TITLE:
A Duo 4-Plex Real Time PCR for Detection of Eight Tick-Borne Zoonoses in Kenya
AUTHORS:
Beth Mutai, Kariuki Njaanake, Kimita Gathii, Benson B. Estambale, John N. Waitumbi
KEYWORDS:
Tick-Borne-Zoonoses, Multiplex Real Time PCR, Acute Febrile Illness
JOURNAL NAME:
Open Journal of Clinical Diagnostics,
Vol.9 No.1,
March
6,
2019
ABSTRACT: 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 Rickettsia species (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 tested Assay 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 variation of less than two
cycles. Multiplex assays performance was comparable to respective singleplex
assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from
acute febrile illness patients attending hospitals in different counties in
Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C.
burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high
analytical sensitivity suggests potential for the duo 4-plex qPCR for detection
of common TBZ.