Advertisement

BMC Veterinary Research

, 15:428 | Cite as

A novel PCR-based point-of-care method enables rapid, sensitive and reliable diagnosis of Babesia gibsoni infection in dogs

  • I-Li Liu
  • Nai-Yu Chi
  • Chia-Ling Chang
  • Ming-Long Hung
  • Chun-Ta Chiu
  • Hui-Wen ChenEmail author
Open Access
Methodology article
Part of the following topical collections:
  1. Parasitology

Abstract

Background

Babesia gibsoni (B. gibsoni) is an intraerythrocytic protozoan parasite of dogs that causes fever and hemolytic illness. A timely diagnosis is essential for the disease management.

Results

Here, we report a QubeMDx PCR system which enables a rapid, sensitive and reliable diagnosis of B. gibsoni near the dog patient. Within 30 min, this diagnostic assay was able to detect as low as 0.002% parasitemia of the dog blood. Using clinical samples, this new assay was validated to demonstrate 100% agreement with real-time PCR.

Conclusions

This novel diagnostic method provides a reliable point-of-care test to assist in the identification of B. gibsoni.

Keywords

Babesia gibsoni Point-of-care QubeMDx PCR system Real-time PCR 

Abbreviations

B. gibsoni

Babesia gibsoni

LAMP

Loop-mediated isothermal amplification

LF-RPA

Recombinase polymerase amplification-lateral flow

PCR

Polymerase chain reaction

PCV

Packed cell volume

POC

Point-of-care

Background

Babesia gibsoni (B. gibsoni) is a widespread tick-transmitted protozoan blood parasite that was first identified in 1910 in dogs and jackals from India and now prevalent in many regions, including Taiwan [1, 2] and other Asian countries such as Japan [3], Korea [4], China [5], Bangladesh [6], India [7] and Malaysia [8]. B. gibsoni infection is frequently found in companion dogs and presents a serious clinical problem in the world. The clinical severity of B. gibsoni infection in dogs is age-dependent and potentially life-threatening with clinical symptoms ranging from remittent fever and severe hemolytic illness. Therefore, the diagnosis of this disease and the detection of dogs that carry this blood parasite are very important [9, 10, 11, 12].

Generally, the standard method for definitive means of diagnosis of babesiosis is microscopic examination of blood smears following Giemsa-staining for visual identification of parasites. Although this microscopic method is an inexpensive diagnostic test, the detection of B. gibsoni in erythrocytes on blood smears is sometimes difficult and may be error-prone. As B. gibsoni is a small and pleomorphic organism (1–3 μm in diameter), unlike the large, paired and teardrop-shaped Babesia canis, a highly experienced diagnostic skill is required to distinguish it from artifacts under the microscope, and it becomes even more challenging to find the parasites when an infected dog has low levels of parasitemia [12]. On the other hand, the molecular diagnosis detecting the DNA of B. gibsoni in canine blood samples by polymerase chain reaction (PCR) offers high degrees of detection sensitivity and specificity. Various conventional [13], nested [14] and real-time [15] PCR assays detecting B. gibsoni infection in dogs have been developed. However, these assays mostly require a relatively expensive thermo cycler and well-trained personnel, which may delay results and add costs. As a result, a rapid, affordable and user-friendly platform is needed for a point-of-care (POC) detection of B. gibsoni in daily veterinary practice to facilitate animal care and disease management.

The recently developed QubeMDx (Credo Biomedical Pte. Ltd., Singapore) is a portable diagnostic system (12.1 × 10.9 × 14.7 cm, H × W × D) using reaction tubes prefilled with fluorescence-based reagents to facilitate the real-time PCR detection process within 15 min. The programmed device allows users to perform four different targeted DNA or RNA molecular diagnosis in a given round of reaction with minimal training. The purpose of the present study is to evaluate the QubeMDx PCR system as a practical and accurate method for the diagnosis of B. gibsoni infection in dog by using clinical canine blood samples and comparing to a standard real-time PCR assay.

