Tick-borne relapsing fever (TBRF) is a bacterial infection; the caused by spirochaetes of the genus Borrelia. The disease is widely distributed in the Old and New World with many different species reported. In Africa, the main species are B. duttonii and B. crocidurae, the former being the predominant species endemic in Tanzania. B. recurrentis, which is closely related to B. duttonii, is the agent in louse-borne relapsing fever that causes epidemics in Ethiopia.

The vector is the soft tick, genus Ornithodoros, of which the species complex Ornithodoros moubata is prevalent in sub-Saharan Africa. Ornithodoros ticks live in traditional housing and are mainly nocturnal feeders. The disease is transmitted either by saliva during tick feeding, or in coxal fluid excreted during feeding. The tick feeds for a short time only (less than half an hour) then returns to the earth floor or walls of the house. Humans are believed to be the only natural reservoir for B. duttonii, unlike B. crocidurae in West Africa where rodents are important.

TBRF patients present with fever, headache, muscle pains, arthralgia and abdominal pain. Without treatment the fever will spontaneously subside after approximately 3 days, followed by an afebrile period of around a week before a relapse of the fever. Up to 13 febrile episodes may occur. The majority of cases are children: 75% in Dodoma region and 55% in Mwanza region, Tanzania. Greatest severity, or even fatality occurs in young children and pregnant women. In the latter, infection may lead to premature labour, stillbirths and early neonatal death. Case fatality rates of 1.6% in children and 1.5% in pregnant women have been reported. The perinatal mortality of babies born to women with TBRF in pregnancy is extremely high: 436 per 1,000 in a series reported in Tabora region.

Diagnosis is by microscopy of blood smears stained with Giemsa for detection of spirochaetes. Fluorescence microscopy with acridine-orange stains and phase contrast/ dark field microscopy are alternatives. PCR may also be used to detect Borrelia DNA.

Antimicrobial treatment is with Procaine Penicillin (PPF) injections intramuscularly daily for 5 days (dose 30,000 MU/kg) or in adults tetracycline orally 500mg q.i.d for 5 days. In pregnant women, PPF 0.8MU IM daily for 5 days or erythromycin 500mg q.i.d for 5 days are used.

Jarisch-Herxheimer reactions may occur upon treatment, but are less frequent in TBRF than in louse-borne relapsing fever. Typically this may start 1 hour after commencement of treatment with a chill phase of 30 minutes, rigors, rise in temperature, pulse, respiration rate and blood pressure. The peak temperature occurs 2-3 hours after treatment and coincides with disappearance of spirochaetes from the blood. The flush phase follows with intense sweating and fall in blood pressure. There is a risk of cardiovascular collapse and death. There is no specific drug treatment and supportive measures are needed to prevent hypotension: IV fluids (normal saline) and rest in bed.

There are no accurate data of the number of cases of TBRF in Tanzania as it is not reportable in the Ministry of Health's Health Management Information System (MTUHA). In 1996, the TBRF was reported from most regions of the mainland (see map in presentation) excluding the coastal regions and Kilimanjaro and Mara and Ruvuma.

Recent work by Kisinza in Dodoma region, found 5 % of febrile children have positive blood slides for Borrelia. The annual incidence in under 5's has been reported as 163/1000 by Barclay and Coulter and 59/1000 by Talbert. The estimated number of cases in Dodoma region is thus of the order of tens of thousands per year! The situation in other regions is not known and no widespread survey of TBRF vectors or disease has been done since that of Phipps in 1950. He looked at 18 sites throughout Tanzania and found the highest density of ticks and highest proportion of infested households in Itigi, Singida, Kahama, Dodoma, Kondoa, and Iringa. He also reported that there was no direct correlation between tick numbers and recorded cases from health facility data.

It is known that TBRF is widely spread throughout mainland Tanzania, but is grossly under-reported and forgotten in funding priorities for disease control and research. It is likely that many cases of classified as malaria are in fact TBRF because it is difficult to distinguish clinically between these two diseases. There is a need for up-to-date information on the burden of disease in order to lobby for action on this public health problem.

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Vector research Group, Liverpool School of Tropical Medicine.

