Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

Ticks are arachnid ectoparasites that rank second only to mosquitoes in the transmission of human diseases including bacteria responsible for anaplasmosis, ehrlichiosis, spotted fevers, and Lyme disease among other febrile illnesses. Due to the paucity of data on bacteria transmitted by ticks in Kenya, this study undertook a bacterial metagenomic-based characterization of ticks collected from Isiolo, a semi-arid pastoralist County in Eastern Kenya, and Kwale, a coastal County with a monsoon climate in the southern Kenyan border with Tanzania. A total of 2,918 ticks belonging to 3 genera and 10 species were pooled and screened in this study. Tick identification was confirmed through the sequencing of the Cytochrome C Oxidase Subunit 1 (COI) gene. Bacterial 16S rRNA gene PCR amplicons obtained from the above samples were sequenced using the MinION (Oxford Nanopore Technologies) platform. The resulting reads were demultiplexed in Porechop, followed by trimming and filtering in Trimmomatic before clustering using Qiime2-VSearch. A SILVA database pretrained naïve Bayes classifier was used to classify the Operational Taxonomic Units (OTUs) taxonomically. The bacteria of clinical interest detected in pooled tick assays were as follows: Rickettsia spp. 59.43% of pools, Coxiella burnetii 37.88%, Proteus mirabilis 5.08%, Cutibacterium acnes 6.08%, and Corynebacterium ulcerans 2.43%. These bacteria are responsible for spotted fevers, query fever (Q-fever), urinary tract infections, skin and soft tissue infections, eye infections, and diphtheria-like infections in humans, respectively. P. mirabilis, C. acnes, and C. ulcerans were detected only in Isiolo. Additionally, COI sequences allowed for the identification of Rickettsia and Coxiella species to strain levels in some of the pools. Diversity analysis revealed that the tick genera had high levels of Alpha diversity but the differences between the microbiomes of the three tick genera studied were not significant. The detection of C. acnes, commonly associated with human skin flora suggests that the ticks may have contact with humans potentially exposing them to bacterial infections. The findings in this study highlight the need for further investigation into the viability of these bacteria and the competency of ticks to transmit them. Clinicians in these high-risk areas also need to be appraised for them to include Rickettsial diseases and Q-fever as part of their differential diagnosis.

Malaria Transmission Dynamics in a High-Transmission Setting of Western Kenya and the Inadequate Treatment Response to Artemether-Lumefantrine in an Asymptomatic Population.

BACKGROUND: Assessing the infectious reservoir is critical in malaria control and elimination strategies. We conducted a longitudinal epidemiological study in a high-malaria-burden region in Kenya to characterize transmission in an asymptomatic population. METHODS: 488 study participants encompassing all ages in 120 households within 30 clusters were followed for 1 year with monthly sampling. Malaria was diagnosed by microscopy and molecular methods. Transmission potential in gametocytemic participants was assessed using direct skin and/or membrane mosquito feeding assays, then treated with artemether-lumefantrine. Study variables were assessed using mixed-effects generalized linear models. RESULTS: Asexual and sexual parasite data were collected from 3792 participant visits, with 903 linked with feeding assays. Univariate analysis revealed that the 6-11-year-old age group was at higher risk of harboring asexual and sexual infections than those <6 years old (odds ratio [OR] 1.68, P < .001; and OR 1.81, P < .001), respectively. Participants with submicroscopic parasitemia were at a lower risk of gametocytemia compared with microscopic parasitemia (OR 0.04, P < .001), but they transmitted at a significantly higher rate (OR 2.00, P = .002). A large proportion of the study population who were infected at least once remained infected (despite treatment) with asexual (71.7%, 291/406) or sexual (37.4%, 152/406) parasites. 88.6% (365/412) of feeding assays conducted in individuals who failed treatment the previous month resulted in transmissions. CONCLUSIONS: Individuals with asymptomatic infection sustain the transmission cycle, with the 6-11-year age group serving as an important reservoir. The high rates of artemether-lumefantrine treatment failures suggest surveillance programs using molecular methods need to be expanded for accurate monitoring and evaluation of treatment outcomes.

The spleen bacteriome of wild rodents and shrews from Marigat, Baringo County, Kenya.