Results

As illustrated in Fig. 1a, the DNA extraction can be completed within 8 min. The resultant DNA solution was subsequently assayed using the QubeMDx PCR device (Fig. 1b). After 15-min reaction time, the test result for B. gibsoni was displayed in the panel A, either positive (+) or negative (−) on the screen, and the test validity (internal control) was displayed in the panel B (Fig. 1c). Evaluated using 28 reference test (real-time PCR)-positive clinical samples, the sensitivity of the QubeMDx PCR was 100%. The specificity was also 100%, calculated using 27 reference test-negative samples. The Kappa value was 1, indicating highly reproducible results between these two methods. Furthermore, among different levels of parasitemia analyzed from 28 positive dog samples (Table 1), the QubeMDx PCR system successfully detected the B. gibsoni ranging from 94 to 398,000 parasites/μL of blood (0.002 to 8% of parasitemia). Importantly, no discrepancy between these two methods was found among these clinical samples containing low levels of parasitemia (16/28) (< 5000 parasites/μL of blood or parasitemia < 0.1%). Using ten-fold serially diluted blood sample (dog #25), the QubeMDx PCR was shown to be able to detect 0.00576%, but not 0.000576% of parasitemia (Fig. 2). Collectively, DNA rapid extraction and QubeMDx PCR allow for detection of the B. gibsoni DNA with a sensitivity limit in between 0.000576 and 0.002% of parasitemia. The analytical specificity of the QubeMDx PCR for B. gibsoni detection was further assessed using blood samples positive for Anaplasma platys, Ehrlichia canis or Babesia canis. As indicated in Additional file 1: Table S1, no false positive detection results were obtained from canine blood samples infected with other three pathogens.
Fig. 1

QubeMDx PCR assay for B. gibsoni. a Graphical illustration of the DNA extraction procedures using the Credo Rapid Blood Nucleic Acid Extraction Kit. b A photo of the QubeMDx PCR device. c The reading interface and interpretations of the QubeMDx PCR device. The test result for B. gibsoni is displayed in the panel A, either positive (+) or negative (−) on the screen, and the test validity (internal control) is displayed in the panel B

Table 1

B. gibsoni-positive dog blood samples included in this study

No.

Age (yr)

Sex

Breed

PCVa (%)

Test results

Parasites/μL of bloodb

QubeMDx

Real-time PCR

1

8

M

Mixed

n/a

+

+

94

2

7

M

n/a

32.3

+

+

98

3

12

F

Mixed

28.8

+

+

103

4

13

M

Mixed

21.3

+

+

188

5

4.3

F

Mixed

50.5

+

+

249

6

1.7

M

Miniature Schnauzer

23.6

+

+

261

7

14

F

Mixed

27.3

+

+

422

8

9

F

Mixed

31.8

+

+

798

9

2

F

Mixed

n/a

+

+

1030

10

5

M

Poodle

37.8

+

+

1150

11

8

M

Mixed

48.7

+

+

1290

12

6

F

Mixed

n/a

+

+

1360

13

5

M

Poodle

37.7

+

+

1870

14

6

F

Mixed

n/a

+

+

2180

15

5

F

Mixed

n/a

+

+

3610

16

9

M

Labrador Retriever

25.9

+

+

3840

17

4

F

Mixed

43.9

+

+

5410

18

6

M

Poodle

47.3

+

+

8140

19

3

M

Poodle

14.6

+

+

8140

20

2

F

Mixed

51.2

+

+

8760

21

0.2

F

Mixed

30.3

+

+

10,100

22

12

F

Miniature Schnauzer

16.7

+

+

10,800

23

9

F

Mixed

n/a

+

+

13,700

24

6

M

Corgi

10.5

+

+

14,200

25

2

M

Pomeranian

40.6

+

+

28,800

26

12

M

Dachshund

16

+

+

30,400

27

0.5

M

Bichon Frisé

18.6

+

+

149,000

28

10

M

Maltese

14.6

+

+

398,000

aPacked cell volume

bParasites was determined by real-time PCR and calculated from the standard curve constructed with the plasmid DNA according to Matsuu et al., 2005 [15]

