Louse-borne relapsing fever—A systematic review and analysis of the literature: Part 1—Epidemiology and diagnostic aspects

Louse-borne relapsing fever (LBRF) is a classical epidemic disease, which in the past was associated with war, famine, poverty, forced migration, and crowding under poor hygienic conditions around the world. The disease’s causative pathogen, the spirochete bacterium Borrelia recurrentis, is confined to humans and transmitted by a single vector, the human body louse Pediculus humanus. Since the disease has had its heyday before the days of modern medicine, many of its aspects have never been formally studied and to date, remain incompletely understood. In order to shed light on some of these aspects, we have systematically reviewed the accessible literature on LBRF, since the recognition of its mode of transmission in 1907, and summarized the existing data on epidemiology and diagnostic aspects of the disease. Publications were identified by using a predefined search strategy on electronic databases and a subsequent review of the reference lists of the obtained publications. All publications reporting patients with a confirmed diagnosis of LBRF published in English, French, German, and Spanish since 1907 were included. Data extraction followed a predefined protocol and included a grading system to judge the certainty of the diagnosis of reported cases. Historically, Ethiopia is considered a stronghold of LBRF. The recognition of LBRF among East African migrants (originating from Somalia, Eritrea, and Ethiopia) arriving to Europe in the course of the recent migration flow from this region suggests that this epidemiological focus ostensibly persists. Currently, there is neither evidence to support or refute active transmission foci of LBRF elsewhere on the African continent, in Latin America, or in Asia. Microscopy remains the most commonly used method to diagnose LBRF. Data are lacking on sensitivity and specificity of most diagnostic methods.


Introduction
Louse-borne relapsing fever (LBRF) is an ancient epidemic disease, with descriptions dating back to Hippocrates' times [1]. Linked to war, famine, poverty, forced migration, and crowding under poor hygienic conditions, the disease has accompanied mankind throughout history and was once even described as the "most epidemic among the epidemic diseases" [2]. The use of the name "relapsing fever" was first documented by Craigie and Henderson during the epidemic which occurred in Edinburgh from 1843 until 1848 [3,4], reviewed by Greig 100 years later [5]. Milestones in the disease's history were the discovery of the causative organism by Obermeier in Berlin in 1873 [6], the discovery of the organism in the vector by Mackie in India in 1907 [7], and the description of the mode of transmission by Sergent and Foley in Algeria in the same year [8,9]. In history, LBRF had a massive impact, especially following political crisis, socioeconomic disaster, and war [10,11].
Since the disease had its peak incidence and prevalence before the days of modern medicine, many aspects of the disease have never been formally studied and remain incompletely understood to date. In order to shed light on epidemiological and diagnostic aspects, we reviewed and analyzed the available published data on LBRF since its transmission was identified in 1907.

Epidemiology
With the reduction of the vector Pediculus humanus, due to improved living standards along with the introduction of the insecticide dichlorodiphenyltrichloroethane (DDT) in the 1940s, LBRF declined and finally disappeared from most regions of the world, as well as from most medical text books, over the past century [10,12].
In order to shed light on the global epidemiology of LBRF over the past 100 years, we reviewed all available published reports on LBRF cases and summarized their number, their time of occurrence, and the grade of evidence for their correct diagnosis. Additionally, we region) rather supports TBRF. Nevertheless, microscopic examination has been the gold standard for diagnosing relapsing fever [37,38].
Serology has been used as alternative diagnostic method, but shows limited specificity due to cross-reactivity among Borrelia spp., including TBRF, as well as with other spirochetes (e.g., Treponema pallidum) [25,35,36]. Moreover, within endemic regions, the interpretation of serological assays is often complicated by high background reactivity and sero-scars (persistence of detectable antibodies after infection) in addition to the fact that the respective laboratory capacity and expertise are mostly unavailable. Furthermore, since seroconversion demands time, serology is not helpful to diagnose acute infection. Thus, serology was never developed into commercial available assays, and microscopy remains the sole and most widely available diagnostic tool to date [40]. With the introduction of PCR-based methods, the diagnostic sensitivity improved markedly, and species differentiation of relapsing fever Borrelia became available [41,42]. However, the availability is still largely restricted to affluent countries and research settings. Table 1 lists the advantages and disadvantages of the laboratory diagnostic and research methods applied in LBRF.
In order to shed light on the evolution of LBRF diagnostics over the past 100 years and the accuracy of different test methods, we reviewed the available literature and summarized the available data.