Since Walton's work on the Ornithodoros moubata complex in East Africa during the 1940s and 1950s, few studies of any kind have been made on the vectors of TBRF in Africa. In particular, the taxonomy of this group has been ignored. Walton's classification of O. moubata into four major species, varying in their distribution and host preference, has been disputed but based as it is on comprehensive collection of material and observation of tick natural history from a wide geographical area, it remains the most thorough investigation.

Walton recognised O apertus (feeding on porcupines, Lake Naivasha), O. compactus (feeding on tortoises, South Africa), O. moubata and O. porcinus (humans and warthogs occurring across a wide geographical range). Only the latter two were considered vectors. Both of these further comprised two subspecies or forms: a domestic form that lived in houses and fed on man and a wild form that typically lived in animal burrows and preferred animals. However, the 'wild' form of O. porcinus, named O. p. porcinus also lived in human habitations and would readily feed on humans. This species and the domestic forms of both O. porcinus and O. moubata were believed to be the vectors of TBRF.

Since then the complex has not been investigated further. Current molecular taxonomic methods could reveal the true structure of the species complex and facilitate the elucidation of the vectorial capacity of its member species. Superimposed on a thorough knowledge of the natural history of these sibling species, this would enable the distribution of the group throughout Tanzania to be determined. Plans for developing these studies are underway.

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Mvumi Hospital, 40 km. south east of Dodoma, which serves over 200,000 population in the south and east part of Dodoma Rural District, serves as the base for tick studies in this area. The laboratory reports 200- 400 positive blood slides for Borrelia duttonii each year and TBRF is the fifth commonest reason for admission on the children's ward (164 cases in under 5s in 2002).

Tick surveys of 501 houses in 7 villages revealed that overall 87% houses were infested, (range for different villages 62% to 98% of households). Sleeping areas were most heavily tick infested.

Vector identification:

PCR for tick 16s mitochondrial DNA was used to identify the vector of TBRF in Mvumi (Fukunaga et al. 2001 Vector Borne & Zoonotic Dis 2001 1: 331-338). 16s gene sequences from 13 ticks collected from 1 house in Mvumi village were analysed and compared with published sequences of Ornithodoros moubata complex ticks. The sequences showed greatest homology with those of Ornithodoros porcinus domesticus.

Borrelia detection

A nested PCR for borrelial flagellin gene detected DNA in 8 out of 13 ticks (62%). These were sequenced, aligned and grouped into 3 types: opA, found in 1 tick, identical to B. duttonii strain Ly; opB, in 4 ticks, differing by 1 base, but with the same aminoacids as B. duttonii strain Ly; and opC, in 1 tick, differing by 24 base substitutions and 3 base deletions, resulting in 6 amino acid substitutions and 1 amino acid deletion. Phylogenetic analysis showed this to be closer to the New World species B. hermsii than any of the Old World Borrelia, raising the question: is this a new species of Borrelia?

A second series of 67 ticks from 6 houses in Mvumi were studied and PCR performed for borrelial flagellin gene. Of these, 30 ticks (45%) were infected and from individual households, tick infection rates varied from 19-80%. Sequencing of 18 of the PCR products revealed 5 types: Type 1 identical to B. duttonii; Type 2 the same amino acid sequence but 1 base substitution (opB); and types 3, 4 and 5 with the same amino acid sequence as opC "new species" each having 3 base deletions but differing in number of base substitutions (22-24bp) compared to B. duttonii strain Ly. To date, attempts to cultivate the new borreliae from ticks have failed.

Borrelia in human blood samples:

During a current study of insecticide-treated nets on the incidence of TBRF in children in Muungano village (12 km from Mvumi), Kisinza has found this "new species" of Borrelia (types 3 and 5) in blood samples from afebrile children and type 5 in febrile children (Lancet in press). More work is needed to determine if the new Borrelia is pathogenic in humans. Clinical isolates obtained from patients at Mvumi Hospital were found to be B. duttonii, not the new species (Cutler et al. Int. J. Syst. Bact. 1999 49 1793-1799). We hope that current research will resolve these issues.

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Like Dodoma region, Mwanza in the North of Tanzania, is an endemic area for TBRF. Information on incidence of TBRF was sought from the 8 districts of Mwanza using existing health facility records. Data was received from 2 districts, one claimed no TBRF cases and the remainder were unable to provide information on TBRF incidence. The information below summarises the TBRF data received from two districts in Mwanza over a three-year period:

This data is likely to be an underestimation of TBRF in the Mwanza region as data was not provided from 5 districts and those cases attending local dispensaries will not be included. Despite this, it is still apparent that TBRF is a significant health problem in the Mwanza Region.

Control measures against the soft tick vectors of TBRF can be divided into physical and chemical methods. Physical methods include short term measures such as sprinkling of hot water onto floors and walls to kill the ticks, and mixing water and earth to plaster the surfaces of the earth walls and floor to render them smooth and eliminate the cracks and soft earth where the ticks prefer to hide. Lack of water in Dodoma region is a major constraint.

The long-term solution is to make floors and walls solid with cement mixtures and to use corrugated iron roofing instead of traditional tembe wood and earth construction. Many families are unable to afford the costs of cement and corrugated iron building materials. Some also prefer traditional roofs that are cooler in hot weather.

Chemical measures involve acaricides of the synthetic pyrethroid group. Locally used insecticides that have found to be active against ticks are lambdacyhalothrin (ICON), permethrin and deltamethrin. Three methods of application have been tried. Interior residual spraying of houses with lambdacyhalothrin requires training of technical personnel to apply. Insecticide treated mosquito nets have also been found to protect against tick bites and householders report finding dead ticks after using the treated nets. Some householders have used the deltamethrin, supplied in net treatment kits, diluted with water, to sprinkle on the floor and walls. Inadequate concentrations may lead to the emergence of tick resistance to the insecticide.

William N. Kisinza, Alison Talbert & Philip J McCall

Tick-borne relapsing fever (TBRF) is a disease of man caused by spirochaetes (Borrelia spp), transmitted to man by Ornithodoros soft ticks either through biting (saliva) or contamination of bite wounds with coxal fluid. Ticks acquire the spirochaete through feeding on an infected host. Once the tick is infected, the spirochaete can be maintained for the lifetime of that tick, or to subsequent generations through transovarial transmission.

Although TBRF occurs throughout the country, the extent of the disease in Tanzania, and indeed elsewhere in Africa, remains to be determined. An epidemiological baseline survey carried out in Muungano village in Dodoma, rural central Tanzania, between October and December 2002 to determine the prevalence of the disease and tick infestation rate. This data was an essential prerequisite for a study on the use of ITN's for reduction of TBRF. 361 under five children were screened for Borrelia spirochaetes by blood slide examination and PCR. Tick survey conducted in 200 randomly selected households. PCR was twice as sensitive in detecting infections, revealing Borrelia species in 6/54 (11·1%) of children with fever, and 13/307 (4·2%) of otherwise healthy children. Of the 17 infections genotyped, 11 were B. duttonii and 6 were a recently described new species. Phylogenetic analysis of this yet unnamed Borrelia species revealed that it differed from B. duttonii and the other Afro-tropical species (B. recurrentis & B. crocidurae). The new species is phylogenetically closer to the Nearctic's Borrelia species (B. anserina, B. parkeri, B. turicatae, and B. hermsii). House-tick infestations with the Ornithodoros soft tick vectors ranged from 50% to 87% and the distribution of soft ticks in house was heterogeneous. Bedrooms were heavily infested (76%) followed by sitting rooms (13%), poultry area (7%) and kitchen (4%).

ITNs have been in use to protect against malaria transmission and the promotion of their usage forms the basis of malaria control in the majority of endemic regions worldwide. To date, no systematic studies have been undertaken on their use to control TBRF. If successful for prevention of TBRF, ITNs would be the most cost-effective method to break the transmission cycle between human and ticks. We are assessing the efficacy of insecticide treated nets, introduced in Muungano village in January 2003, for reduction of TBRF. We assessed TBRF incidence after three months use of ITN's (January - April 2003). 1,800 under five children (900 from the households with nets and 900 from control households, no nets. These were screened for Borrelia using both thick blood films and blood spots for PCR analysis. Tick survey conducted from 400 randomly selected households (200 ITN households and 200 controls to determine the effect of ITNs on tick reduction. Data analysis is currently in progress.

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Evaluating the clinical signs and symptoms and the factors associated with risk of TBRF in Mvumi, Central Tanzania

Vector Research Group, Liverpool School of Tropical Medicine and ¹ Mvumi Hospital

In many TBRF cases, blood slides are parasite negative. As differentiation of the symptoms of TBRF from malaria is difficult or impossible, TBRF is commonly misclassified as

malaria and treated as such. This can contribute to the overuse of antimalarials, failure to treat TBRF and undoubtedly leads to the

underestimation of the problem of TBRF. To overcome this problem, reliable simple symptom-based means of differentiating these two common illnesses are needed. A study was carried out on patients presenting at Mvumi hospital and diagnosed by blood slide with either TBRF or malaria (or both) or with a fever of some other, unknown, cause (the slide-based diagnosis was subsequently confirmed by PCR analysis). A range of clinical signs and symptoms were recorded and monitored from the individuals recruited to the study over the following days.

A second study followed up the patients to determine if there were any risk factors associated with the infection acquired, by visiting their homes. A questionnaire survey of a range of parameters that might have been involved (ranging from presence of ticks, animal ownership, etc to sleeping arrangements and house construction type), economic indicators and other factors were all recorded.

At the time of the meeting, analyses have not been completed in either of these studies.

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Borrelia duttonii tick-borne relapsing fever, like louse-borne relapsing fever, is a disease of man with no known other reservoirs. Tanzania is highly endemic for TBRF where this spirochaete results in significant mortality and morbidity. Mvumi is centrally located within this endemic area and through local interest and collaborative links, is ideally suited as a location in which to study TBRF.

Relapsing fever was once a disease of worldwide importance. Cragie made initial descriptions of clinical relapsing fevers in Edinburgh in 1843. From this time, the term relapsing fever has been used to describe this clinical condition. The spirochaetal aetiology was resolved by Otto Obermeire in 1867. Following failed attempts to reproduce the disease in animal models, he resorted to self-inoculation, but was also unsuccessful! The vector association with ticks for B. duttonii and lice for B. recurrentis, were determined in the early 1900's. Attempts to cultivate the spirochaete were made by Robert Koch, but were unsuccessful. Noguchi claimed success in 1912, but unfortunately others could not reproduce his work. Later, Kelly described media formulations capable of supporting growth of B. hispanica and B. hermsii. This medium was later further developed for cultivation of Lyme borreliosis spirochaetes. The author was able to successfully use this for isolation of B. recurrentis and later, for B. duttonii. Prior to this, both of these spirochaetes were considered non-cultivable!

TBRF persists in Tanzania where people live in traditional "Tembe" dwellings. Many of these are infested with ticks (between 76-98%), despite efforts through use of boiling water, "cementing" of walls and floors, and use of acaricides. Traditionally the occupants will sleep on animal hides on the mud floors, where they are frequently bitten by Ornithodoros moubata ticks.

Numerous names have been given to relapsing fever-associated spirochaetes found in the New World, typically based on the vector and geographical location of occurrence. The phylogeny of these should be re-addressed using molecular phylogenetic techniques. All but B. duttonii and the louse-borne B. recurrentis, have rodent reservoirs.

Antigenic variation may serve several functions. It provides a mechanism of rapid adjustment from the different environments found in arthropod vectors and mammalian hosts. Additionally, this provides a method for immune evasion, and may even provide a means of persistence with different variants showing differential ability to stimulate TNF! This however, remains speculative. We are currently investigating the molecular mechanisms of antigenic variation in B. recurrentis and intend to compare the findings with B. duttonii. The cultivated strains show either large variable membrane proteins of approximately 35-45 kDa (vlp) or small proteins of 20-25 kDa (vsp). The genes are located on large linear plasmids that comprise part of the "segmented genomes" of these spirochaetes. Correlation between plasmid profiles and sizes of variable membrane proteins suggests that alteration of plasmid profiles could be associated with antigenic variation. Investigations are currently on going with creation of sub-genomic libraries, use of "family" specific primers and with suppressive subtractive hybridisation.

