Tick-borne Pathogens as Etiologies of Undifferentiated Acute Febrile Illness in Kenya: Geographical Distribution, Risk Factors and the Tick Bacteriome as a Bio-indicator of Tick - Borne Zoonosis
Abstract
. Background:
Vector-borne pathogens, which include viruses, bacteria and protozoa, are among the most
common etiological agents of acute febrile illness (AFI) worldwide and have reportedly
caused epidemics of human disease in recent decades. Ticks are important vectors and they
are ranked only second to mosquitoes, in vector-borne disease transmission. Studies carried
out at Kenya Medical Research Institute / United States Army Medical Research Directorate
– Africa (KEMRI/USAMRD-A) have shown a wide distribution of ticks in Kenya indicating
potential for transmission of tick-borne pathogens including Rickettsia spp., Borrelia spp.,
Coxiella burnetii, Ehrlichia spp., Babesia spp., Anaplasma spp. and Crimean-Congo virus.
Although these pathogens have been reported in Kenya, there has been no systematic study of
the disease burden in the country. Within Kenya, AFI is a reason common for patients to go
to health care facilities and patients are often empirically treated without laboratory evaluation
for the causal agent. It is therefore, imperative to determine etiologies of acute febrile illness
other than malaria and associated risk factors.
Objective: The present study aimed at optimizing two multiplexed real time polymerase chain
reaction assays for detection of eight tick-borne pathogens and using them to evaluate the
distribution, risk factors and associated signs and symptoms of tick-borne pathogen infections
as etiological agents of acute febrile illness in different geographical regions in Kenya. The
study also aimed at determining whether ticks infesting domestic animals in Kenya harbor
bacteria of zoonosis relevance that can be transmitted to human.
Methodology: The present study was conducted on 6,207 archived samples that were
collected from patients seeking healthcare aged one year and above with fever of equal or
greater than 38⁰C without a diagnosis of the causative agent of fever after routine clinical
diagnosis. Samples used in this study were collected between 2008 and 2017 in county and
sub-county hospitals located in diverse geographical regions in Kenya including Lake Victoria
region (Kisumu County hospital, Kombewa sub-county hospital, Alupe sub-county hospital,
Busia), Kisii Highlands (Kisii county hospital), Rift valley region (Marigat sub-county
hospital Baringo, Lodwar county hospital and Gilgil sub-county hospital, Nakuru) North
Eastern Region (Iftin sub-county hospital, Garissa, Garissa police line dispensary, Garissa and
Isiolo county hospital), Coastal region (Malindi sub-county hospital, Kilifi) and Cosmopolitan
urban region (Eastleigh health center, Nairobi). To determine bacteria community in ticks that
can be potentially transmitted to humans, archived genomic DNA samples from 460 adult tick
samples were used. These ticks were collected between 2007 and 2008 from domestic animals
including cattle sheep and goats from diverse geographical regions in Kenya. Two multiplex
Real Time PCR assays and 16s rRNA metagenomics were used for detection of tick-borne
pathogens in the human and tick specimens, respectively. Demographic and clinical data
including age, gender, place of residence, signs and symptoms, contact with animals and type
of animals, tick bite, collected at the time of sample collection was used to determine risk
factors for human tick-borne infections.
Results:
Two real time PCR assays in a multiplex format were optimized and evaluated for eight tickborne
zoonosis pathogens. The assays were organized in duo assays of 4-plex each. Assay 1
was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and
Ehrlichia chaffeensis. Assay 2 was optimized for Rickettsia spp., Bartonella spp., other
Borrelia spp. which are not B. burgdoferi and Babesia spp. Using synthetic plasmids it was
shown these assays can specifically detect all the target sequences in the same reaction tube.
Assay 1 had a limit of detection of 2 copies for all target genes. Assay 2 was less sensitive and
on average had a limit of detection of 18 copies of target genes. In replicate tests, both assays
had intra-assay variation of less than two cycles. Using Bland-Altman analysis, the
performance of the two 4-plex assay was similar to that of the singleplex assays qualifying
them for simultaneous detection of four tick-borne zoonosis pathogens per assay. Whole blood
samples collected from undifferentiated acute febrile illness patients were tested for presence
of eight tick-borne zoonosis using the two 4-plex real time PCR. Of the patients accessed
3,082 (50.9%) were male and 2,979 949.2%) were female. The age of the patients ranged 1 to
80 years with a median of 5 years. Of these, all 6,207 were tested for Rickettsia, 4,017 for
Coxiella, 3,615 for A. phagocytophilum, 3,659 for E. chaffeensis, and 3,657 for B. burgdoferi,
3,480 for Babesia spp., 3,463 for Bartonella spp and 3,463 for Borrelia spp. which are not B.
burgdoferi. Overall, 1,017 (16%) were positive for at least 1 tick - borne zoonosis pathogen
(confidence interval (CI :) 15.3 – 17.1). Of these 475 (8%) (CI: 7.9-8.3) were positive for
Rickettsia, 271 (6%) were positive for Coxiella (CI: 5.6-7.0), 55 (1.5%) were positive for A.
phagocytophilum (CI: 1.1 – 1.9), 111 (3%) were positive for E. chaffeensis (CI: 2.5 – 3.6), 56
(1.5%) were positive for B. burgdoferi, (CI: 1.1 – 1.9), 36 (1%) were positive for Babesia spp.
