- Email: [email protected]
Description of “yaaf”, the vesicular fever caused by acute Rickettsia felis infection in Senegal
Journal of Infection (2013) 66, 536e540
www.elsevierhealth.com/journals/jinf
CASE REPORT
Description of “yaaf”, the vesicular fever caused by acute Rickettsia felis infection in Senegal Oleg Mediannikov a,b, Florence Fenollar a,b, Hubert Bassene b, Adama Tall c, Cheikh Sokhna b, Jean-Franc ¸ois Trape b, Didier Raoult a,b,* a
URMITE UMR 7278, Aix Marseille Universite´, Faculte´ de Me´decine, 27 Bd Jean Moulin, 13005 Marseille, France URMITE IRD 198, Institut de Recherche pour le De´veloppement (IRD), Campus commun UCAD-IRD of Hann, BP 1386, CP 18524 Dakar, Senegal c Institut Pasteur de Dakar, 36 av. Pasteur, BP 220, Dakar, Senegal b
Accepted 7 October 2012 Available online 13 October 2012
KEYWORDS Vesicular fever; Rickettsia felis; Senegal; Primary infection
Summary Rickettsiosis caused by Rickettsia felis is an emerging infection in Africa and may account for 3e4% of ambulatory febrile fevers. We report herein a case of R. felis infection, for which we propose the name “yaaf”, meaning vesicle, in an 8-month-old girl who was diagnosed in the field by real-time PCR analysis of a skin lesion; these PCR analysis was performed at a local experimental point-of-care laboratory. The clinical presentation was polymorphous skin lesions, including papules, vesicles, erosions and ulcers. The patient did not produce antibodies against Rickettsia. We suggest that this disease may be a primary infection caused by R. felis. ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Introduction The aetiologies of ambulatory fevers have been the subject of few investigations. In Africa, most fevers have been considered to be of malarial origin. However, with malaria on the decline, other infectious diseases are posing new public health challenges.1,2 The recent (2010) independent
studies performed by our team in Senegal3 and Kenya4 demonstrated the dramatic incidence of Rickettsia felis infection as a cause of fever in Africa (4.4% and 3.7%, respectively). R. felis infection was thought to be rare, but it may be one of the commonest causes of mild febrile diseases in rural subSaharan Africa. In fact, in the two studied villages in Senegal, the incidence of R. felis infection is higher than that of
* Corresponding author. URMITE UMR, IRD 198, AMU, Faculte ´ de Me ´decine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Tel.: þ33 (0) 4 91 32 43 75; fax: þ33 (0) 4 91 83 03 90. E-mail address: [email protected] (D. Raoult). 0163-4453/$36 ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2012.10.005
Vesicular fever caused by Rickettsia felis malaria.3e7 However, little is known regarding the clinical presentation, geographical distribution, epidemiology (including vectors and reservoirs) and morbidity of this infection. Moreover, most of the reported cases are diagnosed retrospectively5; therefore, the onset and the major symptoms of this infection are unknown because patients are not carefully observed at the time of diagnosis or during treatment. Here, following the establishment of an experimental rural point-of-care laboratory in Senegal, we diagnosed a case of acute infection with R. felis in a child. This case allowed the careful analysis and description of this infection during the course of treatment.
Case report An eight-month-old girl was brought by her mother to the rural dispensary in Ndiop in the Sine-Saloum region of Senegal in November 2011. For several days, the girl had suffered from severe cutaneous eruptions that evolved from small 3e5 mm vesicles to ulcers up to 5 cm in diameter. Her entire body was affected, with the most prominent areas of ulceration being located on her back and right leg. Parents noticed that the girl had never before such lesions. A day before the medical visit, the mother noted high fever and cough. No traditional treatment was used. At admission, the girl had a fever of 38.1 C. A physical examination revealed no abnormalities of the internal organs. The eruptions on the skin were at different stages, including small papules, small vesicles filled with opalescent liquid, superficial erosions and ulcers. The two most prominent areas of erosion were on the patient’s back and right leg. At a nearby point-of-care (POC) laboratory, a thick blood smear was performed, but microscopy revealed no Plasmodium, other parasites or Borrelia. Capillary blood and a swab from a cutaneous lesion were tested at the experimental point-of-care laboratory in the nearby village of Dielmo. There, rapid diagnostic immunochromatographic tests for malaria and dengue fever were negative. Quantitative PCR (qPCR) failed to detect the DNA of Coxiella burnetii, Bartonella spp., Tropheryma whipplei, Borrelia spp., Rickettsia conorii or Rickettsia africae. However, qPCR specific for R. felis was positive for the cutaneous lesion swab but negative for the blood samples. These results were later confirmed in Marseille, France.6 Because this case was the first suspected case of R. felis infection diagnosed in the field at our POC by qPCR analysis of a swab sample from a cutaneous lesion, one of the authors (O.M.) immediately visited the patient to carefully observe the patient and complete a questionnaire, to take photographs of the skin lesions and to obtain parental consent for future inclusion of the case in publications (Fig. 1). Due to the participation of the patient and her mother in a longitudinal project based in Dielmo, monthly serum samples collected prior to the infection were available for this patient.7 We tested the sera of the patient collected at the ages of 5, 7 and 8 months and 40 days after the infection for the presence of antibodies against R. felis by IFA and Western blot analysis.8 All of the sera were
537 negative. The treatment (25 mg doxycycline twice per day for 5 days and paracetamol) was administered and it resulted in the normalisation of the patient’s temperature on the next day. No new vesicles appeared during treatment, and the skin lesions gradually regressed. Local depigmentation and light scars appeared where ulcers had developed.