Methods
A systematic review protocol established for this review is available in the Supporting information section (S1 Text). The electronic databases BIOSIS, CINAHL, Cochrane Library, Current Contents Connect, Elsevier, EMBASE Ovid, Ovid MEDLINE, PMC, PubMed, Scopus, and Web of Science were searched on October 4, 2017 using the search term ((Relapsing AND fever AND (Louse OR Lice OR (Pediculus AND humanus))) OR (Borrelia AND recurrentis) OR LBRF). A second and third search, using the same search term on the same databases, was conducted on August 7, 2018 and June 17, 2019, respectively. After checking for and removing duplicates (using EndNote software and manually [43]), publications were prescreened by checking titles and abstracts if they concerned patient(s) with the diagnosis of LBRF. Publications not reporting patient(s) with the diagnosis of LBRF were excluded. The remaining publications were then full-text assessed for fulfillment of the inclusion criteria: reporting conclusively diagnosed case(s) of LBRF and published after 1907 (the year when the disease's mode of transmission was discovered) and published in English, French, German, or Spanish. Publications not fulfilling the inclusion criteria were excluded. Publications that could neither be retrieved through their respective journals, nor by contacting libraries, or after contacting the authors, were classified as "not retrievable" and excluded. Additional relevant publications identified when reading the full-text articles or checking their reference lists were reviewed and included if they fulfilled the inclusion criteria ("snowball" search strategy).
A data extraction sheet for screening and selecting eligible publications was developed and is available in the Supporting information section (S2 Text). The following data were extracted from eligible publications using a standardized excel spreadsheet: patient characteristics (number of patients, age, gender, origin, occupation, social status, and way and duration of migration), diagnostic method (microscopy and molecular method), symptoms and signs (fever, chills, myalgia, headache, hepatomegaly, splenomegaly, signs of hemorrhage, and others), treatment (number of treated and untreated patients, drug, dosage, and duration and route of administration), and outcome (Jarisch-Herxheimer reaction (JHR), abortion/stillbirth, premature delivery, and mortality).
To minimize bias, the same reviewer conducted a second full data extraction more than 1 month after the first extraction. Discrepant results and unclear cases were resolved by consulting a second reviewer.
In order to consider the probability of a correct diagnosis of LBRF, all reviewed cases were graded according to the used diagnostic method and respectively classified ( Table 2). To visualize the worldwide epidemiology of LBRF over the past century, data of all identified cases were entered into a geographic information system (GIS) application (https://www. qgis.org/en/site/) and graphically displayed using geodata from Natural Earth (https://www. naturalearthdata.com/about/terms-of-use/).
The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (S1 Checklist).

Results
Our search strategy identified 4,943 publications of which 184 finally proved eligible for being included and analyzed (Fig 3, S1 Fig). A list of included and excluded publications is available in the Supporting information section (S3 Text).

Epidemiology
The geographic localisation of all included LBRF cases is displayed in Fig 4 according to the following criteria: (i) time of occurrence; (ii) autochthonous versus imported; and (iii) underlying diagnostic certainty.
Publications reporting only indirect evidence for the presence of LBRF (but no clinical cases) are summarized in Table 4.
Reports on imported cases of LBRF are summarized in Tables 5 and 6. Reports on the occurrence of LBRF in Latin America, Asia, and the Middle East are summarized in Tables 7-13. In Fig 5, the number of published LBRF cases is visualized in relation to the number of publications.  summarize the milestones of LBRF diagnostics.