Fig. 2

Detection limit of QubeMDx PCR assay for B. gibsoni. B. gibsosi-positive dog blood (0.576% of parasitemia according to the real-time PCR) was 10-fold serially diluted and tested onto the QubeMDx PCR system to determine the detection limit. The test result for B. gibsoni is displayed in the panel A, with positive (+) or negative (−) result. The test validity (internal control) is displayed in the panel B

Discussion

In this study, a novel and reliable POC diagnostic method for B. gibsoni detection was reported. By coupling the Credo Rapid Blood Nucleic Acid Extraction Kit and the QubeMDx PCR system, the entire diagnostic procedures for B. gibsoni can be completed within 30 min in clinic, where veterinarians primarily rely on microscopic examination as the diagnosis method. The simplified assay procedures and saved time from laboratory identification may expedite the prescription medications and lead to effective disease management for the affected dogs.

The detection limit of diagnostic tests is a critical parameter for accurate and timely pathogen identification. It has been suggested that B. gibsoni infection with low parasitemia (0–0.75%) is easily misdiagnosed as immune-mediated hemolytic anemia and sometimes inappropriately treated with immune suppressive drugs [16, 17]. For dogs with babesiosis, either during the early or chronic stages of infection or under medical treatment, the parasite numbers in the blood may be low. Previous experimental infection study has shown that, not until 10 days after infection, dogs inoculated with B. gibsoni revealed a significantly decreased level of packed cell volume (PCV) (< 37%) or with a parasitemia greater than 0.1% [15]. By using this sensitive QubeMDx PCR system, the detection limit of 0.002% of parasitemia (94 parasites/μL of blood) allows for early diagnosis of infected dogs. Although greater detection limit was previously demonstrated by the real-time PCR (9 parasites/μL of blood) [15], recombinase polymerase amplification-lateral flow (LF-RPA) dipstick method (0.5 parasites/μL of blood) [5], loop-mediated isothermal amplification (LAMP)(0.0000135% of parasitemia) [18], the differences can be attributed to the use of rapid DNA extraction procedures in the QubeMDx system, leading to a compromised DNA yield.

In addition to a low detection limit, a 100% of sensitivity and specificity for B. gibsoni detection was achieved by this QubeMDx PCR based on real-time PCR confirmed positive/negative samples, while previously 86.67 and 57.33% of positive rate were obtained by the LF-RPA dipstick method [5] and the LAMP method [18], respectively, evaluated from clinically suspected dogs. Furthermore, the internal control included in each reaction of QubeMDx PCR provides an additional source for quality control. Taken together, compared to other available molecular methods, QubeMDx PCR system is a reliable POC method with advantages of rapidity and reaction simplicity.

Conclusions

This reported QubeMDx PCR system offers a rapid (< 30 min), sensitive, and effective diagnosis for B. gibsoni in clinical infections. The present point-of-care testing allows for treatment prescription in the early stages of the infection, facilitating the recovery of affected dogs.

Methods

A total of 57 EDTA-anticoagulated canine whole blood samples were collected at the National Taiwan University Veterinary Hospital. For the reference test of B. gibsoni detection, total DNA was extracted from 100 μL of blood sample with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A real-time PCR for B. gibsoni detection was performed to define the infection status and the standard curve was constructed with plasmid DNA for the conversion of parasitemia [15]. Through the reference test, 28 samples were defined as positive and used for the sensitivity evaluation for QubeMDx PCR system, whereas other 29 samples were negative and used for the specificity determination. In addition, the detection limit of the QubeMDx PCR system was determined using serially-diluted dog blood from one of the real-time PCR-positive blood sample. Furthermore, six canine whole blood samples diagnosed as Ehrlichia canis, Anaplasma platys or Babesia canis positive by PCR or real-time PCR [19, 20, 21] were also included in this study to evaluate the specificity of the QubeMDx PCR system.