The ecology of TBRF is believed to involve a man-tick cycle, however, for this to sustain the cycle, prolonged blood persistence would be required. In addition to antigenic variation, these spirochaetes have recently been demonstrated to bind factor H and factor H-like protein and C4 of the complement pathway. Furthermore, the phenomenon of rosetting of red blood cells has been described with B. duttonii. Collectively, these mechanisms may enable prolonged blood persistence. Clinically, the disease is seen in children and during pregnancy, begging the question of whether this is sufficient to sustain the ecological cycle. This raises the question of possible alternative reservoirs. Other borrelial species have rodent and avian reservoirs. Both chickens and rats frequently share traditionally Tembe houses, raising the question of whether these may also be part of the reservoir for B. duttonii. Investigation of their role as a reservoir is currently underway. Expanding on the role of infection reservoir, could man be a reservoir in the absence of overt disease? This too is currently under investigation. Samples taken from asymptomatic villagers, rats and chickens are being investigated using a hemi-nested flagellin PCR. Products will be compared by RFLP and representatives of different profiles examined by DNA sequencing.

Areas to be addressed by future research should focus on the epidemiology and incidence of disease; vector competence for the spirochaete; the reservoirs of infection; and host-microbial interactions. Once we have data on these aspects, a mathematical model could be established through which intervention strategies could be explored, prior to testing in the field. Evaluation of the success of these methods will only be possible through comparison with sound baseline incidence data. To undertake such a programme of research will require significant funding. Efforts should be made in this direction utilising the network of experts in the field attending this meeting.

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Tick-borne relapsing fever (TBRF) caused by the spirochaete, Borrelia duttonii, endemic in the Dodoma region. A question exists of whether man is the only reservoir of this spirochaete. All other tick-borne relapsing fever borreliae have rodent reservoirs, leaving the question of whether rodents could be an additional reservoir for B. duttonii? Further evidence comes from the finding of Borrelia spp in the blood of locally trapped Crocidura spp. This presentation reports our findings of a recent study in which samples from 250 rodents; 250 ticks and 7 human clinical samples were analysed. Methods included cultivation from human blood, rodent blood and ticks; Giemsa stained films of blood and haemolymph and PCR analysis using a 668 bp 16sRNA target.

Human blood samples yielded two borrelial cultures, but no growth was found in 50 rodent blood samples or from 50 ticks. No spirochaetes were demonstrated by microscopic analysis of 250 rodent bloods. PCR demonstrated products of expected size from 2/7 human bloods tested. A PCR band was also demonstrated in one Rattus spp. One tick similarly yielded a positive PCR for Borrelia.

Ornithodoros moubata ticks are able to feed on rats. The finding of Borrelia spp DNA in a rat suggests that rodents may serve as a reservoir for this spirochaete. Further investigations are necessary to confirm these findings.

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Issues raised at the last meeting held in 2001 were reviewed and are summarised below:

Raise awareness - NIMR have now included TBRF on its list of research priority areas.

Distribution, potential reservoirs, sub-clinical infection and understanding pathogenic mechanisms, effect of ITN's for TBRF reduction and tick identification, are all priority areas with aspects currently under investigation.

Vector bionomics - funding for work is currently being sought.

Climatic conditions, social behaviour, disease in pregnancy, differential diagnosis and algorithms, treatment guidelines, extending microscopic diagnostic facilities all require further work, but require funding to allow further progress.

Determination of the incidence and breadth of TBRF infection must be a priority area. Currently we have no funds to permit comprehensive surveillance, however, much data already exists in different health facilities. We should seek to extract and collate data from different areas within Tanzania in order to raise awareness of the problem and hopefully secure funding for more in depth surveillance. The following agreed to produce data from districts in their regions for October 2003:

William Kisinza will request a letter from NIMR to support collection of this data and request that some funds to cover transportation costs are made available.

Alison Talbert and William Kisinza to act as co-ordinators for data collation. This information should be published (East African Medical Journal?).

Training should be offered to "trainers" of laboratory staff who could then disseminate this information locally.

Rubber stamps should be made available stating "No Borrelia seen" to focus attention on the need to look for more than malaria when examining blood films.

Sally Cutler to contact Wellcome Trust to see if Malaria teaching materials could include under differential diagnosis, the possibility of TBRF.

Radio interview to be arranged by Elizabeth Nchimbi to publicise the meeting and its recommendations. We need to encourage reporting of cases to allow collection of data.

We need to have knowledge of epidemiology and incidence; vector competence; reservoirs; host-microbial interactions; to be able to create mathematical models to test intervention strategies, to field test reduction/intervention methods and evaluate these against baseline incidence data. To achieve these aims will require significant funding. Appropriate funding organisations to be contacted as calls are made.

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We are grateful to the Wellcome Foundation for their generous support for this meeting.