Epidemiology
Latin America. The last conclusive evidence of the occurrence of LBRF in Latin America dates back almost a century and is restricted to Peru. This notably contrasts the assumed occurence of the disease in South America in the mid-20 th century published by Felsenfeld in  (Fig 1) (Note: The method used to create the map is not known to us; the question marks within the map suggest some degree of uncertainty). We did not find any evidence supporting the persistent occurence of LBRF foci in Peru beyond the 1920s, nor did we find any reports on the occurrence of LBRF in other countries of Latin America.
Asia and the Middle East. Reports from Asia often coincide with times of colonialization as well as wars that western countries were involved in, when medical officers published their observations during their service. Subsequently, publications ceased shortly after the ends of conflicts and colonialization. The most recent examples were the Second World War and the Korean War. However, endemic foci may have persisted among a certain population at risk, as it currently does in Ethiopia, which simply were not detected or not published in western languages. It seems unlikely that the disease vanished as abruptly as publications ceased. A publication bias seems likely for these regions. However, our findings strikingly contrast the assumed occurence of LBRF in Asia in the mid-20th century published by Felsenfeld in 1971 (Fig 1).
China: The last published report dealing with microscopically diagnosed LBRF cases linked to China dates back to 1946, when relapsing fever was reported in Chinese soldiers just flown into Assam, India (Table 9). No reports on cases of LBRF exist from China thereafter.
Korea and Japan: Scarce data are available from Korea, where LBRF occurred and was confirmed until the end of the Korean War (Table 10). A retrospective description suggests a far wider spread than assumed in English literature and describes existing publications in Japanese or Korean language before the Korean War [90]. Thereafter, no information is available.
Southeast Asia: Data from Southeast Asia, where LBRF was reported in Vietnam and Cambodia, are very limited, and no information is available after 1912 and 1958, respectively (Table 11). Remaining residua past that can neither be confirmed nor excluded.
Indian subcontinent: Many accounts were published from the Indian subcontinent, where the disease often occurred in epidemics and endemic foci (Table 12). Investigations in the North-West Frontier province, nowadays located in Pakistan, led to the belief that both the louse-borne and the tick-borne variety are endemic in these areas, suggesting that careful watch should be kept for a sufficient differentiation of LBRF to TBRF [107]. The diagnosis of the reported sporadic cases was established after careful consideration of TBRF as a differential

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Louse-borne relapsing fever -part 1 Grd., grade of diagnostic certainty according to Grd., grade of diagnostic certainty according to

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Louse-borne relapsing fever -part 1 diagnosis. The author notes further that in most cases, blood was taken after symptoms resolved. Therefore, most microscopic examinations were negative. As there was no further data on the positive results, all cases were considered as clinically diagnosed. A worker informed the authors in a personal communication that LBRF had been known in the area for many years [107]. The last published report from 1990 microscopically diagnosed LBRF in 2 patients in 1984 to 1985 [111]. Despite the authors titling this discovery B. recurrentis, there is neither sufficient clinical evidence nor conclusive case histories to retrospectively comprehend how the differentiation was achieved. The authors noted that the disease is common in the Karachi region; however, their work lacks discussion as to whether these cases belong to the tick-borne or louse-borne species [111]. The study was included due to positive blood smears and the authors publishing the data as B. recurrentis. However, considering the circumstances, the evidence should be regarded with caution. Further studies are needed from this area to either confirm or rebut the presence of LBRF in these areas. Afghanistan and Middle East: Accounts on LBRF from former Persia and Mesopotamia are closely related to the World Wars and the medical officers who published their reports, with no further information published thereafter (Table 13). Migration of refugees, prisoners of war, and the movement of Russian, Turkish, and Indian troops were often reported to be the main cause for the infection of western troops with the disease [113,114,116]. Many of the occurrences mentioned were traced back to troop movements, close contact with the native population or the recruiting of refugees for labor corps [113,114,[116][117][118]. A report from Iran in 1976 found borreliae in a febrile patient, suggesting a new species in the area, although noting a similarity to the malady known from Ethiopia. Despite the suspicions, no further Grd., grade of diagnostic certainty according to Grd., grade of diagnostic certainty according to

