1887

Abstract

The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

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2010-11-01
2019-11-13
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References

  1. Archer, G. L., Coleman, P. H., Cole, R. M., Duma, R. J. & Johnston, C. L., Jr ( 1979; ). Human infection from an unidentified erythrocyte-associated bacterium. N Engl J Med 301, 897–900.[CrossRef]
    [Google Scholar]
  2. Aubouy, A. & Carme, B. ( 2004; ). Plasmodium DNA contamination between blood smears during Giemsa staining and microscopic examination. J Infect Dis 190, 1335–1337.[CrossRef]
    [Google Scholar]
  3. Barker, E. N., Tasker, S., Day, M. J., Warman, S. M., Woolley, K., Birtles, R., Georges, K. C., Ezeokoli, C. D., Newaj-Fyzul, A. & other authors ( 2010; ). Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs. Vet Microbiol 140, 167–170.[CrossRef]
    [Google Scholar]
  4. Bauer, N., Balzer, H. J., Thure, S. & Moritz, A. ( 2008; ). Prevalence of feline haemotropic mycoplasmas in convenience samples of cats in Germany. J Feline Med Surg 10, 252–258.[CrossRef]
    [Google Scholar]
  5. Bosnic, D., Baresic, M., Anic, B., Sentic, M., Cerovec, M., Mayer, M. & Cikes, N. ( 2010; ). Rare zoonosis (hemotrophic mycoplasma infection) in a newly diagnosed systemic lupus erythematosus patient followed by a Nocardia asteroides pneumonia. Braz J Infect Dis 14, 92–95.[CrossRef]
    [Google Scholar]
  6. Clark, K. G. ( 1975; ). A basophilic micro-organism infecting human red cells. Br J Haematol 29, 301–304.[CrossRef]
    [Google Scholar]
  7. Dooley, J. R. ( 1980; ). Haemotropic bacteria in man. Lancet 2, 1237–1239.
    [Google Scholar]
  8. dos Santos, A. P., dos Santos, R. P., Biondo, A. W., Dora, J. M., Goldani, L. Z., de Oliveira, S. T., de Sa Guimaraes, A. M., Timenetsky, J., de Morais, H. A. & other authors ( 2008; ). Hemoplasma infection in HIV-positive patient, Brazil. Emerg Infect Dis 14, 1922–1924.[CrossRef]
    [Google Scholar]
  9. Duarte, M. I., Oliveira, M. S., Shikanai-Yasuda, M. A., Mariano, O. N., Takakura, C. F., Pagliari, C. & Corbett, C. E. ( 1992; ). Haemobartonella-like microorganism infection in AIDS patients: ultrastructural pathology. J Infect Dis 165, 976–977.
    [Google Scholar]
  10. Grétillat, S. & Konarzewski, B. ( 1978; ). Presence of a prokaryote of the genus Haemobartonella Tyzzer and Weinman, 1939, in the blood of Nigerians in the Niamey region. Bull Soc Pathol Exot Filiales 71, 412–416 (in French ).
    [Google Scholar]
  11. Hoelzle, L. E. ( 2008; ). Haemotrophic mycoplasmas: recent advances in Mycoplasma suis. Vet Microbiol 130, 215–226.[CrossRef]
    [Google Scholar]
  12. Hofmann-Lehmann, R., Meli, M. L., Dreher, U. M., Gonczi, E., Deplazes, P., Braun, U., Engels, M., Schupbach, J., Jorger, K. & other authors ( 2004; ). Concurrent infections with vector-borne pathogens associated with fatal hemolytic anemia in a cattle herd in Switzerland. J Clin Microbiol 42, 3775–3780.[CrossRef]
    [Google Scholar]
  13. Hu, Z., Yin, J., Shen, K., Kang, W. & Chen, Q. ( 2009; ). Outbreaks of hemotrophic mycoplasma infections in China. Emerg Infect Dis 15, 1139–1140.[CrossRef]
    [Google Scholar]
  14. Kallick, C. A., Levin, S., Reddi, K. T. & Landau, W. L. ( 1972; ). Systemic lupus erythematosus associated with haemobartonella-like organisms. Nat New Biol 236, 145–146.
