There were no specific selection criteria for choosing the samples included in this study. Rather, the samples were available due to an ongoing surveillance project in these two areas, which provided the necessary dialogue with affected families to enable sample collection and to undertake follow up. All five deaths described resulted from exposures by domestic dogs and a lack of, or inappropriate, PEP administration (Table 1). Four of the five patients were children who were less than 15 years old. Three of the children were bitten on the head or neck, sites that are at highest risk for progression to rabies in the absence of PEP [22].

Case 1 was vaccinated against tetanus, but not advised on rabies post-exposure vaccination despite attending a health facility the same day as being bitten multiple times by an unknown dog, including one bite to the forehead. Twenty-eight days after being bitten, Case 1 started to show rabies symptoms. From symptoms onset the patient was treated for malaria, initially at home, then at a local hospital. The patient’s condition deteriorated rapidly, leading to their transfer to a major referral hospital where they died upon arrival. Although Case 2 had no bite history, the patient had killed his two dogs after they manifested signs of rabies one month prior to his death. After presenting to a nearby health facility with rabies symptoms the patient was transferred to a major referral hospital and pronounced dead 6 days later. Case 3 reported to a local hospital with bites to the lips from an unknown dog, and was vaccinated against rabies following an off-label intramuscular (IM) regimen (1 ml on days 0, 7 and 18). WHO recommendation for IM in use in the country is day 0, 3, 7, 14 and 28. Rabies symptoms began 19 days later, i.e., one day after the third vaccination; and the patient died four days later following transfer to a major referral hospital. Case 4 started post-exposure vaccination, via the intramuscular route, one day after being bitten multiple times on the head and arm by an unknown dog, but did not receive further vaccinations as relatives reported that they were not advised to do so. After symptoms onset (69 days later) the patient was taken back to the health facility where the patient was initially vaccinated, then transferred to a major referral hospital where the patient died shortly thereafter. Case 5 was referred to a major hospital from a health facility where he presented with symptoms of rabies 19 days after exposure. The patient had sought treatment from a traditional healer after being bitten on the leg by his own dog, but otherwise did not receive formal health care after the bite. Palliative care was given until death 7 days after hospital admission. RIG was not administered to any of these patients, despite the site and severity of bites (multiple bites on the forehead and lips) for cases 1, 3 and 4. Further details about each case are as follows:

Case 1 On 23rd February 2018, a 10-year-old boy from Nyawara village, Gem sub-county, Siaya county, Kenya, presented at a local health facility with fever, headache, and general body weakness. Suspected of having malaria, he was initially treated at home with a single dose of the antimalarial Coartem. His condition worsened the next day, with symptoms including headache, dizziness, restlessness, vomiting, and incoherent speech. At the health facility, a rapid diagnostic test confirmed malaria, and he was given intramuscular Artesunate, with a repeat dose after four hours. The nurse noticed restlessness, aggression to touch, and abnormal vocalisation. Upon inquiry, it was revealed that the boy had been bitten three times on his left forearm and forehead by an unknown dog a month earlier but had not received PEP. The child’s parents reported that on the day of the bite, they had taken him to a local health facility where he received painkillers (Paracetamol), a tetanus vaccination, and wound cleaning with paraffin, but no rabies-related treatment or advice was provided. With evident rabies symptoms, he was referred to the nearest hospital and given more painkillers (Diclofenac). That night, he experienced difficulty swallowing, uncontrollable salivation, and extreme agitation at the sight of liquids. His condition deteriorated further on 25th February, leading to his referral to Siaya County Referral Hospital for palliative care, where he was declared dead on arrival.

Case 2 On 3rd July 2018, a 37-year-old man from Rarieda village, Gem sub-county, Siaya county, Kenya, visited a local health facility with symptoms of paralysis, abnormal vocalisation, and difficulty breathing. He was given Paracetamol. His condition worsened the following day, and he was transferred to Siaya County Referral Hospital for further treatment. The exact nature of the treatment he received there is unclear. On 8th July 2018, he was pronounced dead due to suspected rabies. Tracing back his exposure history, the family reported no knowledge of any bites from rabid animals. However, it was noted that the man had killed his two dogs after they exhibited signs of rabies, one month and three weeks prior to his own death, respectively. Details on whether the dogs were vaccinated during the mass vaccination campaign conducted in Siaya in 2018 were not disclosed.