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Louse-borne relapsing fever -part 1 research on the means of transmission has been conducted. For this reason, the species of borreliae found in Iran remains inconclusive [139]. Caution should be taken regarding sporadic cases, as the authors themselves and others noted the presence of TBRF in these areas [119,139]. Imported cases. Living in cramped and poor hygienic conditions provides favorable conditions for the transmission of LBRF [19,20,45,134,[140][141][142][143][144][145]. As this is the case for many immigrants, refugees, and seasonal workers, human migration is a critical component in the development of LBRF epidemics. In the last century, when the presence of LBRF was almost worldwide, only sporadic cases of imported LBRF were reported. In recent years, the proportional discrepancy between the number of reports from endemic regions and reports on imported cases to non-endemic countries is striking: Between 2010 and 2019, 25 publications reported 78 imported cases of LBRF in non-endemic countries, whereas from endemic regions, 7 publications reported 431 autochthonous cases (Fig 5). This increase in reports on imported cases of LBRF is primarily attributable to the increased migration flow from Africa to Europe observed in 2015 and 2016. Most of these cases were diagnosed using PCR-based methods.
Persistence of LBRF and factors perpetuating the disease. The vector: The transmitting vector P. humanus [146] is a specialized human ectoparasite that flourishes in hygienically poor and overcrowded conditions [11,146]. Besides B. recurrentis, it also transmits R.

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Louse-borne relapsing fever -part 1 prowazekii and B. quintana, the causative agents of epidemic typhus and trench fever, respectively [146]. Experiments demonstrated that B. recurrentis is not transmitted by ticks [147]. The body louse is currently the only proven vector, and humans are the only known reservoir [11]. Body lice live and lay eggs in clothing and only approach the human body for an obligate blood meal [40]. A factor that perpetuates transmission, especially during epidemics, is that lice are temperature sensitive and tend to leave patients clothes during febrile episodes [22]. Interestingly, body lice were reported on secondhand clothing found on a street market in Italy in 2018, making it the first report of human body lice since 1945 in Italy [148]. This finding challenges the paradigm that body lice die quickly once off the host. Transmission: Unlike most vector borne infections, B. recurrentis is not transmitted to the human host during the blood sucking act of the vector, as the digestive tract and the salivary glands of the body louse are not affected by the infection [11,86,149,150]. Since the louse's hemolymph was found to harbor B. recurrentis, human infection is traditionally considered to result from damaging or crushing the louse, thus liberating the insect's hemolymph. The coliberated bacteria were then considered to enter the human host through microlesions of the skin, which are either caused by the bite itself or by scratching induced by the itchiness of the bites [11]. This transcutaneous route of infection is supported by animal [80] as well as human experiments [86,151]. In 1938, Chung and colleagues identified B. recurrentis in the feces of lice and suggested this as an additional source of infection, with the caveat that in their experiments, the excreted spirochetes were dead [86]. The issue remained dormant until 2005, when Houhamdi and Raoult reported the detection of living B. recurrentis in excreted feces of an infected louse, which revived the discussion [152]. However, whether the fecal excretion of B. recurrentis is relevant for the pathogens transmission still remains to be clarified.
Unnoticed reservoir? Asymptomatic cases: Asymptomatic cases of LBRF in Sudan were observed by Atkey in 1930 [153]. These cases appeared toward the end of an epidemic, and spirochetes were readily found in their blood. The author further observed a general milder course of the disease toward the end of the epidemic [153]. Another author reported latent and atypical LBRF infections [154]. During an outbreak survey conducted in Khartoum in 1969, 22 microscopically positive but asymptomatic cases of LBRF were identified among 979 immigrant laborers from southern Sudan [134]. The fact that such cases have been reported may imply a high number of overlooked cases. Further research is needed to confirm the Grd., grade of diagnostic certainty according to PLOS NEGLECTED TROPICAL DISEASES existence of asymptomatic cases and to assess the rate of asymptomatic infections since they may contribute to the persistence of the disease in certain areas. Residual brain infection: Residual brain infection (RBI) describes the tendency of spirochetes to persist in the brain after they have cleared from the blood and was first described by Buschke and Kroo with TBRF in an animal model [38]. Data for B. recurrentis are scarce. One study demonstrated infection of squirrels using cerebrospinal fluids of LBRF patients [87]. Other studies demonstrated B. recurrentis in the cerebrospinal fluid of patients [155] and 1923-1924 Retrospective description of an epidemic in Madras NA [107] 1923-1926 Account on diseases in India suggesting LBRF to be far more widely spread at the time than formerly suspected, with affected areas scattered in both central and southern areas with a few spots in eastern areas NA [108] 1924 Description of microscopically diagnosed cases in Nilgiri Hills, Madras Presidency, and further research with sera of patients B [107,109] 1925-1929 Description of sporadic cases and the occurrence of small outbreaks in Punjab between 1925 and 1929, indicating endemic residua in the North after the epidemic. Following the description of sporadic LBRF cases in the North-West Frontier, in present day Pakistan, the report further suggests both the louse-borne and the tick-borne variety to be endemic in these areas E [107] 1948 Report of microscopically diagnosed LBRF cases in North-East Bengal among military personnel B [110] 1984-1985 Report of 2 microscopically diagnosed LBRF cases in the Karachi region. Finding was called "B. recurrentis" by authors; however, the publication lacks data on differentiation B [111] Grd., grade of diagnostic certainty according to

