Ticks harbor multiple pathogens, most of which can
be transmitted to humans. The ensuing zoonoses display non-specific symptoms
that make definitive diagnosis difficult. We report here the development and
evaluation of multiplex real time polymerase
chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The
assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was
optimized for Rickettsiaspecies (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays
can specifically detect all target sequences in the same reaction tube. Assays
were assayed eight times to determine assay performance and the limit of
detection was determined as the lowest
plasmid concentration that was amplified for all the targets. Standard curves
of threshold cycle (Ct) versus copy numbers were generated and used to
determine linearity and efficiency of the assays. Pairwise comparison of
singleplex and multiplex assays was done using Bland-Altman plots.
Prevalence was calculated as overall percentage of positive patients to each
TBZ testedAssay 1 had a limit of detection of 2 copy numbers for all targets. Assay
2 was less sensitive and on average had a limit of detection of 18 gene copies.
In replicate tests, both assays had intra-assay
References
[1]
Sonenshine, D.E. (1992) Biology of Ticks. 1st Edition, Oxford University Press, Oxford.
[2]
Coipan, E.C., Jahfari, S., Fonville, M., Maassen, C.B., van der Giessen, J., Takken, W., et al. (2013) Spatiotemporal Dynamics of Emerging Pathogens in Questing Ixodes Ricinus. Frontiers in Cellular and Infection Microbiology, 3, 36.
https://doi.org/10.3389/fcimb.2013.00036
[3]
Moutailler, S., Valiente Moro, C., Vaumourin, E., Michelet, L., Tran, F.H., Devillers, E., et al. (2016) Co-Infection of Ticks: The Rule Rather than the Exception. PLoS Neglected Tropical Diseases, 10, e0004539.
https://doi.org/10.1371/journal.pntd.0004539
[4]
Raileanu, C., Moutailler, S., Pavel, I., Porea, D., Mihalca, A.D., Savuta, G., et al. (2017) Borrelia Diversity and Co-Infection with Other Tick Borne Pathogens in Ticks. Frontiers in Cellular and Infection Microbiology, 7, 36.
https://doi.org/10.3389/fcimb.2017.00036
[5]
Mutai, B.K., Wainaina, J.M., Magiri, C.G., Nganga, J.K., Ithondeka, P.M., Njagi, O.N., et al. (2013) Zoonotic Surveillance for Rickettsiae in Domestic Animals in Kenya. Vector-Borne and Zoonotic Diseases, 13, 360-366.
https://doi.org/10.1089/vbz.2012.0977
[6]
Halliday, J.E.B., Knobel, D.L., Agwanda, B., Bai, Y., Breiman, R.F., Cleaveland, S., et al. (2015) Prevalence and Diversity of Small Mammal-Associated Bartonella Species in Rural and Urban Kenya. PLoS Neglected Tropical Diseases, e0003608.
[7]
Jowi, J.O. and Gathua, S.N. (2005) Lyme Disease: Report of Two Cases. East African Medical Journal, 82, 267-269. https://doi.org/10.4314/eamj.v82i5.9318
[8]
Richards, A.L., Jiang, J., Omulo, S., Dare, R., Abdirahman, K., Ali, A., et al. (2010) Human Infection with Rickettsia felis, Kenya. Emerging Infectious Diseases, 16, 1081-1086. https://doi.org/10.3201/eid1607.091885
[9]
Njiiri, N.E., deC. Bronsvoort, B.M., Collins, N.E., Steyn, H.C., Troskie, M., Vorster, I., et al. (2015) The Epidemiology of Tick-Borne Haemoparasites as Determined by the Reverse Line Blot Hybridization Assay in an Intensively Studied Cohort of Calves in Western Kenya. Veterinary Parasitology, 210, 69-76.
https://doi.org/10.1016/j.vetpar.2015.02.020
[10]
Adjou Moumouni, P.F., Aboge, G.O., Terkawi, M.A., Masatani, T., Cao, S., Kamyingkird, K., et al. (2015) Molecular Detection and Characterization of Babesia bovis, Babesia bigemina, Theileria Species and Anaplasma marginale Isolated from Cattle in Kenya. Parasites and Vectors, 8, 496.
https://doi.org/10.1186/s13071-015-1106-9
[11]
Prasad, N., Murdoch, D.R., Reyburn, H. and Crump, J.A. (2015) Etiology of Severe Febrile Illness in Low- and Middle-Income Countries: A Systematic Review. PLoS ONE, 10, e0127962. https://doi.org/10.1371/journal.pone.0127962
[12]
Courtney, J.W., Kostelnik, L.M., Zeidner, N.S. and Massung, R.F. (2004) Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi. Journal of Clinical Microbiology, 42, 3164-3168.
