Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Clinical Blood Samples for Parallel IFA and FISH Tests
2.2. Indirect Immunofluorescence Assays (IFA)
2.3. Babesia Genus-Specific FISH Assay
2.4. Specificity Controls for the B. duncani and B. microti IFA Tests with Sera from Associated Diseases
2.5. Ethics Statement
3. Results
3.1. Specificity of the B. duncani and B. microti IFA Assays Tested with Sera Positive for Antibodies in Bartonellosis, Ehrlichiosis and Lyme Borreliosis
3.2. Parallel IFA and FISH Tests Performed on Clinical Blood Samples
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Disclaimer
References
- Vannier, E.; Gewurz, B.E.; Krause, P.J. Human babesiosis. Infect. Dis. Clin. N. Am. 2008, 22, 469–488. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ord, R.L.; Lobo, C.A. Human babesiosis: Pathogens, prevalence, diagnosis, and treatment. Curr. Clin. Microbiol. Rep. 2015, 2, 173–181. [Google Scholar] [CrossRef] [PubMed]
- Krause, P.J. Human babesiosis. Int. J. Parasitol. 2019, 49, 165–174. [Google Scholar] [CrossRef] [PubMed]
- Centers for Disease Control and Prevention (CDC). Surveillance for babesiosis—United States, 2018 Annual Summary; U.S. Department of Health and Human Services, CDC: Atlanta, GA, USA, 2020.
- Swei, A.; O’Connor, K.E.; Couper, L.I.; Thekkiniath, J.; Conrad, P.A.; Padgett, K.; Burns, J.; Yoshimizu, M.H.; Gonzales, B.; Munk, B.; et al. Evidence for transmission of the zoonotic apicomplexan parasite Babesia duncani by the tick Dermacentor albipictus. Int. J. Parasitol. 2019, 49, 95–103. [Google Scholar] [CrossRef]
- Brennan, M.B.; Herwaldt, B.L.; Kazmierczak, J.J.; Weiss, J.W.; Klein, C.L.; Leith, C.P.; He, R.; Oberley, M.J.; Tonnetti, L.; Wilkins, P.P.; et al. Transmission of Babesia microti parasites by solid organ transplantation. Emerg. Infect. Dis. 2016, 22, 1869–1876. [Google Scholar] [CrossRef] [Green Version]
- Conrad, P.; Kjemtrup, A.; Carreno, R.A.; Thomford, J.; Wainwright, K.; Eberhard, M.; Quick, R.; Iii, S.R.T.; Herwaldt, B.L. Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. Int. J. Parasitol. 2006, 36, 779–789. [Google Scholar] [CrossRef]
- Scott, J.D.; Scott, C.M. Human babesiosis caused by Babesia duncani has widespread distribution across Canada. Healthcare 2018, 6, 49. [Google Scholar] [CrossRef] [Green Version]
- Shah, J.S.; Mark, O.; Caoili, E.; Poruri, A.; Horowitz, R.I.; Ashbaugh, A.D.; Ramasamy, R. A Fluorescence in situ Hybridization (FISH) test for diagnosing babesiosis. Diagnostics 2020, 10, 377. [Google Scholar] [CrossRef]
- Wang, G.; Villafuerte, P.; Zhuge, J.; Visintainer, P.; Wormser, G.P. Comparison of a quantitative PCR assay with peripheral blood smear examination for detection and quantitation of Babesia microti infection in humans. Diagn. Microbiol. Infect. Dis. 2015, 82, 109–113. [Google Scholar] [CrossRef]
- Grabias, B.; Clement, J.; Krause, P.J.; Lepore, T.; Kumar, S. Superior real-time polymerase chain reaction detection of Babesia microti parasites in whole blood utilizing high-copy BMN antigens as amplification targets. Transfusion 2018, 58, 1924–1932. [Google Scholar] [CrossRef]
- Souza, S.S.; Bishop, H.S.; Sprinkle, P.; Qvarnstrom, Y. Comparison of Babesia microti real-time polymerase chain reaction assays for confirmatory diagnosis of babesiosis. Am. J. Trop. Med. Hyg. 2016, 95, 1413–1416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ward, S.J.; Stramer, S.L.; Szczepiorkowski, Z.M. Assessing the risk of Babesia to the United States blood supply using a risk-based decision-making approach: Report of AABB’s ad hoc Babesia policy working group (original report). Transfusion 2018, 58, 1916–1923. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- O’Connor, K.E.; Kjemtrup, A.; Conrad, P.A.; Swei, A. An improved PCR protocol for detection of Babesia duncani in wildlife and vector samples. J. Parasitol. 2018, 104, 429–432. [Google Scholar] [CrossRef] [PubMed]