Methods The DNA from the blood and swab samples was extracted using the BioRobot EZ1 Workstation (Qiagen, Courtaboeuf, France) with a customised extraction protocol following the manufacturer’s instructions. The extracted DNA was stored at 4 C until use in PCR amplifications. We used the following sets of primers and fluorescent probes for conventional and real-time PCR: R. felis-specific sets of primers and probes,3 a Borrelia genus-specific set9; a C. burnetii-specific set of primers and a probe based on the IS1111 repeat sequence10; a 16Se23S internal transcribed spacer (ITS)-based set of qPCR primers and a probe specific for the Bartonella genus; and T. whipplei-specific multiple repeat sequence-based qPCR systems.11 Field qPCR assays were performed at a point-of-care laboratory in Dielmo. Ready-to-use freeze-dried qPCR mixes were prepared in the Laboratory of Molecular Biology in URMITE, Marseille. A total 0.2 M trehalose12 was added to stabilise and enhance the qPCR reaction. Each tube containing the lyophilised mixture contained a sufficient volume for 4 reactions (2 reactions for the patient sample [undiluted and 1/10 diluted] and positive and negative controls). All of the molecular data were confirmed in Marseille6 with freshly prepared mixtures. In both Senegal and France, the reactions were performed using a SmartCycler (Cepheid, Sunnyvale, CA). Fresh master mixes were prepared according to the instructions of the manufacturer. Malaria tests were performed with Core Malaria rapid tests (common antigens for all Plasmodium and Plasmodium vivax- and Plasmodium falciparum-specific antigens) (Core Diagnostics, Birmingham, UK). Assays for dengue fever (ICT for antigens and antibodies) using serum samples were performed with Dengue Duo tests (SD, Korea). The titres of IgG, IgM, and IgA antibodies in serum samples were determined using an indirect immunofluorescence assay with R. felis California 2 strain antigens generated in-house. All of the serum samples were diluted at ratios of 1:25, 1:50 and 1:100 and screened for total immunoglobulin. WB was performed as described elsewhere.13
Ethic statement Written informed consent was obtained from the patient mother for the use of picture. Our project was initially approved by the Ministry of Health of Senegal and the assembled village population. Approval was then renewed on a yearly basis. Audits were done regularly by the National Ethics Committee of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institute (Dakar, Senegal), and the Institut de Recherche pour le Developpement (Marseille, France).
538
Figure 1
O. Mediannikov et al.
Skin lesions of the patient with a primary infection with R. felis. a e general appearance; b e ulcerative lesions; c e vesicles.