https://doi.org/10.1128/JCM.42.7.3164-3168.2004
[13]
Pripuzova, N., Wang, R., Tsai, S., Li, B., Hung, G.C., Ptak, R.G., et al. (2012) Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens. PLoS ONE, 7, e43246.
https://doi.org/10.1371/journal.pone.0043246
[14]
Gowin, E., Bartkowska-Sniatkowska, A., Jończyk-Potoczna, K., Wysocka Leszczyńska, J., Bobkowski, W., Fichna, P., et al. (2017) Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience. BioMed Research International, 2017, Article ID: 8037963. https://doi.org/10.1155/2017/8037963
[15]
Staroscik, A. (2004) Calculator for Determining the Number of Copies of a Template. http://cels.uri.edu/gsc/cndna.html
Schneeberger, P.M., Hermans, M.H.A., Van Hannen, E.J., Schellekens, J.J.A., Leenders, A.C.A.P. and Wever, P.C. (2010) Real-Time PCR with Serum Samples Is Indispensable for Early Diagnosis of Acute Q Fever. Clinical and Vaccine Immunology, 17, 286-290. https://doi.org/10.1128/CVI.00454-09
[18]
Loftis, A.D., Massung, R.F. and Levin, M.L. (2003) Quantitative Real-Time PCR Assay for Detection of Ehrlichia chaffeensis. Journal of Clinical Microbiology, 41, 3870-3872. https://doi.org/10.1128/JCM.41.8.3870-3872.2003
[19]
Jiang, J., Stromdahl, E.Y. and Richards, A.L. (2012) Detection of Rickettsia parkeri and Candidatus Rickettsia Andeanae in Amblyomma maculatum Gulf Coast Ticks Collected from Humans in the United States. Vector-Borne and Zoonotic Diseases, 12, 175-182. https://doi.org/10.1089/vbz.2011.0614
[20]
Radzijevskaja, J., Paulauskas, A. and Rosef, O. (2008) Prevalence of Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus Ticks from Lithuania and Norway. International Journal of Medical Microbiology, 298, 218-221.
https://doi.org/10.1016/j.ijmm.2008.01.008
[21]
Jiang, J., Temenak, J.J. and Richards, A.L. (2003) Real-Time PCR Duplex Assay for Rickettsia prowazekii and Borrelia recurrentis. Annals of the New York Academy of Sciences, 990, 302-310. https://doi.org/10.1111/j.1749-6632.2003.tb07380.x
[22]
Billeter, S.A., Gundi, V.A.K.B., Rood, M.P. and Kosoy, M.Y. (2011) Molecular Detection and Identification of Bartonella Species in Xenopsylla cheopis Fleas (Siphonaptera: Pulicidae) Collected from Rattus norvegicus Rats in Los Angeles, California. Applied and Environmental Microbiology, 77, 7850-7852.
https://doi.org/10.1128/AEM.06012-11
[23]
Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J. and Sayers, E.W. (2016) GenBank. Nucleic Acids Research, 44, D67-D72. https://doi.org/10.1093/nar/gkv1276
[24]
Schwartz, J.J., Gazumyan, A. and Schwartz, I. (1992) rRNA Gene Organization in the Lyme Disease Spirochete, Borrelia burgdorferi. Journal of Bacteriology, 174, 3757-3765. https://doi.org/10.1128/jb.174.11.3757-3765.1992
[25]
Lin, Q., Rikihisa, Y., Felek, S., Wang, X., Massung, R.F. and Woldehiwet, Z. (2004) Anaplasma phagocytophilum Has a Functional msp2 Gene That Is Distinct from p44. Infection and Immunity, 72, 3883-3889.
https://doi.org/10.1128/IAI.72.7.3883-3889.2004
[26]
Seshadri, R., Paulsen, I.T., Eisen, J.A., Read, T.D., Nelson, K.E., Nelson, W.C., et al. (2003) Complete Genome Sequence of the Q-Fever Pathogen Coxiella burnetii. Proceedings of the National Academy of Sciences of the United States, 100, 5455-5460. https://doi.org/10.1073/pnas.0931379100
[27]
Reif, K.E., Stout, R.W., Henry, G.C., Foil, L.D. and Macaluso, K.R. (2008) Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas. PLoS ONE, 3, e2805. https://doi.org/10.1371/journal.pone.0002805
[28]
Wallich, R., Moter, S.E., Simon, M.M., Ebnet, K., Heiberger, A. and Kramer, M.D. (1990) The Borrelia burgdorferi Flagellum-Associated 41-Kilodalton Antigen (Flagellin): Molecular Cloning, Expression, and Amplification of the Gene. Infection and Immunity, 58, 1711-1719.
[29]
Guy, L., Nystedt, B., Toft, C., Zaremba-Niedzwiedzka, K., Berglund, E.C., Granberg, F., et al. (2013) A Gene Transfer Agent and a Dynamic Repertoire of Secretion Systems Hold the Keys to the Explosive Radiation of the Emerging Pathogen Bartonella. PLoS Genetics, 9, e1003393. https://doi.org/10.1371/journal.pgen.1003393
[30]
Stańczak, J., Cieniuch, S., Lass, A., Biernat, B. and Racewicz, M. (2015) Detection and Quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus Ticks from Urban and Rural Environment, Northern Poland, by Real-Time Polymerase Chain Reaction. Experimental and Applied Acarology, 66, 63-81.
https://doi.org/10.1007/s10493-015-9887-2
[31]
Ian, M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Poole.
[32]
Altman, D.G. and Bland, J.M. (1983) Measurement in Medicine: The Analysis of Method Comparison Studies. The Statistician, 32, 307.
https://doi.org/10.2307/2987937
[33]
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic Local Alignment Search Tool. Journal of Molecular Biology, 215, 403-410.
https://doi.org/10.1016/S0022-2836(05)80360-2
[34]
Parola, P. and Raoult, D. (2001) Molecular Tools in the Epidemiology of Tick-Borne Bacterial Diseases. Annales de biologie Clinique, 59, 177-182.
[35]
Parola, P. and Raoult, D. (2001) Ticks and Tickborne Bacterial Diseases in Humans: An Emerging Infectious Threat. Clinical Infectious Diseases, 32, 897-928.
https://doi.org/10.1086/319347
[36]
Kimita, G., Mutai, B., Nyanjom, S.G., Wamunyokoli, F. and Waitumbi, J. (2016) Phylogenetic Variants of Rickettsia africae, and Incidental Identification of “Candidatus Rickettsia Moyalensis” in Kenya. PLOS Neglected Tropical Diseases, 10, e0004788. https://doi.org/10.1371/journal.pntd.0004788
[37]
Dunster, L., Dunster, M., Ofula, V., Beti, D., Kazooba-Voskamp, F., Burt, F., et al. (2002) First Documentation of Human Crimean-Congo Hemorrhagic Fever, Kenya. Emerging Infectious Diseases, 1005-1006. https://doi.org/10.3201/eid0809.010510
[38]
Kitaa, J.M.A., Mulei, C.M., Mande, J.D. and Wabacha, J. (2014) A Retrospective Study of Canine Ehrlichiosis in Kenya. International Journal of Veterinary Science and Medicine, 3, 122-124.
[39]
Wang, H.-Y., Kim, S., Kim, J., Park, S.-D., Uh, Y. and Lee, H. (2014) Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures. Journal of Clinical Microbiology, 52, 1911-1920. https://doi.org/10.1128/JCM.00389-14
[40]
Yu, D., Chen, Y., Wu, S., Wang, B., Tang, Y.W. and Li, L. (2012) Simultaneous Detection and Differentiation of Human Papillomavirus Genotypes 6, 11, 16 and 18 by AllGlo Quadruplex Quantitative PCR. PLoS ONE, 7, e48972.
https://doi.org/10.1371/journal.pone.0048972
[41]
Fenollar, F. and Raoult, D. (2007) Molecular Diagnosis of Bloodstream Infections Caused by Non-Cultivable Bacteria. International Journal of Antimicrobial Agents, 30, 7-15. https://doi.org/10.1016/j.ijantimicag.2007.06.024
[42]
Nilsson, A.C., Bjorkman, P., Welinder-Olsson, C., Widell, A. and Persson, K. (2010) Clinical Severity of Mycoplasma pneumoniae (MP) Infection Is Associated with Bacterial Load in Oropharyngeal Secretions But Not with MP Genotype. BMC Infectious Diseases, 10, 39. https://doi.org/10.1186/1471-2334-10-39
[43]
Swanson, S.J., Neitzel, D., Reed, K.D. and Belongia, E.A. (2006) Coinfections Acquired from Ixodes Ticks. Clinical Microbiology Reviews, 19, 708-727.
https://doi.org/10.1128/CMR.00011-06
[44]
Levin, M.L. and Fish, D. (2001) Interference between the Agents of Lyme Disease and Human Granulocytic Ehrlichiosis in a Natural Reservoir Host. Vector-Borne and Zoonotic Diseases, 1, 139-148. https://doi.org/10.1089/153036601316977741