    [Google Scholar]
  15. Kallick, C. A., Thadhani, K. C. & Rice, T. W. ( 1980; ). Identification of Anaplasmataceae (Haemobartonella) antigen and antibodies in systemic lupus erythematosus. Arthritis Rheum 23, 197–205.[CrossRef]
    [Google Scholar]
  16. Neimark, H., Johansson, K. E., Rikihisa, Y. & Tully, J. G. ( 2001; ). Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus Mycoplasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, ‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyonii’. Int J Syst Evol Microbiol 51, 891–899.[CrossRef]
    [Google Scholar]
  17. Neimark, H., Barnaud, A., Gounon, P., Michel, J. C. & Contamin, H. ( 2002; ). The putative haemobartonella that influences Plasmodium falciparum parasitaemia in squirrel monkeys is a haemotrophic mycoplasma. Microbes Infect 4, 693–698.[CrossRef]
    [Google Scholar]
  18. Neimark, H., Hoff, B. & Ganter, M. ( 2004; ). Mycoplasma ovis comb. nov. (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats. Int J Syst Evol Microbiol 54, 365–371.[CrossRef]
    [Google Scholar]
  19. Peters, I. R., Helps, C. R., Willi, B., Hofmann-Lehmann, R. & Tasker, S. ( 2008a; ). The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays. Vet Microbiol 126, 142–150.[CrossRef]
    [Google Scholar]
  20. Peters, I. R., Helps, C. R., McAuliffe, L., Neimark, H., Lappin, M. R., Gruffydd-Jones, T. J., Day, M. J., Hoelzle, L. E., Willi, B. & other authors ( 2008b; ). RNase P RNA gene (rnpB) phylogeny of hemoplasmas and other Mycoplasma species. J Clin Microbiol 46, 1873–1877.[CrossRef]
    [Google Scholar]
  21. Pretorius, A. M., Sommer, A. P., Aho, K. M. & Kajander, E. O. ( 2004; ). HIV and nanobacteria. HIV Med 5, 391–393.[CrossRef]
    [Google Scholar]
  22. Puntaric, V., Borcic, D., Vukelic, D., Jeren, T., Burek, V., Wikerhauser, T. & Richter, B. ( 1986; ). Eperythrozoonosis in man. Lancet 2, 868–869.
    [Google Scholar]
  23. Puntaric, V., Borcic, D., Bejuk, D., Vrhovec, B., Madic, J., Busch, K. & Richter, B. ( 1994; ). Haemotropic bacteria in man. Lancet 343, 359–360.
    [Google Scholar]
  24. Stoffregen, W. C., Alt, D. P., Palmer, M. V., Olsen, S. C., Waters, W. R. & Stasko, J. A. ( 2006; ). Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus). J Wildl Dis 42, 249–258.[CrossRef]
    [Google Scholar]
  25. Sykes, J. E., Owens, S. D., Terry, J. C., Lindsay, L. L. & Pusterla, N. ( 2008; ). Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction. J Vet Diagn Invest 20, 616–620.[CrossRef]
    [Google Scholar]
  26. Tasker, S. ( 2010; ). Haemotropic mycoplasmas: what's the real significance in cats? J Feline Med Surg 12, 369–381.[CrossRef]
    [Google Scholar]
  27. Tasker, S., Helps, C. R., Day, M. J., Gruffydd-Jones, T. J. & Harbour, D. A. ( 2003a; ). Use of real-time PCR to detect and quantify Mycoplasma haemofelis and “Candidatus Mycoplasma haemominutum” DNA. J Clin Microbiol 41, 439–441.[CrossRef]
    [Google Scholar]
  28. Tasker, S., Binns, S. H., Day, M. J., Gruffydd-Jones, T. J., Harbour, D. A., Helps, C. R., Jensen, W. A., Olver, C. S. & Lappin, M. R. ( 2003b; ). Use of a PCR assay to assess prevalence and risk factors for Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ in cats in the United Kingdom. Vet Rec 152, 193–198.[CrossRef]