Discussion R. felis is an obligate intracellular gram-negative bacterium belonging to the spotted fever group (SFG) of Rickettsia. The species R. felis was formally validated using molecular criteria in 2001, and the reference strain was isolated and definitively characterised in 2002.14 Since the definitive description of R. felis, the interest in this pathogen and its association with arthropods has increased. The use of molecular assays, including regular and quantitative real-time polymerase chain reaction (qPCR), allows the rapid and reproducible detection of R. felis in arthropods and human samples throughout the world, but the facilities and equipment required to perform these assays are not available everywhere and are particularly scarce in developing countries. R. felis has been found in arthropods, including Anopheles mosquitoes (GenBank accession number JN620082), from more than 20 countries on five continents, cats’ and dogs’ fleas of Ctenocephalides genus are generally considered as vectors.15,16 The first evidence of the pathogenicity of R. felis in humans was published in 1994, when R. felis DNA fragments were detected in blood samples obtained from a patient in Texas in 199117 and from other patients in Mexico.15,18 Indeed, in one of three cases diagnosed by PCR in Mexico, the clinical presentation of the patient closely resembled that of our case, with a rash consisting of papules, furuncles and necrotic lesions. Unfortunately, no sequence analysis or specific molecular test can confirm this pioneering study. Since then, approximately 100 isolated cases or small clusters of cases have been reported worldwide, including in Africa.15 The reported clinical features are unremarkable
and include fever, marked fatigue, and headache and sometimes generalised maculopapular rash and a cutaneous eschar. Rarely, neurological signs (including polyneuropathylike syndrome and subacute meningitis), digestive symptoms, cough and pneumonia have been reported. In Senegal, our team investigated the origin of fever in Senegalese patients who tested negative for malaria.3,9,11,19 The prevalence of R. felis infection in the tested samples was 4.4%dhigher than the prevalence of malaria.7 In addition to fever and nonspecific symptoms (weakness, headache with sleep disorders, and digestive and respiratory signs), no rashes or eschars were found in infected Senegalese patients.3 Surprisingly, we identified a patient who had suffered two successive episodes of infection associated with R. felis, showing that patients may relapse or be reinfected. In Kenya, a similar study has been conducted by another team that aimed to determine the aetiologic agents of non-malaria acute febrile illnesses in the semi-arid North Eastern Province of Kenya during an inter-epidemic period for Rift Valley fever that lasted 23 months during 2006e2008. A total of 163 patients with fever were included. Two quantitative real-time PCR (qPCR) assays were used to screen for rickettsial nucleic acids in the patients’ serum samples. An R. felis-specific qPCR was assay also used, and six patients were subsequently confirmed by molecular sequencing. The symptoms included headache, nausea, and muscle, back and joint pain. None of the patients reported skin rash, and no information regarding possible eschars was available. These results were the first evidence of R. felis infection in humans in Kenya and the first such evidence from eastern sub-Saharan Africa.4
Vesicular fever caused by Rickettsia felis These strikingly similar case series revealed the high incidence of R. felis infection in sub-Saharan Africa. The clinical presentation described in these two series is vague and lacks details. Patients did not present with rash or eschars, clinical signs typical of rickettsioses, in either Senegal or Kenya. Indeed, in all of these cases, the diagnoses were performed post factum, and the clinical data analysed were acquired from questionnaires. The high incidence of R. felis infection in these places together with one reported case presenting a second episode of documented infection3 suggests that patients may suffer severe episodes of R. felis infection (relapse or reinfection). The case presented herein is unique because it represents a rare case of R. felis infection in which the diagnosis was made several hours after admission to a dispensary. The young age of the patient (8 months) suggests that this infection was primary. The absence of rickettsial antibodies in patients with PCR-diagnosed R. felis infection, as observed here, has been repeatedly reported.18e20 This absence of antibodies may explain why some people relapse (as for borrelioses) or are reinfected. Moreover, in this particular case the early treatment could prevent the development of the full immune response. Another explanation may be that the only available strain of R. felis originates from fleas; no clinical isolates exist up to date. The antigenic differences among human and vector strains may also play role in the absence of rickettsial antibodies in R. felis infection. A clinical description of primary infection by R. felis is not available in the literature, except for one case18 with similar cutaneous lesions described in Yucatan, Mexico, and most likely, the presented clinical characteristics of specific cutaneous lesions may be pathognomonic. In the previous work we reported R. felis infection as “uneruptive fever”3; however, in this work the clinical description was based on the case report forms that may lack precision. Moreover, the typical skin lesions may be the symptom of the primary infection in children; the secondary infections may be milder and without skin eruptions. Indeed, having discussed this case and shared the pictures with the elders from Senegalese villages (Ndiop, Dielmo and Niakhar), we found that this type of skin lesion is not rare and is observed sporadically but regularly in young children, usually at the end of the rainy season (AugusteNovember). The disease has no particular name in the local languages; therefore, we decided to call it “yaaf”, which means vesicle in Wolof (the language of most Senegalese). Interestingly, that previously performed epidemiological studies in the same village failed to identify the R. felis in the Ctenocephalides fleas, considered as the main vector of this infection.21 The high prevalence of R. felis infection in this region and the absence of this bacterium in fleas suggest that it may be transmitted by another vector. We were able to perform the patient observation described herein due to the ability to diagnose a spectrum of infectious diseases in a field rural dispensary directly after the admission of the patient. The clinical and economic efficacy of the POC22 approach has already been proven for large hospitals; here, the POC approach serves a role in local healthcare and for medical researchers. We are currently experimenting with its role in the diagnosis of mild fevers in Africa.
539 Overall, this case of R. felis infection presents a unique clinical picture. This infection may correspond to a primary infection with R. felis, which had not been described in Africa previously. The Wolof word “yaaf” is proposed to identify this clinical entity.
Financial support Supported by the Agence National de Recherche (ANR) grant 2010 MALEMAF (Research of Emergent Pathogens in Africa).