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Louse-borne relapsing fever -part 1 infection of the brain of primates [121]. The involvement of the central nervous system in the acute phase of LBRF infection was suggested in up to 30% of cases [36]. However, a review article suggested that most of neurologic symptoms in LBRF infection may be due to hemorrhages in the central nervous system, rather than direct involvement of the spirochetes [38]. In animal experiments with Borrelia turicatae, RBI was found in 19% of immunocompentent mice [156].
Another study using a murine model to investigate RBI using B. turicatae, Borrelia crocidurae, Borrelia hermsii, and B. duttonii found limited brain persistence in B. crocidurae and longest persistence for B. duttonii. Additionally, reactivation of the infection was demonstrated in the case of immunosuppression. The authors suggested the brain as reservoir [157]. Further studies are needed to investigate RBI. Historic point of view: Many hypotheses were discussed regarding the question of persistence and maintenance of the disease between large epidemics. As an example, in North Africa, despite systematic research, no LBRF cases were identified between the 2 large epidemics of 1908 to 1920 and 1943 to 1945 [2]. Considering that LBRF has no currently known host Grd., grade of diagnostic certainty according to Year Comment Ref.
1873 Discovery of the causative organism [6,120] 1907 Discovery of the organism in the vector [7] 1947 A publication reported 65% of suspected relapsing fever cases to yield a positive result on microscopic examination [121] 1969 A retrospective review of 2,825 cases of relapsing fever suggested a sensitivity of approximately 70% on initial blood smears to find spirochetes [36] 1983 Enhancement of sensitivity was reported when using either fluorescence microscopy on acridine orange-stained blood smears [122] 1996 A review suggested animal inoculation or culture to enhance sensitivity and specificity [27] 2001 Enhancement of sensitivity was reported using QBC fluorescent technique. (QBC: detection of spirochetes down to 10 organisms/mm3, Wright-stained blood film: did not detect organisms <82 organisms/mm3; positive readings found by means of blood film fell significantly after dilutions below 3,263 organisms/mm3, in contrast to the QBC, the accuracy of which fell only at dilutions of <82 organisms/mm3) [123] 2009 A publication suggested a centrifugation-based method to concentrate spirochetes, followed by Giemsa staining, to be equally sensitive to PCR-based methods. According to the study results, detection at a bacterial concentration of 1 bacteria/ml was achieved [124] PCR, polymerase chain reaction; QBC, quantitative buffy coat; Ref., reference. https://doi.org/10.1371/journal.pntd.0008564.t014

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Louse-borne relapsing fever -part 1 other than humans, that there is currently no known reservoir, that lice do not transmit B. recurrentis to their progeny, and that transmission requires injury or crushing of the louse, the persistence of the disease was a mystery. Two hypotheses were formulated around 1960 concerning the origins of epidemics: (i) the epidemics of LBRF are only a temporary phenomenon due to a mutation of a tick-borne species by passage through the human into the louse; and (ii) the existence of reservoirs in an endemic focus from where epidemics originate [2]. Regarding the first hypothesis, it was experimentally achieved to infect lice with tick-borne species [2]. In one study, lice were infected with B. duttonii and successfully transmitted back to primates [158]. However, no mutations or changes in pathogenic character were observed, and no evidence supporting this hypothesis was found in vivo. Regarding the second hypothesis, Ethiopia was recognized to be an endemic focus by Sparrow [133]. Since then, Ethiopia has remained an endemic focus. It seems likely that the persistence of endemic foci were the origins of the ?-2019 Antigenic variation and cross-reactivity within the species, and with other closely related organisms, were 2 major issues in the development of reliable serological diagnostic methods [25,34,38,[125][126][127][128] 1996 and 2000 Publications suggest the use of glpQ gene, which was found to be absent in Lyme disease borreliae, and very different in amino acid sequences in Treponema pallidum, hence enabling the serological distinction from relapsing fever cases [34,127] 2000 A report demonstrated seroconversion 1 to 2 weeks after clinical presentation, hence recommending the use of paired acute-phase and convalescent-phase sera. Persistence of detectable antibodies to recombinant GlpQ in a serum sample taken 27 years after infection with LBRF. Immunoblotting with recombinant GlpQ was found to be more sensitive than ELISA with purified His-tagged GlpQ