- Dao, A.H.; Eberhard, M.L. Pathology of acute fatal babesiosis in hamsters experimentally infected with the WA-1 strain of Babesia. Lab. Investig. 1996, 74, 853–869. [Google Scholar] [PubMed]
- O’Connor, R.M.; Allred, D.R. Selection of Babesia bovis-infected erythrocytes for adhesion to endothelial cells co-selects for altered variant erythrocyte surface antigen isoforms. J. Immunol. 2000, 164, 2037–2045. [Google Scholar] [CrossRef] [Green Version]
- Ramasamy, R. Molecular basis for evasion of host immunity and pathogenesis in malaria. Biochim. Biophys. Acta (BBA) Mol. Basis Dis. 1998, 1406, 10–27. [Google Scholar] [CrossRef] [Green Version]
- Chisholm, E.S.; Ruebush, T.K.; Sulzer, A.J.; Healy, G.R.; Ii, T.K.R. Babesia Microti infection in man: Evaluation of an indirect immunofluorescent antibody test. Am. J. Trop. Med. Hyg. 1978, 27, 14–19. [Google Scholar] [CrossRef]
- Krause, P.J.; Telford, S.R.; Ryan, R.; Conrad, P.A.; Wilson, M.; Thomford, J.W.; Spielman, A. Diagnosis of babesiosis: Evaluation of a serologic test for the detection of Babesia microti antibody. J. Infect. Dis. 1994, 169, 923. [Google Scholar] [CrossRef]
- Levin, A.; Williamson, P.C.; Bloch, E.M.; Clifford, J.; Cyrus, S.; Shaz, B.H.; Kessler, D.; Gorlin, J.; Erwin, J.L.; Krueger, N.X.; et al. Serologic screening of United States blood donors for Babesia microti using an investigational enzyme immunoassay. Transfusion 2016, 56, 1866–1874. [Google Scholar] [CrossRef] [Green Version]
- Tayebwa, D.S.; Beshbishy, A.M.; Batiha, G.E.-S.; Komugisha, M.; Joseph, B.; Vudriko, P.; Yahia, R.; Alkazmi, L.; Hetta, H.F.; Yokoyama, N.; et al. Assessing the immunochromatographic test strip for serological detection of bovine babesiosis in Uganda. Microorganisms 2020, 8, 1110. [Google Scholar] [CrossRef]
- Gray, E.B.; Herwaldt, B.L. Babesiosis surveillance—United States, 2011-MMWR. Surveill. Summ. 2019, 68, 1–11. [Google Scholar] [CrossRef] [PubMed]
- Krause, P.J.; Ryan, R.; Telford, S.; Persing, D.; Spielman, A. Efficacy of immunoglobulin M serodiagnostic test for rapid diagnosis of acute babesiosis. J. Clin. Microbiol. 1996, 34, 2014–2016. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bloch, E.M.; Kumar, S.; Krause, P.J. Persistence of Babesia microti infection in humans. Pathogens 2019, 8, 102. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Harrison, T.E.; Mørch, A.M.; Felce, J.H.; Sakoguchi, A.; Reid, A.J.; Arase, H.; Dustin, M.L.; Higgins, M.K. Structural basis for RIFIN-mediated activation of LILRB1 in malaria. Nature 2020, 1–7. [Google Scholar] [CrossRef]
- Lemieux, J.E.; Tran, A.D.; Freimark, L.; Schaffner, S.F.; Goethert, H.; Andersen, K.G.; Bazner, S.; Li, A.; McGrath, G.; Sloan, L.; et al. A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse. Nat. Microbiol. 2016, 1, 16079. [Google Scholar] [CrossRef] [Green Version]
- Babesiosis and Tick-Borne Pathogens Subcommittee Report to the Tick-Borne Disease Working Group 2020; U.S. Department of Health & Human Services: Washington, DC, USA, 2020. Available online: https://www.hhs.gov/ash/advisory-committees/tickbornedisease/reports/babesiosis-subcomm-2020/index.html (accessed on 31 July 2020).