    [Google Scholar]
  29. Tasker, S., Helps, C. R., Day, M. J., Harbour, D. A., Shaw, S. E., Harrus, S., Baneth, G., Lobetti, R. G., Malik, R. & other authors ( 2003c; ). Phylogenetic analysis of hemoplasma species – an international study. J Clin Microbiol 41, 3877–3880.[CrossRef]
    [Google Scholar]
  30. Tasker, S., Caney, S. M. A., Day, M. J., Dean, R. S., Helps, C. R., Knowles, T. G., Lait, P. J. P., Pinches, M. D. G. & Gruffydd-Jones, T. J. ( 2006a; ). Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Vet Microbiol 117, 169–179.[CrossRef]
    [Google Scholar]
  31. Tasker, S., Caney, S. M. A., Day, M. J., Dean, R. S., Helps, C. R., Knowles, T. G., Lait, P. J. P., Pinches, M. D. G. & Gruffydd-Jones, T. J. ( 2006b; ). Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on ‘Candidatus Mycoplasma haemominutum’ infection. Microbes Infect 8, 653–661.[CrossRef]
    [Google Scholar]
  32. Tasker, S., Peters, I. R., Day, M. J., Willi, B., Hofmann-Lehmann, R., Gruffydd-Jones, T. J. & Helps, C. R. ( 2009; ). Distribution of feline haemoplasmas in blood and tissue following experimental infection. Microb Pathog 47, 334–340.[CrossRef]
    [Google Scholar]
  33. Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R. & Leunissen, J. A. M. ( 2007; ). Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35, W71–W74.[CrossRef]
    [Google Scholar]
  34. Westfall, D. S., Jensen, W. A., Reagan, W. J., Radecki, S. V. & Lappin, M. R. ( 2001; ). Inoculation of two genotypes of Haemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin. Am J Vet Res 62, 687–691.[CrossRef]
    [Google Scholar]
  35. Willi, B., Filoni, C., Catao-Dias, J. L., Cattori, V., Meli, M. L., Vargas, A., Martinez, F., Roelke, M. E., Ryser-Degiorgis, M. P. & other authors ( 2007; ). Worldwide occurrence of feline hemoplasma infections in wild felid species. J Clin Microbiol 45, 1159–1166.[CrossRef]
    [Google Scholar]
  36. Willi, B., Meli, M. L., Luthy, R., Honegger, H., Wengi, N., Hoelzle, L. E., Reusch, C. E., Lutz, H. & Hofmann-Lehmann, R. ( 2009; ). Development and application of a universal hemoplasma screening assay based on the SYBR Green PCR principle. J Clin Microbiol 47, 4049–4054.[CrossRef]
    [Google Scholar]
  37. Yang, D., Tai, X., Qiu, Y. & Yun, S. ( 2000; ). Prevalence of Eperythrozoon spp. infection and congenital eperythrozoonosis in humans in Inner Mongolia, China. Epidemiol Infect 125, 421–426.[CrossRef]
    [Google Scholar]
  38. Yang, Z., Yuan, C., Yu, F. & Hua, X. ( 2007; ). Haemotrophic mycoplasma: review of aetiology and prevalence. Rev Med Microbiol 18, 1–3.[CrossRef]
    [Google Scholar]
  39. Yuan, C., Liang, A., Yu, F., Yang, Z., Li, Z., Zhu, J., Cui, L., Han, Y. & Hua, X. ( 2007; ). Eperythrozoon infection identified in an unknown aetiology anaemia patient. Ann Microbiol 57, 467–469.[CrossRef]
    [Google Scholar]
  40. Yuan, C. L., Liang, A. B., Yao, C. B., Yang, Z. B., Zhu, J. G., Cui, L., Yu, F., Zhu, N. Y., Yang, X. W. & other authors ( 2009; ). Prevalence of Mycoplasma suis (Eperythrozoon suis) infection in swine and swine-farm workers in Shanghai, China. Am J Vet Res 70, 890–894.[CrossRef]
    [Google Scholar]
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