1996
Phylogenetic analysis, using PCR-based methods targeting 16S ribosomal DNA sequences, reported a close relationship between the different relapsing fever borreliae, and researchers have suggested this strategy for diagnosis [129] 2003 Report of successful discrimination using a real-time PCR assay targeting the flagellin gene in B. recurrentis, which differs only by a single nucleotide from the sequence in B. duttonii. Successful recognition has been achieved at annealing/extension temperatures of 64.5˚C, 65˚C, and 66˚C. Sensitivity of 3 copies of the target sequence was reported

2008-2012
Phylogenetic analysis has led to the concept that B. recurrentis is a degraded subset of B. duttonii. Using a MLSA approach, gene sequence identities greater than 99% were reported [130][131][132] 2012 Using MST method, 3% sequence divergence was observed when using the MST7 spacer to discriminate between B. duttonii and B. recurrentis [131]

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Louse-borne relapsing fever -part 1 former epidemics, rather than the first mentioned, or any other hypothesis. Asymptomatic infections and RBIs most probably serve as factors that further perpetuate the persistence of LBRF in an endemic focus. New endemic foci? Most of the recently imported cases originated from the Horn of Africa. In one refugee from Mali, contact with people migrating from this area was reported. The location of a current endemic focus has been suspected in Libya, which reportedly serves as a focal point for smugglers to bring refugees across the Mediterranean Sea [15,32,159]. Even though there is no published evidence to confirm the suspicions, a temporary unnoticed endemic focus in certain refugee camps or places where migrants have gathered is likely. The duration of a migrant's journey from East Africa to Europe largely exceeds the reported incubation periods of LBRF. It is probable that refugees go through the first attack and the following relapses during the first month of their journey. The fact that these patients have showed first symptoms upon arrival in Europe suggests an endemic focus around the Mediterranean Sea. This could have been a temporary focus, as it vanished after 2016 without further reports from imported cases thereafter. However, the epidemiological investigation in this review has shown that there is currently no other known endemic focus than Ethiopia.
Migrants and the homeless population, overlooked small outbreaks in Europe? Since the Second World War, there has been no reported outbreak of LBRF in Europe. In 2005, one study indicated that there may have been a small outbreak among the homeless population in Marseille based on the detection of immunoglobulin G (IgG) antibodies to B. recurrentis [29]. In Marseille, about 60% of the homeless population notably consists of migrants [29,160]. It is possible that an imported, unnoticed case could have caused a small epidemic among the high-risk population of homeless people. According to several studies, these people are commonly found infested with body lice, and in some instances further infected with other louseborne pathogens, such as B. quintana [161][162][163][164]. A case report from Saudi-Arabia described B. recurrentis in a homeless man [165]. A report from Italy suggested that 2 migrants acquired the disease in a housing facility of newly arrived refugees. The authors noted that the disease Table 17. Milestones in animal inoculation and culture.

Year
Comment Ref.

1954
The inoculation of guinea pigs was suggested for use in differential diagnosis of TBRF and LBRF: Adult rodents are susceptible to TBRF borreliae, but refractory to B. recurrentis infection [120] 1958 and 1969 Susceptibility tests in adult rodents were reported to be the most reliable method for the differentiation between tick-borne and louse-borne borreliae [133,134] 1965 and 1968 Reviews on LBRF describing that monkeys are susceptible to infection with B. recurrentis, while adult mice and rats have limited susceptibility. Young mice and rats were found to be susceptible [125,135] 1969 and 1971 Reviews retrospectively describing that maintenance and arguably even limited growth of B. recurrentis was achieved in vitro