Potential conflict of interest No potential conflict of interest relevant to this article was reported.
Acknowledgements We thank all of the participants in the Dielmo project for their collaboration and Aliou Diallo for technical assistance.
References 1. Gething PW, Kirui VC, Alegana VA, Okiro EA, Noor AM, Snow RW. Estimating the number of paediatric fevers associated with malaria infection presenting to Africa’s public health sector in 2007. PLoS Med 2010;7(7):e1000301. 2. Amexo M, Tolhurst R, Barnish G, Bates I. Malaria misdiagnosis: effects on the poor and vulnerable. Lancet 2004;364(9448): 1896e8. 3. Socolovschi C, Mediannikov O, Sokhna C, Tall A, Diatta G, Bassene H, et al. Rickettsia felis-associated uneruptive fever, Senegal. Emerg Infect Dis 2010;16(7):1140e2. 4. Richards AL, Jiang J, Omulo S, Dare R, Abdirahman K, Ali A, et al. Human infection with Rickettsia felis, Kenya. Emerg Infect Dis 2010;16(7):1081e6. 5. Renvoise A, Joliot AY, Raoult D. Rickettsia felis infection in man, France. Emerg Infect Dis 2009;15(7):1126e7. 6. Renvoise A, Rolain JM, Socolovschi C, Raoult D. Widespread use of real-time PCR for rickettsial diagnosis. FEMS Immunol Med Microbiol 2012;64(1):126e9. 7. Trape JF, Tall A, Diagne N, Ndiath O, Ly AB, Faye J, et al. Malaria morbidity and pyrethroid resistance after the introduction of insecticide-treated bednets and artemisinin-based combination therapies: a longitudinal study. Lancet Infect Dis 2011; 11(12):925e32. 8. Perez-Arellano JL, Fenollar F, Angel-Moreno A, Bolanos M, Hernandez M, Santana E, et al. Human Rickettsia felis infection, Canary Islands, Spain. Emerg Infect Dis 2005;11(12):1961e4. 9. Parola P, Diatta G, Socolovschi C, Mediannikov O, Tall A, Bassene H, et al. Tick-borne relapsing fever borreliosis, rural Senegal. Emerg Infect Dis 2011;17(5):883e5. 10. Mediannikov O, Fenollar F, Socolovschi C, Diatta G, Bassene H, Molez JF, et al. Coxiella burnetii in humans and ticks in rural Senegal. PLoS Negl Trop Dis 2010;4(4):e654. 11. Fenollar F, Mediannikov O, Socolovschi C, Bassene H, Diatta G, Richet H, et al. Tropheryma whipplei bacteremia during fever in rural West Africa. Clin Infect Dis 2010;51(5):515e21. 12. Spiess AN, Mueller N, Ivell R. Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem 2004;50(7):1256e9.
540 13. Raoult D, Dasch GA. The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J Immunol Methods 1989;125(1e2): 57e65. 14. La Scola B, Meconi S, Fenollar F, Rolain JM, Roux V, Raoult D. Emended description of Rickettsia felis (Bouyer et al. 2001), a temperature-dependent cultured bacterium. Int J Syst Evol Microbiol 2002;52(Pt 6):2035e41. 15. Parola P. Rickettsia felis: from a rare disease in the USA to a common cause of fever in sub-Saharan Africa. Clin Microbiol Infect 2011;17(7):996e1000. 16. Abdad MY, Stenos J, Graves S. Rickettsia felis, an emerging fleatransmitted human pathogen. Emerg Health Threats J 2011; 4(7168):1e7. 17. Schriefer ME, Sacci Jr JB, Dumler JS, Bullen MG, Azad AF. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol 1994;32(4):949e54.
O. Mediannikov et al. 18. Zavala-Velazquez JE, Ruiz-Sosa JA, Sanchez-Elias RA, BecerraCarmona G, Walker DH. Rickettsia felis rickettsiosis in Yucatan. Lancet 2000;356(9235):1079e80. 19. Mediannikov O, Diatta G, Fenollar F, Sokhna C, Trape JF, Raoult D. Tick-borne rickettsioses, neglected emerging diseases in rural Senegal. PLoS Negl Trop Dis 2010;4(9). 20. Zavala-Velazquez JE, Yu XJ, Walker DH. Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico. Am J Trop Med Hyg 1996;55(2):157e9. 21. Roucher C, Mediannikov O, Diatta G, Trape JF, Raoult D. A new Rickettsia species found in fleas collected from human dwellings and from domestic cats and dogs in Senegal. Vector Borne Zoonotic Dis 2012;12:360e5. 22. Cohen-Bacrie S, Ninove L, Nougairede A, Charrel R, Richet H, Minodier P, et al. Revolutionizing clinical microbiology laboratory organization in hospitals with in situ point-of-care. PLoS One 2011;6(7):e22403.