BACKGROUND: There is a global increase in reports of emerging diseases, some of which have emerged as spillover events from wild animals. The spleen is a major phagocytic organ and can therefore be probed for systemic microbiome. This study assessed bacterial diversity in the spleen of wild caught small mammals so as to evaluate their utility as surveillance tools for monitoring bacteria in an ecosystem shared with humans. METHODS: Fifty-four small mammals (rodents and shrews) were trapped from different sites in Marigat, Baringo County, Kenya. To characterize their bacteriome, DNA was extracted from their spleens and the V3-V4 regions of the 16S rRNA amplified and then sequenced on Illumina MiSeq. A non-target control sample was used to track laboratory contaminants. Sequence data was analyzed with Mothur v1.35, and taxomy determined using the SILVA database. The Shannon diversity index was used to estimate bacterial diversity in each animal and then aggregated to genus level before computing the means. Animal species within the rodents and shrews were identified by amplification of mitochondrial cytochrome b (cytb) gene followed by Sanger sequencing. CLC workbench was used to assemble the cytb gene sequences, after which their phylogenetic placements were determined by querying them against the GenBank nucleotide database. RESULTS: cytb gene sequences were generated for 49/54 mammalian samples: 38 rodents (Rodentia) and 11 shrews (Eulipotyphyla). Within the order Rodentia, 21 Acomys, eight Mastomys, six Arvicanthis and three Rattus were identified. In the order Eulipotyphyla, 11 Crucidura were identified. Bacteria characterization revealed 17 phyla that grouped into 182 genera. Of the phyla, Proteobacteria was the most abundant (67.9%). Other phyla included Actinobacteria (16.5%), Firmicutes (5.5%), Chlamydiae (3.8%), Chloroflexi (2.6%) and Bacteroidetes (1.3%) among others. Of the potentially pathogenic bacteria, Bartonella was the most abundant (45.6%), followed by Anaplasma (8.0%), Methylobacterium (3.5%), Delftia (3.8%), Coxiella (2.6%), Bradyrhizobium (1.6%) and Acinetobacter (1.1%). Other less abundant (<1%) and potentially pathogenic included Ehrlichia, Rickettsia, Leptospira, Borrelia, Brucella, Chlamydia and Streptococcus. By Shannon diversity index, Acomys spleens carried more diverse bacteria (mean Shannon diversity index of 2.86, p = 0.008) compared to 1.77 for Crocidura, 1.44 for Rattus, 1.40 for Arvicathis and 0.60 for Mastomys. CONCLUSION: This study examined systemic bacteria that are filtered by the spleen and the findings underscore the utility of 16S rRNA deep sequencing in characterizing complex microbiota that are potentially relevant to one health issues. An inherent problem with the V3-V4 region of 16S rRNA is the inability to classify bacteria reliably beyond the genera. Future studies should utilize the newer long read methods of 16S rRNA analysis that can delimit the species composition.

Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Comparative efficacy of existing surveillance tools for Aedes aegypti in Western Kenya.

All traditional surveillance techniques for Aedes aegypti have been developed for the cosmopolitan domestic subspecies Ae. aegypti aegypti, and not the sylvatic subspecies, Ae. aegypti formosus. The predominant form in Western Kenya is Ae. aegypti formosus that is rarely associated with human habitations but is linked to transmission of sylvatic dengue virus strains. We compared five surveillance methods for their effectiveness in sampling Ae. aegypti formosus with the goal of determining a sustainable surveillance strategy in Kenya. The methods included larval and pupal surveys, oviposition trapping, BG-Sentinel trapping, resting boxes, and backpack aspirations. Larval and pupal surveys collected the highest number of Ae. aegypti formosus (51.3%), followed by oviposition traps (45.7%), BG-Sentinel traps (3.0%), and zero collected with either backpack aspiration or resting box collections. No Ae. aegypti formosus larvae or pupae were found indoors. The results indicate that oviposition traps and outdoor larval and pupal surveys were better surveillance methods for Ae. aegypti formosus in Western Kenya.

The AFHSC-Division of GEIS Operations Predictive Surveillance Program: a multidisciplinary approach for the early detection and response to disease outbreaks.

The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program's ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia.