anorexia, chills, emotional lability and splenomegaly were more frequently reported in B. microti-B. burgdorferi coinfected individuals with symptoms’ duration lasting up to three months compared to those individuals monoinfected with B. burgdorferi s.l.. No cardiac abnormalities have been described in these coinfected American patients, though ⁷ .

A recent report described twenty-nine Swedish patients bitten by ticks infected with both B. microti or B. venatorum. Ten of them (34.5%) had minor symptoms such as headache, muscle pain, fatigue, neck pain, dizziness, concentration difficulties, numbness, radiating pain, joint pain and nausea. However these symptoms were not relevant enough to need medical attention. No cardiac symptoms were reported in these patients, either ¹⁹ .

To our knowledge this is the first report regarding clinical aspects of Babesia divergens-Borrelia burgdorferi s.l. double infection in the literature and the second in which B. divergens- B .burgdorferi s.l. human double infections were observed. Svensson et al., reported five Swedish patients with B. divergens.-Borrelia B. burgdorferi double infections ⁸ A recent French meta-analysis found eight reports of Babesia spp.-Borrelia B. burgdorferi s.l. coinfection in the literature but all of them were due to B. microti ²⁰ .

We observed a prevalence rate of 39.2% of B. divergens-B.burgdorferi s.l. double infection in Asturias patients with a serologically-confirmed borreliosis which is more than twofold higher (16.3%) than the B. microti/B. divergens–B. burgderfori infection rate found in a geographical- area endemic for Lyme disease in Sweden ⁸.

Overall, 53.2% of B. divergens- B. burgdorferi s.l. doubly infected patients had minor neurologic symptoms, and only 22.2% reported constitutional complains (fever, asthenia, anorexi, weight loss). In addition their clinical course was mild and short unlike the complicated clinical course of those with B. microti-B. burgdorferi s.l. coinfections ^{7, 12}. A possible explanation for this discordance might be differences in the infectivity of each Babesia species, as well as in the susceptibility of individual patients to infection.

It is intriguing that B. divergens-B. burgdorferi s.l. doubly infected patients had more frequently cardiorespiratory symptoms, mostly dyspnea compared to B. burgdorferi s.l. monoinfected patients. These were the only significant differential clinical symptoms between mono and doubly infected individuals. One possible explanation for the cardiac dysfunction in double infected individuals might be a summative potential myocardial damage of both microorganisms manifested by ECG AV block present in five doubly infected individuals. Both B. burgdorferi s.l. and B. divergens might affect AV conduction leading to AV block. B. burgdorferi AV block is a well-known complication of borreliosis ²¹. Babesia bigemina infection induced changes in cardiac function biomarkers and D-dimer in cattle ²². Babesia canis infection induced cardiac disorders in dogs with 32% of AV block ²³. Two reports showed myocardial damage and serious arrhtymia associated with severe babesiosis in two American patients ^{24, 25}. A recent report showed that 19.6% of hospitalized American patients with acute babesiosis due to B. microti developed cardiac complications, mostly atrial fibrillation, heart failure, QT interval prolongation and cardiac ischemia ²⁶. No AV block I in that study was observed, though. All the patients had high parasitemia and had received antimicrobials (macrolides, quinine) which might enhance heart arrythmia. Interestingly cardiac complications were not worse in 12 of them with confirmed B. microti-B. burgdorferi s.l. coinfection. We assume that B. divergens might induce a myocardial damage similar to B. microti. Unluckily, neither troponin, creatin kinase (CK), natriuretic peptides (B-type natriuretric peptide [BNT] or N-terminal pro-B-type natriuretic peptide [NT-proBNT]), other cardiac function biomarkers or echocardiograms were done in our study being retrospective. In addition ECG was performed in only 58.4% of our patients.

The strength of our study relies in being the first report studing clinical differences, analytical, imaging and ECG between patients mono or doubly-infected with B.divergens-B.burgdorferi. Their B.divergens infection had been previously diagnosed using some novel diagnostic techniques ¹¹. The mean weakness of the study is derived of its retrospective design lacking universal ECG, cardiac biomarkers and echocardiograms testing that might provide deeper insight into the mechanism of cardiac complications in these doubly infected patients. Other weakness of the study is the uncertainty of the timing, simultaneous or sequential, of both infections, by B. burgdoferi s. l and B. divergens. Patients could be firstly infected by B. divergens and had a spontaneous recovery to become infected by B. burgdoferi subsequently. If this be the case we might be comparing virtually two groups with a B. burgdoferi monoinfection. The fact that even so clinical differences between mono and doubly-infected individuals were observed suggests that disparity might be higher in cases of a true simultaneous coinfection.

In summary patients with B. burgdorferi s. l. and B. divergens double infections had a mild clinical course,and did not develop anemia or other analytical abnormalities. Their only differential symptoms were cardiorespiratory manifested as dyspnea and ECG AV block.

Patients with suspected Lyme disease could be screened for babesiosis as well to obtain relevant information on epidemiology and prevalence of both concurrent diseases in endemic areas for both zoonosis. Detection of coinfections might also provide useful information to the physician to monitor and treat coinfected patients living in these areas. This concern is especially important in those immunocompromised in whom babesiosis has a more severe evolution and could lead even to a fatal outcome.

Data Availability Statement

All data generated or analysed during this study are included in this published article

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Clinical, analytical, imaging and ECG differences in adult patients infected by Borrelia burgdorferi and doubly-infected by Babesia divergens-Borrelia burgdorferi

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