Case 3 On 13th September 2019, a six-year-old girl from Tarakea-Rombo village, Moshi district, Kilimanjaro region, Tanzania, was taken to her local health facility with a headache. She was given painkillers (Paracetamol) and discharged the same day. The next day, her condition worsened with high fever, headache, and hallucinations. She was referred to Huruma district hospital and then to Kilimanjaro Christian Medical Centre (KCMC) referral hospital in Moshi town. On 16th September 2019, 22 days after the bite, she died. Investigation of her exposure history indicated that on 26th August 2019, the girl was bitten multiple times on the upper lip by an unknown dog that ran away after the bite. She reported immediately to her local health facility, received proper wound cleaning, and was administered a first dose of rabies vaccine on day 0 (26th August 2019) via the intramuscular route as well as an anti-tetanus injection. She returned for her second rabies vaccine dose on 2nd September 2019 (day 7), followed by her third dose on 12th September (day 18). The family reported paying 30,000 Tanzania Shillings (Tsh) per vaccination, excluding transportation fees to the health facility, which was 16 km away. Despite receiving these vaccine doses, her symptoms progressed, leading to her death.

Case 4 On 29th August 2022, a six-year-old boy from Alara village, South West Sakwa ward, Bondo sub-county, Siaya county, Kenya, was taken to a local health facility with complaints of fever, insomnia, abnormal vocalisation, difficulty breathing and swallowing, hallucinations, and restlessness. He was given normal saline intravenous and then transferred to the referral hospital in Bondo, where he received palliative care and died four hours later. According to his exposure history, on 22nd June 2022, the boy was bitten and scratched multiple times on the head and arm by an unknown dog while walking home. The dog was chased away by villagers. The boy received first aid at home, where his wounds were washed with soap and water, and he was then rushed to a traditional herbalist, where he received a concoction of herbs. Upon hearing this news, the community health worker advised the family to take the child to a hospital for PEP. The family took the boy to a local health facility in West Sakwa, Bondo sub-county, where he received the first dose of the rabies vaccine on 23rd June 2022 via the intramuscular route. The family reported paying 1000 Kenya shillings (Ksh) for the vaccine, excluding transportation to the health facility, which was 15 km away. They were given no further advice on the follow-up course of vaccination nor the severity of rabies. Despite receiving the initial vaccine dose, his symptoms progressed, leading to his death.

Case 5 On 27th September 2022, a thirteen-year-old boy from Bulati village, Ngorongoro district, Arusha region, Tanzania, was admitted to Fame Hospital after being referred from Bulati Health Facility on the same day. The boy exhibited signs of rabies, including excessive salivation, paralysis, abnormal vocalisation, and restlessness. Tracing back his exposure history, the boy had been bitten by his own dog on the left leg on 8th September 2022. Despite regularly attending the hospital for other medical treatments, he did not report the dog bite or receive any treatment from the health facility or hospital. Instead, the boy was brought to a healer where his wound was washed with milk, and a traditional treatment was initiated by placing a coin on the wound to suck out the poison. Upon presenting at the health facility, the medical staff discovered that the boy had been bitten by the dog 19 days prior. He was referred to a major hospital on the same day, where he received palliative care until he died on 3rd October 2022, 25 days after the bite.

The rabies incubation period varies; symptoms typically develop days to weeks after infection, but can take months depending on factors such as the bite location and severity [1]. Three of the patients in this case series progressed to rabies within one month of exposure (the date of exposure was not possible to confirm for Case 2, although was recalled to be around one month prior to death), whereas Case 4 developed symptoms more than two months later. Each patient displayed common clinical signs of rabies: fever, abnormal vocalisation, difficulty breathing and swallowing, hallucinations, paralysis, hydrophobia, aggressiveness, excessive salivation and restlessness. All patients except Case 2 had a clear history of a dog bite making the clinical diagnosis straightforward. A history of close contact with two suspect rabid dogs assisted in reaching a diagnosis for Case 2. Samples from four of the five cases were positive by rapid diagnostic test. Case 3 had a negative test result; however, the presence of rabies virus antigen was confirmed by immunohistochemistry, using the streptavidin–biotin complex staining method (Fig. 1).