www.elsevierhealth.com/journals/jinf
CASE REPORT
Description of “yaaf”, the vesicular fever caused by acute Rickettsia felis infection in Senegal Oleg Mediannikov a,b, Florence Fenollar a,b, Hubert Bassene b, Adama Tall c, Cheikh Sokhna b, Jean-Franc ¸ois Trape b, Didier Raoult a,b,* a
URMITE UMR 7278, Aix Marseille Universite´, Faculte´ de Me´decine, 27 Bd Jean Moulin, 13005 Marseille, France URMITE IRD 198, Institut de Recherche pour le De´veloppement (IRD), Campus commun UCAD-IRD of Hann, BP 1386, CP 18524 Dakar, Senegal c Institut Pasteur de Dakar, 36 av. Pasteur, BP 220, Dakar, Senegal b
Accepted 7 October 2012 Available online 13 October 2012
KEYWORDS Vesicular fever; Rickettsia felis; Senegal; Primary infection
Summary Rickettsiosis caused by Rickettsia felis is an emerging infection in Africa and may account for 3e4% of ambulatory febrile fevers. We report herein a case of R. felis infection, for which we propose the name “yaaf”, meaning vesicle, in an 8-month-old girl who was diagnosed in the field by real-time PCR analysis of a skin lesion; these PCR analysis was performed at a local experimental point-of-care laboratory. The clinical presentation was polymorphous skin lesions, including papules, vesicles, erosions and ulcers. The patient did not produce antibodies against Rickettsia. We suggest that this disease may be a primary infection caused by R. felis. ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Introduction The aetiologies of ambulatory fevers have been the subject of few investigations. In Africa, most fevers have been considered to be of malarial origin. However, with malaria on the decline, other infectious diseases are posing new public health challenges.1,2 The recent (2010) independent
studies performed by our team in Senegal3 and Kenya4 demonstrated the dramatic incidence of Rickettsia felis infection as a cause of fever in Africa (4.4% and 3.7%, respectively). R. felis infection was thought to be rare, but it may be one of the commonest causes of mild febrile diseases in rural subSaharan Africa. In fact, in the two studied villages in Senegal, the incidence of R. felis infection is higher than that of
* Corresponding author. URMITE UMR, IRD 198, AMU, Faculte ´ de Me ´decine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Tel.: þ33 (0) 4 91 32 43 75; fax: þ33 (0) 4 91 83 03 90. E-mail address: [email protected] (D. Raoult). 0163-4453/$36 ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2012.10.005
Vesicular fever caused by Rickettsia felis malaria.3e7 However, little is known regarding the clinical presentation, geographical distribution, epidemiology (including vectors and reservoirs) and morbidity of this infection. Moreover, most of the reported cases are diagnosed retrospectively5; therefore, the onset and the major symptoms of this infection are unknown because patients are not carefully observed at the time of diagnosis or during treatment. Here, following the establishment of an experimental rural point-of-care laboratory in Senegal, we diagnosed a case of acute infection with R. felis in a child. This case allowed the careful analysis and description of this infection during the course of treatment.
Case report An eight-month-old girl was brought by her mother to the rural dispensary in Ndiop in the Sine-Saloum region of Senegal in November 2011. For several days, the girl had suffered from severe cutaneous eruptions that evolved from small 3e5 mm vesicles to ulcers up to 5 cm in diameter. Her entire body was affected, with the most prominent areas of ulceration being located on her back and right leg. Parents noticed that the girl had never before such lesions. A day before the medical visit, the mother noted high fever and cough. No traditional treatment was used. At admission, the girl had a fever of 38.1 C. A physical examination revealed no abnormalities of the internal organs. The eruptions on the skin were at different stages, including small papules, small vesicles filled with opalescent liquid, superficial erosions and ulcers. The two most prominent areas of erosion were on the patient’s back and right leg. At a nearby point-of-care (POC) laboratory, a thick blood smear was performed, but microscopy revealed no Plasmodium, other parasites or Borrelia. Capillary blood and a swab from a cutaneous lesion were tested at the experimental point-of-care laboratory in the nearby village of Dielmo. There, rapid diagnostic immunochromatographic tests for malaria and dengue fever were negative. Quantitative PCR (qPCR) failed to detect the DNA of Coxiella burnetii, Bartonella spp., Tropheryma whipplei, Borrelia spp., Rickettsia conorii or Rickettsia africae. However, qPCR specific for R. felis was positive for the cutaneous lesion swab but negative for the blood samples. These results were later confirmed in Marseille, France.6 Because this case was the first suspected case of R. felis infection diagnosed in the field at our POC by qPCR analysis of a swab sample from a cutaneous lesion, one of the authors (O.M.) immediately visited the patient to carefully observe the patient and complete a questionnaire, to take photographs of the skin lesions and to obtain parental consent for future inclusion of the case in publications (Fig. 1). Due to the participation of the patient and her mother in a longitudinal project based in Dielmo, monthly serum samples collected prior to the infection were available for this patient.7 We tested the sera of the patient collected at the ages of 5, 7 and 8 months and 40 days after the infection for the presence of antibodies against R. felis by IFA and Western blot analysis.8 All of the sera were
537 negative. The treatment (25 mg doxycycline twice per day for 5 days and paracetamol) was administered and it resulted in the normalisation of the patient’s temperature on the next day. No new vesicles appeared during treatment, and the skin lesions gradually regressed. Local depigmentation and light scars appeared where ulcers had developed.
Methods The DNA from the blood and swab samples was extracted using the BioRobot EZ1 Workstation (Qiagen, Courtaboeuf, France) with a customised extraction protocol following the manufacturer’s instructions. The extracted DNA was stored at 4 C until use in PCR amplifications. We used the following sets of primers and fluorescent probes for conventional and real-time PCR: R. felis-specific sets of primers and probes,3 a Borrelia genus-specific set9; a C. burnetii-specific set of primers and a probe based on the IS1111 repeat sequence10; a 16Se23S internal transcribed spacer (ITS)-based set of qPCR primers and a probe specific for the Bartonella genus; and T. whipplei-specific multiple repeat sequence-based qPCR systems.11 Field qPCR assays were performed at a point-of-care laboratory in Dielmo. Ready-to-use freeze-dried qPCR mixes were prepared in the Laboratory of Molecular Biology in URMITE, Marseille. A total 0.2 M trehalose12 was added to stabilise and enhance the qPCR reaction. Each tube containing the lyophilised mixture contained a sufficient volume for 4 reactions (2 reactions for the patient sample [undiluted and 1/10 diluted] and positive and negative controls). All of the molecular data were confirmed in Marseille6 with freshly prepared mixtures. In both Senegal and France, the reactions were performed using a SmartCycler (Cepheid, Sunnyvale, CA). Fresh master mixes were prepared according to the instructions of the manufacturer. Malaria tests were performed with Core Malaria rapid tests (common antigens for all Plasmodium and Plasmodium vivax- and Plasmodium falciparum-specific antigens) (Core Diagnostics, Birmingham, UK). Assays for dengue fever (ICT for antigens and antibodies) using serum samples were performed with Dengue Duo tests (SD, Korea). The titres of IgG, IgM, and IgA antibodies in serum samples were determined using an indirect immunofluorescence assay with R. felis California 2 strain antigens generated in-house. All of the serum samples were diluted at ratios of 1:25, 1:50 and 1:100 and screened for total immunoglobulin. WB was performed as described elsewhere.13
Ethic statement Written informed consent was obtained from the patient mother for the use of picture. Our project was initially approved by the Ministry of Health of Senegal and the assembled village population. Approval was then renewed on a yearly basis. Audits were done regularly by the National Ethics Committee of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institute (Dakar, Senegal), and the Institut de Recherche pour le Developpement (Marseille, France).
538
Figure 1
O. Mediannikov et al.
Skin lesions of the patient with a primary infection with R. felis. a e general appearance; b e ulcerative lesions; c e vesicles.