- Horowitz, R.I.; Freeman, P.R. Precision medicine: Retrospective chart review and data analysis of 200 patients on dapsone combination therapy for chronic Lyme disease/post-treatment Lyme disease syndrome: Part 1. Int. J. Gen. Med. 2019, 12, 101–119. [Google Scholar] [CrossRef] [Green Version]
- Liu, S.; Du Cruz, I.; Ramos, C.C.; Taleon, P.; Ramasamy, R.; Shah, J.S. Pilot study of immunoblots with recombinant Borrelia burgdorferi antigens for laboratory diagnosis of Lyme disease. Healthcare 2018, 6, 99. [Google Scholar] [CrossRef] [Green Version]
- Primus, S.; Akoolo, L.; Schlachter, S.; Gedroic, K.; Rojtman, A.D.; Parveen, N. Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR. PLoS ONE 2018, 13, e0196748. [Google Scholar] [CrossRef]
- Horowitz, R.I.; Freeman, P.R. Precision medicine: The role of the MSIDS model in defining, diagnosing, and treating chronic Lyme disease/post treatment Lyme disease syndrome and other chronic illness: Part 2. Healthcare 2018, 6, 129. [Google Scholar] [CrossRef] [Green Version]
- Duh, D.; Jelovšek, M.; Avšič-Županc, T. Evaluation of an indirect fluorescence immunoassay for the detection of serum antibodies against Babesia divergens in humans. Parasitology 2006, 134, 179–185. [Google Scholar] [CrossRef]
- Todorovic, R.A.; Long, R.F. Comparison of indirect fluorescent antibody (IFA) with complement fixation (CF) tests for diagnosis of Babesia spp infections in Colombian cattle. Trop. Parasitol. 1976, 27, 169–181. [Google Scholar]
- Prince, H.E.; Lapé-Nixon, M.; Patel, H.; Yeh, C. Comparison of the Babesia duncani (WA1) IgG detection rates among clinical sera submitted to a reference laboratory for WA1 IgG testing and blood donor specimens from diverse geographic areas of the United States. Clin. Vaccine Immunol. 2010, 17, 1729–1733. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mayne, P.J. Emerging incidence of Lyme borreliosis, babesiosis, bartonellosis, and granulocytic ehrlichiosis in Australia. Int. J. Gen. Med. 2011, 4, 845–852. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Young, K.M.; Corrin, T.; Wilhelm, B.; Uhland, C.; Greig, J.; Mascarenhas, M.; Waddell, L.A. Zoonotic Babesia: A scoping review of the global evidence. PLoS ONE 2019, 14, e0226781. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Quick, R.E.; Herwaldt, B.L.; Thomford, J.W.; Garnett, M.E.; Eberhard, M.L.; Wilson, M.; Spach, D.H.; Dickerson, J.W.; Telford, S.R.; Steingart, K.R.; et al. Babesiosis in Washington state: A new species of Babesia? Ann. Intern. Med. 1993, 119, 284. [Google Scholar] [CrossRef]
- Wozniak, E.J.; Lowenstine, L.J.; Hemmer, R.; Robinson, T.; A Conrad, P. Comparative pathogenesis of human WA1 and Babesia microti isolates in a Syrian hamster model. Lab. Anim. Sci. 1996, 46, 507–515. [Google Scholar]
Test Result | Number of Positive Samples (% of Total) | Conclusion on Babesiosis Status |
---|---|---|
FISH +ve and IFA −ve | 5 (2.0%) | Active infection |
FISH +ve and IFA +ve | 31 (12.4%) | Active infection |
FISH −ve and IFA +ve with IgG titer ≥1:160 | 32 (12.9%) | Probable active infection |
FISH −ve and IFA +ve with IgG titer ≤1:80 | 21 (8.4%) | Probable resolved infection |
Total positive in all tests | 89 (35.7%) | Exposure to Babesia |
Total negative in all tests | 160 (64.3%) | No exposure to Babesia |
FISH +ve and/or IFA +ve with IgG titer ≥1:160 | 68 (27.3%) | Active infection or probable active infection |
IFA Test Species | No. of FISH +ve Samples | Antibody Class and Titer | |
---|---|---|---|
IgM | IgG | ||
B. microti | 2 | 1:160 | negative |
1* | 1:160 Bm | 1:320 Bd | |
1* | 1:160 Bm | 1:160 Bd | |
1 | 1:160 | 1:80 | |
5 | 1:80 | negative | |
B. duncani | 1 | 1:160 | 1:40 |
1 | 1:160 | 1:320 | |
1 | 1:80 | 1:160 | |
2 | 1:40 | 1:160 | |
4 | 1:80 | 1:80 | |
3 | 1:80 | 1:40 | |
8 | 1:80 | negative | |
1 | 1:40 | 1:80 | |
IFA negative | 5 | negative | negative |
Total FISH positive | 36 |
Region/Country and Number of Samples | FISH +ve and B. Microti IFA +ve | FISH −ve and B. Microti IFA +ve | FISH +ve and B. Duncani IFA +ve | FISH −ve and B. Duncani IFA +ve | FISH +ve and IFA −ve | Total FISH or IFA +ve (% of Samples) | |
---|---|---|---|---|---|---|---|
Australia | 49 | 2 | 1 | 1 | 10 | 1 | 15 (30.6%) |
Europe | 89 | 1 | 2 | 0 | 8 | 7 | 18 (20.2%) |
USA | 249 | 10 | 14 | 21 | 39 | 5 | 89 (35.7%) |
All | 387 | 13 | 17 | 22 | 57 | 13 | 122 (31.5%) |
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Shah, J.S.; Caoili, E.; Patton, M.F.; Tamhankar, S.; Myint, M.M.; Poruri, A.; Mark, O.; Horowitz, R.I.; Ashbaugh, A.D.; Ramasamy, R. Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia. Diagnostics 2020, 10, 761. https://doi.org/10.3390/diagnostics10100761
Shah JS, Caoili E, Patton MF, Tamhankar S, Myint MM, Poruri A, Mark O, Horowitz RI, Ashbaugh AD, Ramasamy R. Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia. Diagnostics. 2020; 10(10):761. https://doi.org/10.3390/diagnostics10100761
Chicago/Turabian StyleShah, Jyotsna S., Eddie Caoili, Marie Fe Patton, Snehal Tamhankar, Mu Mu Myint, Akhila Poruri, Olivia Mark, Richard I. Horowitz, Alan D. Ashbaugh, and Ranjan Ramasamy. 2020. "Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia" Diagnostics 10, no. 10: 761. https://doi.org/10.3390/diagnostics10100761