Positive immunohistochemistry staining of frozen brain slides of Case 3. (A) Slides at × 40 magnification, and manual zoomed-in of cells of interest B) and C). The red stain zoomed in, indicates the presence of rabies virus antigen detected with specific antibodies (RABV-N, antibody 5DF12) and streptavidin-biotin complex staining

Amplicon-based sequencing was carried out to compare rabies viruses from the five human cases to those from recent animal rabies cases in the region using a previously described protocol [23]. Full details of the laboratory procedures are found in Supplementary File 1. Due to the poor sample conditions and primer mismatches related to the early primer set used (i.e., targeting rabies virus diversity in Tanzania from 2019–2020 but not optimised for samples from Kenya), a few of the sequences generated (14/98) had less than 90% genome coverage. Sequences from Cases 1 and 2 from Kenya both had less than 90% genome coverage (Supplementary Table 1).

All the sequenced viruses were from the Cosmopolitan clade; Cases 2 and 4 belonged to minor clade AF1a (both from Kenya), and Cases 1, 3 and 5 belonged to minor clade AF1b (from Kenya, Tanzania and Tanzania respectively) (Fig. 2). Cases 1, 2, 3 and 5 were from previously reported circulating lineages, with Cases 3 and 5 from the same lineage (AF1b_A1.1), while Case 4 was from a newly designated lineage (AF1a_C1). The most closely related antecedent and subsequent sequences to all the human cases were from domestic dogs, except for the subsequent sequence to Case 2 which was from a cow, indicating likely spill over from the lineage which was circulating in domestic dogs.

Rabies viruses from five human rabies cases and animal rabies cases from the same viral lineages. A Sequenced cases in East Africa and B maximum likelihood tree of sequences (n = 262). Sequences from the Arctic AL1a clade (GenBank accession AB699220, AY956319, EF437215, HE802675, HE802676, KF154996, KY775603, KY775604, LT909539, LT909541 and MG099711) were used as an outgroup (not shown) to root the tree. Tips and points are coloured by lineage, with diamonds and numbers denoting the human cases (Case 1 sequence OR045959 from 27 Feb 2018, Case 2 sequence OR045960 from 18 Jul 2018, Case 3 sequence OR920212 from 16 Sep 2019, Case 4 sequence OR045927 from 29 Aug 2022, and Case 5 sequence OR045947 from 3 Oct 2022) and circles denoting animal cases. Scale in substitutions/site. Ultrafast bootstrap values of lineage-defining nodes shown

All cases except Case 4 represent cross-border lineages, with lineage AF1b_A2, widespread across Africa but only reported from East Africa in 2018 with this human death (Case 1) in Kenya (Fig. 3). The most closely related antecedent sequence to Case 1 is from Bangui, CAR, where over 85% of cases in this lineage were also from (Fig. 2A). The geographic distance and phylogenetic divergence between these cases indicate limited wider sampling of the lineage, which likely originated decades ago (supplementary Table 2) and is now widespread, though largely undetected, across Africa. In contrast, lineage AF1b_A1.1 (Cases 3 and 5) has been seen exclusively in East Africa; first seen in Uganda in 2009, then Tanzania in 2011 followed by Kenya in 2020, where it has been repeatedly detected in Makueni county. Conversely, detection in Tanzania, has been sporadic and near the Kenyan border (Fig. 3A), suggestive of cross-border spread. The closest antecedent sequence is the same for both Cases 3 and 5—a rabid dog from Serengeti District in Tanzania sampled in 2019 (Fig. 3A). Lineage AF1a_A1.1 (Case 2), was originally detected in Ethiopia in 1987, then in Morocco in 1989 where it was seen frequently until 2008. There have also been infrequent detections of AF1a_A1.1 in Algeria since 2000. Virus infections from this lineage were first detected in Kenya in 2013 with human cases in both Nairobi and Siaya (Fig. 3B). Lineage AF1a_C1 (Case 4) is newly designated and highly localised, found exclusively in Siaya County, Kenya since 2021. The detection of three lineages (corresponding to Cases 1, 2 and 4) all within years or months of each other within Siaya County (Fig. 3) highlight the apparently localised co-circulation of lineages.

Geolocations of RABV sequences and subtrees of human and animal cases in Kenya and Tanzania. A The map shows the locations of the sequenced human and animal cases from East Africa coloured by lineage. B Phylogenetic subtrees shown for lineage AF1b_A2 (n = 151, Case 1) in red, for lineage AF1a_A1.1 (n = 42, Case 2) in blue, for lineage AF1b_A1.1 (n = 53, Cases 3 and 5) in green and for lineage AF1a_C1 (n = 16, Case 5) in yellow. Human cases denoted by diamonds (OR045959, OR045960, OR920212, OR045947, and OR045927). Relevant locations labelled. Scale in substitutions/site, and outgroup rooted with ordered nodes