Discussion R. felis is an obligate intracellular gram-negative bacterium belonging to the spotted fever group (SFG) of Rickettsia. The species R. felis was formally validated using molecular criteria in 2001, and the reference strain was isolated and definitively characterised in 2002.14 Since the definitive description of R. felis, the interest in this pathogen and its association with arthropods has increased. The use of molecular assays, including regular and quantitative real-time polymerase chain reaction (qPCR), allows the rapid and reproducible detection of R. felis in arthropods and human samples throughout the world, but the facilities and equipment required to perform these assays are not available everywhere and are particularly scarce in developing countries. R. felis has been found in arthropods, including Anopheles mosquitoes (GenBank accession number JN620082), from more than 20 countries on five continents, cats’ and dogs’ fleas of Ctenocephalides genus are generally considered as vectors.15,16 The first evidence of the pathogenicity of R. felis in humans was published in 1994, when R. felis DNA fragments were detected in blood samples obtained from a patient in Texas in 199117 and from other patients in Mexico.15,18 Indeed, in one of three cases diagnosed by PCR in Mexico, the clinical presentation of the patient closely resembled that of our case, with a rash consisting of papules, furuncles and necrotic lesions. Unfortunately, no sequence analysis or specific molecular test can confirm this pioneering study. Since then, approximately 100 isolated cases or small clusters of cases have been reported worldwide, including in Africa.15 The reported clinical features are unremarkable
and include fever, marked fatigue, and headache and sometimes generalised maculopapular rash and a cutaneous eschar. Rarely, neurological signs (including polyneuropathylike syndrome and subacute meningitis), digestive symptoms, cough and pneumonia have been reported. In Senegal, our team investigated the origin of fever in Senegalese patients who tested negative for malaria.3,9,11,19 The prevalence of R. felis infection in the tested samples was 4.4%dhigher than the prevalence of malaria.7 In addition to fever and nonspecific symptoms (weakness, headache with sleep disorders, and digestive and respiratory signs), no rashes or eschars were found in infected Senegalese patients.3 Surprisingly, we identified a patient who had suffered two successive episodes of infection associated with R. felis, showing that patients may relapse or be reinfected. In Kenya, a similar study has been conducted by another team that aimed to determine the aetiologic agents of non-malaria acute febrile illnesses in the semi-arid North Eastern Province of Kenya during an inter-epidemic period for Rift Valley fever that lasted 23 months during 2006e2008. A total of 163 patients with fever were included. Two quantitative real-time PCR (qPCR) assays were used to screen for rickettsial nucleic acids in the patients’ serum samples. An R. felis-specific qPCR was assay also used, and six patients were subsequently confirmed by molecular sequencing. The symptoms included headache, nausea, and muscle, back and joint pain. None of the patients reported skin rash, and no information regarding possible eschars was available. These results were the first evidence of R. felis infection in humans in Kenya and the first such evidence from eastern sub-Saharan Africa.4
Vesicular fever caused by Rickettsia felis These strikingly similar case series revealed the high incidence of R. felis infection in sub-Saharan Africa. The clinical presentation described in these two series is vague and lacks details. Patients did not present with rash or eschars, clinical signs typical of rickettsioses, in either Senegal or Kenya. Indeed, in all of these cases, the diagnoses were performed post factum, and the clinical data analysed were acquired from questionnaires. The high incidence of R. felis infection in these places together with one reported case presenting a second episode of documented infection3 suggests that patients may suffer severe episodes of R. felis infection (relapse or reinfection). The case presented herein is unique because it represents a rare case of R. felis infection in which the diagnosis was made several hours after admission to a dispensary. The young age of the patient (8 months) suggests that this infection was primary. The absence of rickettsial antibodies in patients with PCR-diagnosed R. felis infection, as observed here, has been repeatedly reported.18e20 This absence of antibodies may explain why some people relapse (as for borrelioses) or are reinfected. Moreover, in this particular case the early treatment could prevent the development of the full immune response. Another explanation may be that the only available strain of R. felis originates from fleas; no clinical isolates exist up to date. The antigenic differences among human and vector strains may also play role in the absence of rickettsial antibodies in R. felis infection. A clinical description of primary infection by R. felis is not available in the literature, except for one case18 with similar cutaneous lesions described in Yucatan, Mexico, and most likely, the presented clinical characteristics of specific cutaneous lesions may be pathognomonic. In the previous work we reported R. felis infection as “uneruptive fever”3; however, in this work the clinical description was based on the case report forms that may lack precision. Moreover, the typical skin lesions may be the symptom of the primary infection in children; the secondary infections may be milder and without skin eruptions. Indeed, having discussed this case and shared the pictures with the elders from Senegalese villages (Ndiop, Dielmo and Niakhar), we found that this type of skin lesion is not rare and is observed sporadically but regularly in young children, usually at the end of the rainy season (AugusteNovember). The disease has no particular name in the local languages; therefore, we decided to call it “yaaf”, which means vesicle in Wolof (the language of most Senegalese). Interestingly, that previously performed epidemiological studies in the same village failed to identify the R. felis in the Ctenocephalides fleas, considered as the main vector of this infection.21 The high prevalence of R. felis infection in this region and the absence of this bacterium in fleas suggest that it may be transmitted by another vector. We were able to perform the patient observation described herein due to the ability to diagnose a spectrum of infectious diseases in a field rural dispensary directly after the admission of the patient. The clinical and economic efficacy of the POC22 approach has already been proven for large hospitals; here, the POC approach serves a role in local healthcare and for medical researchers. We are currently experimenting with its role in the diagnosis of mild fevers in Africa.
539 Overall, this case of R. felis infection presents a unique clinical picture. This infection may correspond to a primary infection with R. felis, which had not been described in Africa previously. The Wolof word “yaaf” is proposed to identify this clinical entity.
Financial support Supported by the Agence National de Recherche (ANR) grant 2010 MALEMAF (Research of Emergent Pathogens in Africa).
Potential conflict of interest No potential conflict of interest relevant to this article was reported.