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Louse-borne relapsing fever -part 1 was only diagnosed because of the microscopic blood smear investigations for malaria conducted due to the patients' recent migration history. They further stated that febrile patients without travel history may receive empirical antibiotic treatment, which may result in the resolution of symptoms without further investigation [60]. Given the susceptibility of borreliae to common empirical antibiotic treatment and the unspecific symptoms of the disease, such minor outbreaks may be easily overlooked. Moreover, vulnerable population groups, such as the homeless, may have limited access to medical care. Under these premises, the introduction of a single case into such surroundings may be sufficient to create a small, unnoticed outbreak. Thus, in regard to LBRF, close attention should be paid to patients from vulnerable population groups, such as migrants or the homeless that display febrile symptoms. Considering asymptomatic cases, publication bias, and possible temporary foci somewhere along migration routes, another reemergence of the disease should neither be neglected nor its epidemic potential be underestimated.
New vector? Interestingly, two studies found DNA of B. recurrentis in head lice obtained from humans in the Republic of Congo and Ethiopia [47,48]. They raise the question whether head lice can transmit human louse-borne pathogens. The evidence suggests that head lice, contrary to former belief, may act as a vector. However, further research is necessary to investigate the role of head lice in the transmission of louse-borne pathogens.

Diagnostic aspects
Microscopy. Microscopy remains the gold standard for diagnosing LBRF since the discovery of the organism in 1867. The sensitivity, however, is directly affected both by the number of spirochetes in the blood and whether the blood sample was taken during a febrile or afebrile period of the infection. Blood should be obtained during a febrile period [19,[36][37][38]124], yet it is possible, though very hard, to find spirochetes during afebrile periods [124]. Although data on sensitivity are scarce, one study reported spirochetes in 38% of patients whose blood had been taken during an afebrile period [155], and another study reported positive microscopy in 5% during afebrile periods [27]. The number of positive results may be increased through the examination of repeated blood smears [36]. Furthermore, the results are often dependant on the fixative and staining method used [25]. Additionally, the level of experience the observer has is another issue that can influence sensitivity [166]. Five cases were identified that reported negative microscopy but positive PCR. In one case, blood was taken the day after initiation of adequate empirical antibiotic therapy [57], and in the other four, reasons remain unknown [69,167]. One study reported positive blood smears, but only after reexamination by an experienced microbiologist [65]. The sensitivity of 70% reported in 1969 was obtained through analysis of both tick-borne and LBRF cases [36], hence may not be fully representative for B. recurrentis specifically. Research into the validation of the enhancement methods is needed and has already been suggested [136].
Animal inoculation, serology, and PCR. Animal vaccination has been used to aid diagnosis, for research purposes and to some extent as a tool to differentiate between TBRF and LBRF. However, animal inoculation has never been routinely used for diagnostic reasons alone. Lack of standardized data and protocol is further limiting this method [25]. Historically, serological methods were extensively investigated, but the development was hampered by antigenetic variability and cross-reactivity [25,27,34,38,[125][126][127][128]. To date, serology is not routinely used for diagnosis and is not recommended. PCR-based methods lack availability in poor countries, however, are unquestionably the best and arguably only means for a certain diagnosis of LBRF. Still, protocols for differentiation of B. recurrentis and B. duttonii need to be established. Due to the proximity of these relapsing fever borreliae, development of specific diagnostic tools and accurate discrimination between the species are challenging [131].
Considering that roughly 90% of all published LBRF cases have been diagnosed by microscopy and that microscopy continues to be the most widely used and available diagnostic method, there is a need to determine its sensitivity and to evaluate how to increase sensitivity by serial investigations and/or enhancement methods. Negative microscopy results should be regarded critically, since the last available sensitivity data from 1969 suggested a sensitivity of 70% on a single blood smear. Positive microscopy should be regarded critically, especially in areas where TBRF is endemic. The data show that microbiological methods were mainly used in Europe. In Ethiopia, the country most affected by the disease, microscopy remains the main diagnostic tool.

Key Learning Points
• Currently, East Africa remains the only endemic focus of louse-borne relapsing fever (LBRF).
• Human migration has repeatedly imported the disease into non-endemic countries.
• Although polymerase chain reaction (PCR)-based methods are the only means of species identification, microscopy remains the gold standard in diagnosing LBRF.

Top Five Papers
Supporting information S1 Checklist. PRISMA Checklist.