To conduct the QubeMDx PCR assay, DNA was prepared by using the Credo Rapid Blood Nucleic Acid Extraction Kit (Credo Biomedical Pte. Ltd.). Briefly, 30 μL of blood sample was added to the tube containing the lysis buffer and the mixture was heated at 98 °C for 6 min. After the heating, the reaction mix was fractionated via centrifugation, and total DNA in the supernatant was collected to a fresh tube. The resultant DNA solution was subsequently assayed using the QubeMDx Babesia gibsoni Detection Kit (Credo Biomedical Pte. Ltd.) according to the manufacturer’s instructions. Briefly, 30 μL of the DNA solution was added to the reaction tube prefilled with lyophilized reagents containing primers and fluorogenic probes targeting the p18 gene of B. gibsoni as well as PCR essential reagents and internal controls. Tubes were then loaded onto the QubeMDx PCR device, and the fluorescent signals during amplification were detected and processed by a built-in algorithm. The video demonstrating assay procedures was filmed and can be found in the following link: https://www.dropbox.com/s/nbdt9ryvuubxsea/QubeMDx.mp4?dl=0.

Notes

Acknowledgments

Not applicable.

Authors’ contributions

ILL, MLH and HWC designed the study. NYC, CLC, MLH and CTC performed the experiments. ILL, MLH and HWC analyzed the data. ILL, MLH and HWC wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Credo Biomedical Pte. Ltd. and National Taiwan University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethics approval and consent to participate

Samples used in this study were derived during medical practice in the National Taiwan University Veterinary Hospital (NTUVH) and the use of samples has been approved by dog owners and NTUVH with written consent. The ethics approval was waived according to the “Guideline for the Care and Use of Laboratory Animals” issued by Council of Agriculture Executive Yuan, Taiwan on June 22nd of 2018 and the official notice made by the Ministry of Health and Welfare, Taiwan on July 5th of 2012 (document number: 1010265083).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

12917_2019_2181_MOESM1_ESM.pptx (38 kb)
Additional file 1: Table S1. Analytical specificity results of the QubeMDx PCR for B. gibsoni.

References

  1. 1.
    Lee CC, Hsieh YC, Huang CC, Tsang CL, Chung YT. Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan. J Vet Med Sci. 2010;72:1329–35.CrossRefGoogle Scholar
  2. 2.
    Liu PC, Lin YL, Lin CN, Su BL. A SimpleProbe((R)) real-time PCR assay for differentiating the cytochrome b M121I mutation in clinical specimens from dogs infected with Babesia gibsoni. Ticks Tick Borne Dis. 2016;7:639–43.CrossRefGoogle Scholar
  3. 3.
    Konishi K, Sakata Y, Miyazaki N, Jia H, Goo YK, Xuan X, et al. Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen. Vet Parasitol. 2008;155:204–8.CrossRefGoogle Scholar
  4. 4.
    Suh GH, Ahn KS, Ahn JH, Kim HJ, Leutenegger C, Shin S. Serological and molecular prevalence of canine vector-borne diseases (CVBDs) in Korea. Parasit Vectors. 2017;10:146.CrossRefGoogle Scholar
  5. 5.
    Cui J, Zhao Y, Sun Y, Yu L, Liu Q, Zhan X, et al. Detection of Babesia gibsoni in dogs by combining recombinase polymerase amplification (RPA) with lateral flow (LF) dipstick. Parasitol Res. 2018;117:3945–51.CrossRefGoogle Scholar
  6. 6.
    Terao M, Akter S, Yasin MG, Nakao R, Kato H, Alam MZ, et al. Molecular detection and genetic diversity of Babesia gibsoni in dogs in Bangladesh. Infect Genet Evol. 2015;31:53–60.CrossRefGoogle Scholar
  7. 7.
    Singh MN, Raina OK, Sankar M, Rialch A, Tigga MN, Kumar GR, et al. Molecular detection and genetic diversity of Babesia gibsoni in dogs in India. Infect Genet Evol. 2016;41:100–6.CrossRefGoogle Scholar
  8. 8.
    Mokhtar AS, Lim SF, Tay ST. Molecular detection of Anaplasma platys and Babesia gibsoni in dogs in Malaysia. Trop Biomed. 2013;30:345–8.PubMedGoogle Scholar
  9. 9.
    Taboada J, Merchant SR. Babesiosis of companion animals and man. Vet Clin North Am Small Anim Pract. 1991;21:103–23.CrossRefGoogle Scholar
  10. 10.
    Conrad P, Thomford J, Yamane I, Whiting J, Bosma L, Uno T, et al. Hemolytic anemia caused by Babesia gibsoni infection in dogs. J Am Vet Med Assoc. 1991;199:601–5.PubMedGoogle Scholar
  11. 11.
    Casapulla R, Baldi L, Avallone V, Sannino R, Pazzanese L, Mizzoni V. Canine piroplasmosis due to Babesia gibsoni: clinical and morphological aspects. Vet Rec. 1998;142:168–9.CrossRefGoogle Scholar
  12. 12.
    Boozer AL, Macintire DK. Canine babesiosis. Vet Clin North Am Small Anim Pract. 2003;33:885–904 viii.CrossRefGoogle Scholar
  13. 13.
    Fukumoto S, Xuan X, Shigeno S, Kimbita E, Igarashi I, Nagasawa H, et al. Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs. J Vet Med Sci. 2001;63:977–81.CrossRefGoogle Scholar
  14. 14.
    Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. J Clin Microbiol. 2003;41:4172–7.CrossRefGoogle Scholar
  15. 15.
    Matsuu A, Ono S, Ikadai H, Uchide T, Imamura S, Onuma M, et al. Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA. J Vet Diagn Invest. 2005;17:569–73.Google Scholar
  16. 16.
    Inokuma H, Okuda M, Yoshizaki Y, Hiraoka H, Miyama T, Itamoto K, et al. Clinical observations of Babesia gibsoni infection with low parasitaemia confirmed by PCR in dogs. Vet Rec. 2005;156:116–8.CrossRefGoogle Scholar
  17. 17.
    Otsuka Y, Yamasaki M, Yamato O, Maede Y. The effect of macrophages on the erythrocyte oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia. J Vet Med Sci. 2002;64:221–6.CrossRefGoogle Scholar
  18. 18.
    Mandal M, Banerjee PS, Kumar S, Ram H, Garg R, Pawde AM. Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs. Vet Parasitol. 2015;209:50–5.CrossRefGoogle Scholar
  19. 19.
    Dalrymple BP, Casu RE, Peters JM, Dimmock CM, Gale KR, Boese R, et al. Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis. Mol Biochem Parasitol. 1993;57:181–92.CrossRefGoogle Scholar
  20. 20.
    da Silva CB, Pires MS, Vilela JA, Peckle M, da Costa RL, Vitari GL, et al. A new quantitative PCR method for the detection of Anaplasma platys in dogs based on the citrate synthase gene. J Vet Diagn Invest. 2016;28:529–35.Google Scholar
  21. 21.
    Nakaghi AC, Machado RZ, Ferro JA, Labruna MB, Chryssafidis AL, Andre MR, et al. Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis. Rev Bras Parasitol Vet. 2010;19:75–9.CrossRefGoogle Scholar

Copyright information

© The Author(s). 2019

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Institute of Veterinary Clinical ScienceNational Taiwan UniversityTaipeiTaiwan
  2. 2.Credo Biomedical Pte. Ltd.SingaporeSingapore
  3. 3.Department of Veterinary MedicineNational Taiwan UniversityTaipeiTaiwan

Personalised recommendations