Map of Kenya showing the locations of various surveillance hospitals.

<p>Lake Victoria basin: Kisumu District Hospital, New Nyanza Provincial Hospital and Alupe District Hospital. Kisii highland: Kisii District Hospital. Semi-arid: Marigat District Hospital on floor of the Rift Valley. Arid north eastern Kenya: Garissa District Hospital and Iftin sub-District Hospital. Coast of Indian Ocean: Malindi District Hospital. The map was generated in house using the ArcView^®10.0 application (Environmental Systems Research Institute, Redlands, CA, USA). Elevation base data was downloaded from the world resources institute website (<a href="http://www.wri.org/" target="_blank">http://www.wri.org/</a>).</p

Frequency of EBV viremia among different age categories.

<p>Frequency of EBV was highest among children under 5 years, and was significantly different between <5 years vs. 5–15 years (p = 0.039) and between <5 years and >15 years (p = 0.002) age categories.</p

EBV viral load in different age groups.

<p>EBV viral load in patients with AFI were determined by quantitative real time PCR as described in the methods section. The geometric mean viral load were significantly different between the <5 year vs. 5–15 year age categories.</p

Distribution of EBV infection in AFI patients from four geographical regions.

<p>The frequency of EBV was highest in patients from Lake Victoria region compared to other sites, with the difference being significant between Lake Victoria basin and Kisii highland (p = 0.001) (*) and between Lake Victoria and coastal region (p = 0.045) (**).</p

Primers used for PCR and sequencing.

<p>Primers used for PCR and sequencing.</p

Bayesian probability tree of study samples with validated <i>Rickettsia</i> species.

<p>The tree is based on partitioned concatenated datasets of <i>gltA</i>, <i>ompA</i>, <i>ompB</i>, 17kDa and <i>sca4</i> partial nucleotide sequences. Amino acid alignments were used to guide the nucleotide alignments. The tree is estimated using a GTR+G substitution model as implemented in MrBayes v3.2. The tree is a consensus of 15,002 trees (post burn-in) pooled from two independent Markov Chain run in parallel. Thin lines indicate posterior probability values of < 1. Lineage diversity within the <i>R</i>. <i>africae</i> study samples is highlighted in red and blue to indicate clades i and ii respectively. Samples previously misclassified as <i>R</i>. <i>africae</i> are now classified as <i>R</i>. <i>aeschlimanii</i> (black diamond). Study sample 176_Moyale branches distinctly from other rickettsiae and is considered a novel rickettsia species and a provisional name "<i>Candidatus</i> rickettsia moyalensis" (black circle) is proposed. NB: Although 293_Migori (open circle) branched as a lone taxon, it clustered with <i>R</i>. <i>aeschlimanii</i> by Maximum Likelihood method. Non-spotted fever group lineages are highlighted orange for transition group and grey for typhus group. The status of <i>R</i>. <i>helvetica</i> (shown in black cross), originally in spotted fever group is now uncertain [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004788#pntd.0004788.ref020" target="_blank">20</a>].</p

Demographic and clinical findings of the patients in this study.

<p>Demographic and clinical findings of the patients in this study.</p

Phylogenetic Variants of <i>Rickettsia africae</i>, and Incidental Identification of "<i>Candidatus</i> Rickettsia Moyalensis" in Kenya

<div><p>Background</p><p><i>Rickettsia africae</i>, the etiological agent of African tick bite fever, is widely distributed in sub-Saharan Africa. Contrary to reports of its homogeneity, a localized study in Asembo, Kenya recently reported high genetic diversity. The present study aims to elucidate the extent of this heterogeneity by examining archived <i>Rickettsia africae</i> DNA samples collected from different eco-regions of Kenya.</p><p>Methods</p><p>To evaluate their phylogenetic relationships, archived genomic DNA obtained from 57 ticks <i>a priori</i> identified to contain <i>R</i>. <i>africae</i> by comparison to <i>ompA</i>, <i>ompB</i> and <i>gltA</i> genes was used to amplify five rickettsial genes i.e. <i>gltA</i>, <i>ompA</i>, <i>ompB</i>, 17kDa and <i>sca4</i>. The resulting amplicons were sequenced. Translated amino acid alignments were used to guide the nucleotide alignments. Single gene and concatenated alignments were used to infer phylogenetic relationships.</p><p>Results</p><p>Out of the 57 DNA samples, three were determined to be <i>R</i>. <i>aeschlimanii</i> and not <i>R</i>. <i>africae</i>. One sample turned out to be a novel rickettsiae and an interim name of “<i>Candidatus</i> Rickettsia moyalensis” is proposed. The bonafide <i>R</i>. <i>africae</i> formed two distinct clades. Clade I contained 9% of the samples and branched with the validated <i>R</i>. <i>africae str ESF-5</i>, while clade II (two samples) formed a distinct sub-lineage.</p><p>Conclusions</p><p>This data supports the use of multiple genes for phylogenetic inferences. It is determined that, despite its recent emergence, the <i>R</i>. <i>africae</i> lineage is diverse. This data also provides evidence of a novel Rickettsia species, <i>Candidatus</i> Rickettsia moyalensis.</p></div

EBV viral load in AFI patients across different geographical regions.

<p>Mann-Whitney test showed that viral load were significantly higher in Lake Victoria basin vs. Kisii highland (p = 0.003), Coastal region vs. Kisii highland (p = 0.005), and Coastal region vs. semi arid (p = 0.024).</p

Phylogeny of Rickettsia sequences from this study and those collected previously in Kenya [23, 31].

<p><i>ompA</i> nucleotide sequences of study isolates and other <i>R</i>. <i>africae</i> reported from previous studies [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004788#pntd.0004788.ref023" target="_blank">23</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004788#pntd.0004788.ref031" target="_blank">31</a>] were analysed by Maximum Likelihood method using MEGA v7 based on the Hasegawa-Kishino-Yano (HKY) model of substitution. The tree has a log likelihood ratio of -1049 and involved all codon positions. Members of clade I, II and III are shown beside the bolded red, blue and green lines respectively. Sequences from Parola et al 2001 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004788#pntd.0004788.ref023" target="_blank">23</a>] are shown as black triangles and those from Macaluso et al 2003 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004788#pntd.0004788.ref031" target="_blank">31</a>] by black circles. Numbers at the nodes are bootstrap proportions with 1000 replicates. Only bootstrap values >50% are shown. The scale bar indicates the number of substitutions per nucleotide position. Clearly, five of our sequences (044, 045 and 164 from Wajir, 176 Moyale and 195 Machakos) are distinct from those described previously.</p