Acknowledgements We thank all of the participants in the Dielmo project for their collaboration and Aliou Diallo for technical assistance.
References 1. Gething PW, Kirui VC, Alegana VA, Okiro EA, Noor AM, Snow RW. Estimating the number of paediatric fevers associated with malaria infection presenting to Africa’s public health sector in 2007. PLoS Med 2010;7(7):e1000301. 2. Amexo M, Tolhurst R, Barnish G, Bates I. Malaria misdiagnosis: effects on the poor and vulnerable. Lancet 2004;364(9448): 1896e8. 3. Socolovschi C, Mediannikov O, Sokhna C, Tall A, Diatta G, Bassene H, et al. Rickettsia felis-associated uneruptive fever, Senegal. Emerg Infect Dis 2010;16(7):1140e2. 4. Richards AL, Jiang J, Omulo S, Dare R, Abdirahman K, Ali A, et al. Human infection with Rickettsia felis, Kenya. Emerg Infect Dis 2010;16(7):1081e6. 5. Renvoise A, Joliot AY, Raoult D. Rickettsia felis infection in man, France. Emerg Infect Dis 2009;15(7):1126e7. 6. Renvoise A, Rolain JM, Socolovschi C, Raoult D. Widespread use of real-time PCR for rickettsial diagnosis. FEMS Immunol Med Microbiol 2012;64(1):126e9. 7. Trape JF, Tall A, Diagne N, Ndiath O, Ly AB, Faye J, et al. Malaria morbidity and pyrethroid resistance after the introduction of insecticide-treated bednets and artemisinin-based combination therapies: a longitudinal study. Lancet Infect Dis 2011; 11(12):925e32. 8. Perez-Arellano JL, Fenollar F, Angel-Moreno A, Bolanos M, Hernandez M, Santana E, et al. Human Rickettsia felis infection, Canary Islands, Spain. Emerg Infect Dis 2005;11(12):1961e4. 9. Parola P, Diatta G, Socolovschi C, Mediannikov O, Tall A, Bassene H, et al. Tick-borne relapsing fever borreliosis, rural Senegal. Emerg Infect Dis 2011;17(5):883e5. 10. Mediannikov O, Fenollar F, Socolovschi C, Diatta G, Bassene H, Molez JF, et al. Coxiella burnetii in humans and ticks in rural Senegal. PLoS Negl Trop Dis 2010;4(4):e654. 11. Fenollar F, Mediannikov O, Socolovschi C, Bassene H, Diatta G, Richet H, et al. Tropheryma whipplei bacteremia during fever in rural West Africa. Clin Infect Dis 2010;51(5):515e21. 12. Spiess AN, Mueller N, Ivell R. Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem 2004;50(7):1256e9.
540 13. Raoult D, Dasch GA. The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J Immunol Methods 1989;125(1e2): 57e65. 14. La Scola B, Meconi S, Fenollar F, Rolain JM, Roux V, Raoult D. Emended description of Rickettsia felis (Bouyer et al. 2001), a temperature-dependent cultured bacterium. Int J Syst Evol Microbiol 2002;52(Pt 6):2035e41. 15. Parola P. Rickettsia felis: from a rare disease in the USA to a common cause of fever in sub-Saharan Africa. Clin Microbiol Infect 2011;17(7):996e1000. 16. Abdad MY, Stenos J, Graves S. Rickettsia felis, an emerging fleatransmitted human pathogen. Emerg Health Threats J 2011; 4(7168):1e7. 17. Schriefer ME, Sacci Jr JB, Dumler JS, Bullen MG, Azad AF. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol 1994;32(4):949e54.
O. Mediannikov et al. 18. Zavala-Velazquez JE, Ruiz-Sosa JA, Sanchez-Elias RA, BecerraCarmona G, Walker DH. Rickettsia felis rickettsiosis in Yucatan. Lancet 2000;356(9235):1079e80. 19. Mediannikov O, Diatta G, Fenollar F, Sokhna C, Trape JF, Raoult D. Tick-borne rickettsioses, neglected emerging diseases in rural Senegal. PLoS Negl Trop Dis 2010;4(9). 20. Zavala-Velazquez JE, Yu XJ, Walker DH. Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico. Am J Trop Med Hyg 1996;55(2):157e9. 21. Roucher C, Mediannikov O, Diatta G, Trape JF, Raoult D. A new Rickettsia species found in fleas collected from human dwellings and from domestic cats and dogs in Senegal. Vector Borne Zoonotic Dis 2012;12:360e5. 22. Cohen-Bacrie S, Ninove L, Nougairede A, Charrel R, Richet H, Minodier P, et al. Revolutionizing clinical microbiology laboratory organization in hospitals with in situ point-of-care. PLoS One 2011;6(7):e22403.