(CI:: 0.2 – 1.4), 8 (0.2%) were positive for Bartonella spp. (CI: 0.07 – 0.4) and 135 (4%) were
positive for other Borrelia spp. which are not B. burgdoferi (CI: 3.3 – 4.5). Out of the 1017
tick-borne zoonosis positive specimens 906 (89.1%) tested positive for a single tick-borne
zoonosis pathogen and 99 (9.73 %) samples were positive for two tick-borne zoonosis
pathogens, 11 (1.1%) samples were positive for three tick-borne zoonosis pathogens and 1
sample was positive for four tick-borne zoonosis pathogens. Majority of the co-infections
were combination of Borrelia spp. with Rickettsia spp. and Coxiella burnetii with Rickettsia
spp. There was a higher prevalence of tick-borne zoonosis pathogen infection in the age
categories 6 to 15 years (17.5%) and ≥ 16 years (19.4%) compared to 14.3% in the ≤ 5 years
category (p-values = 0.02 and < 0.0001, respectively). Patients from a cosmopolitan urban
region of Eastleigh, Nairobi County had higher prevalence of tick-borne zoonosis pathogens
infections of 39% compared to Coastal region with 17%, Kisii highlands with12%, Lake
Victoria region with 16%, and arid and semi – arid region with 17%. Patients who reported
having contact with sheep and had a tick bite had higher odds of tick-borne zoonosis pathogen
infection. In ticks collected from domestic animals, a total of 645 unique operational
taxonomic units (OTUs) (bacteria genera) were detected and grouped into 27 bacteria phyla.
Sequence reads in the phyla were skewed with four phyla contributing 96.2% of the
sequences. Proteobacteria contributed the majority 61.2 % of the sequences that tarried to
33.8% OTUs, 15.9% for Firmicutes (23.4% OTUs), and 15.6% for Actinobacteria (20%
OTUs), 4.7% for Bacteroidetes (11.6% OTUs). The remaining 22 phyla included 0.7% for
Fusobacteria, 0.5% for TM7 (Saccharibacteria) 0.3% for Verrucomicrobia 0.2% for
Acidobacteria, 0.2% for Deinococcus-Thermus, 0.2% for Planctomycetes, 0.1% for
Chloroflexi, OD1 (Parcubacteria), Tenericutes and Gemmatimonadetes, 0.04% for
Armatimonadetes, 0.03% for Spirochaetes, 0.02% for Aquificae, 0.01% for SR1
(Absconditabacteria), Lentisphaerae and BRC1, 0.004% for Chlamydiae and Nitrospira,
0.002% for Chlorobi, Synergistetes, Fibrobacteres, WS3 and Elusimicrobia contributed only
2.5 % of the sequences (11.2% OTUs). Potentially pathogenic bacterial genera identified
include Coxiella 41.8%, Corynebacterium 13.6%, Acinetobacter 4.3%, Staphylococcus 3.9%,
Bacillus 2.7%, Porphyromonas 1.6%, Ralstonia 1.5%, Streptococcus 1.3%, Moraxella 1.3%,
Cloacibacterium 1.3%, Neisseria 1.2%, Escherichia_Shigella 1.2% and Proteus, Aerococcus,
Alloiococcus, Stenotrophomonas 1% each. Coxiella genus was the most abundant constituting
41.8% (15,445,204 out of 36,973,934 total sequences). Other less abundant (<0.1%) but
potentially pathogenic genera included Burkhoderia (0.4%), Klebsiella (0.3%), Escherichia-
Shegella (0.3%), Achromobacter (0.2%), Rickettsia (0.1%), Haemophilus (0.1%), Legionella
(0.1%), Campylobacter (0.04%), Treponema (0.03%), Francisella (0.02), Anaplasma (0.01),
Elizabethkingia (0.006%), Mycoplasma (0.006%), Ehrlichia (0.005%), Bordetella (0.004%),
Vibrio (0.002%), Borrelia (0.0008%) and Brucella (0.0002%). By Shannon diversity index,
A. variegatum carried less diverse bacteria (mean Shannon diversity index of 2.69 ± 0.92)
compared to 3.79 ± 1.10 for Amblyomma gemma, 3.71 ± 1.32 for A. hebraeum, 4.15 ± 1.08
other Amblyomma spp, 3.79 ± 1.37 for Hyalomma truncatum, 3.67 ± 1.38 for other Hyalomma
spp, 3.86 ± 1.27 for Rhipicephalus annulatus, 3.56 ± 1.21 for Rh. appendiculatus, 3.65 ± 1.30
for Rh. Pulchellus, but the difference was not statistically significant (p=0.443).
Conclusion:
Eight tick-borne pathogens were detected in undifferentiated acute febrile illness patients from
different geographical regions in Kenya using the two 4-plex real time PCR assays optimized
in this study. This study has shown up to 16.4 % prevalence of tick-borne zoonosis pathogens
in acute febrile illness patients in Kenya. Prevalence of tick - borne zoonosis infection was
significantly higher in patients of 6 years and above compare to patients of less than 5 years.
Patients living in an overcrowded cosmopolitan urban region and reporting a tick bite were at
higher risk of tick-borne zoonotic infections. Patients with tick-borne zoonosis infections
reported non-descriptive signs and symptoms which can lead to difficulties in definitive
diagnosis of tick-borne zoonosis. Adult ticks collected from livestock animals including cattle,
sheep and goats in the Kenya harbored an array of bacterial community including pathogenic
bacteria genera which suggests potential risk of transmission of tick - borne zoonosis
infections in the country. Pathogens harbored and transmitted by ticks (Tick-borne pathogens)
should be added to the list of etiologies considered for differential diagnosis of acute febrile
illness patients and laboratory capability for their diagnosis should be enhanced for early
detection and appropriate treatment in Kenya. Tick control effort in Kenya should embrace
one health approach to include human, animals and environment factors.
Publisher
University of Nairobi
Rights
Attribution-NonCommercial-NoDerivs 3.0 United StatesUsage Rights
http://creativecommons.org/licenses/by-nc-nd/3.0/us/Collections
The following license files are associated with this item: