• View in gallery

    Prominent maculopapular rash on the patient’s back.

  • View in gallery

    Western immunoblotting of the patient’s serum before and after cross-adsorption. Molecular markers are shown at the left in kilodaltons (kDa). Untreated serum reacted strongly with the 130 kDa R. conorii antigen but weakly with the 130 kDa R. africae antigen. Subsequently, serum adsorbed with R. conorii antigens did not react with either of the R. conorii and R. africae antigens, whereas that adsorbed with R. africae antigens still retained reactivity with the R. conorii antigen, indicating that the patient’s antibodies were produced by R. conorii but not R. africae infection. (A) Untreated serum tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens. (B) Serum adsorbed with R. conorii antigens and tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens. (C) Serum adsorbed with R. africae antigens and tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens.

  • 1

    Jensenius M, Fournier P-E, Kelly P, Myrvang B, Raoult D, 2003. African tick bite fever. Lancet Infect Dis 3 :557–564.

  • 2

    Kelly P, Matthewman L, Beati L, Raoult D, Mason P, Dreary M, Makombe R, 1992. African tick-bite fever: a new spotted fever group rickettsiosis under an old name. Lancet 340 :982–983.

    • Search Google Scholar
    • Export Citation
  • 3

    Furuya Y, Yamamoto S, Otu M, Yoshida Y, Ohashi N, Murata M, Kawabata N, Tamura A, Kawamura A Jr, 1991. Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay. J Clin Microbiol 29 :340–345.

    • Search Google Scholar
    • Export Citation
  • 4

    Amano K, Suzuki N, Fujita M, Nakamura Y, Suto T, 1993. Serological reactivity of sera from scrub typhus patients against Weil-Felix test antigens. Microbiol Immunol 37 :927–933.

    • Search Google Scholar
    • Export Citation
  • 5

    Fournier P-E, Jensenius M, Laferl H, Vene S, Raoult D, 2002. Kinetics of antibody responses in Rickettsia africae and Rickettsia conorii infections. Clin Diagn Lab Immunol 9 :324–328.

    • Search Google Scholar
    • Export Citation
  • 6

    Parola P, Miller RS, McDaniel P, Telford SR III, Rolain J-M, Wongsrichanalai C, Raoult D, 2003. Emerging rickettsioses of the Thai-Myanmar border. Emerg Infect Dis 9 :592–595.

    • Search Google Scholar
    • Export Citation
  • 7

    La Scola B, Raoult D, 1997. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 35 :2715–2727.

    • Search Google Scholar
    • Export Citation
  • 8

    Raoult D, Dasch GA, 1989. Line blot and Western blot immunoassays for diagnosis of Mediterranean spotted fever. J Clin Microbiol 27 :2073–2079.

    • Search Google Scholar
    • Export Citation
  • 9

    Jelinek T, Löscher T, 2001. Clinical features and epidemiology of tick typhus in travelers. J Travel Med 8 :57–59.

  • 10

    McQuiston JH, Paddock CD, Singleton J Jr, Wheeling JT, Zaki SR, Childs JE, 2004. Imported spotted fever rickettsioses in United States travelers returning from Africa: a summary of cases confirmed by laboratory testing at the Centers for Disease Control and Prevention, 1999–2002. Am J Trop Med Hyg 70 :98–101.

    • Search Google Scholar
    • Export Citation
  • 11

    Kimura M, Fujii T, Iwamoto A, 1998. Two cases of spotted fever group rickettsiosis contracted in southern parts of Africa (abstract in English). Kansenshogaku Zasshi 72 :1311–1316.

    • Search Google Scholar
    • Export Citation
  • 12

    Raoult D, Fournier PE, Fenollar F, Jensenius M, Prioe T, de Pina JJ, Caruso G, Jones N, Laferl H, Rosenblatt JE, Marrie TJ, 2001. Rickettsia africae, a tick-borne pathogen in travelers to sub-Saharan Africa. N Engl J Med 344 :1504–1510.

    • Search Google Scholar
    • Export Citation
  • 13

    Raoult D, Weiller PJ, Chagnon A, Chaudet H, Gallais H, Casanova P, 1986. Mediterranean spotted fever: clinical, laboratory and epidemiological features of 199 cases. Am J Trop Med Hyg 35 :845–850.

    • Search Google Scholar
    • Export Citation
  • 14

    Amaro M, Bacellar F, França A, 2003. Report of eight cases of fatal and severe Mediterranean spotted fever in Portugal. Ann N Y Acad Sci 990 :331–343.

    • Search Google Scholar
    • Export Citation
  • 15

    Antón E, Font B, Muñoz T, Sanfeliu I, Segura F, 2003. Clinical and laboratory characteristics of 144 patients with Mediterranean spotted fever. Eur J Clin Microbiol Infect Dis 22 :126–128.

    • Search Google Scholar
    • Export Citation
  • 16

    Teysseire N, Raoult D, 1992. Comparison of Western immunoblotting and microimmunofluorescence for diagnosis of Mediterranean spotted fever. J Clin Microbiol 30 :455–460.

    • Search Google Scholar
    • Export Citation

 

 

 

 

LABORATORY-CONFIRMED MEDITERRANEAN SPOTTED FEVER IN A JAPANESE TRAVELER TO KENYA

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  • 1 Department of Infectious Diseases, Niigata City General Hospital, Niigata, Japan; Infectious Disease Surveillance Center and Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan; Unité des Rickettsies, CNRS UMR6020, Faculté de Médecine, Université de la Méditerranée, Marseilles, France

A Japanese traveler returning from Kenya became ill, presenting with fever and a prominent, generalized rash without an eschar. Results of the immunofluorescence antibody assay of the patient’s sera performed in Japan were compatible with illness due to a spotted fever group (SFG) rickettsia, and a presumptive diagnosis of African SFG rickettsiosis, probably either Mediterranean spotted fever (MSF) or African tick-bite fever (ATBF), was rendered. To further define the disease diagnosis, sera were examined in France by Western immunoblotting combined with cross-adsorption, which confirmed the diagnosis of MSF but not of ATBF. Because of the need to further characterize the epidemiologic and clinical features of the two African SFG rickettsioses, clinicians are encouraged to contact a specialized laboratory when encountering such cases.

INTRODUCTION

Recently, rickettsioses are increasingly highlighted as a significant health risk for international travelers. However, their clinical diagnosis tends to be missed by physicians due to lack of awareness, especially in cases without an inoculation eschar or a skin rash. Another problem is the paucity of specialized laboratories that can confirm the causative rickettsiae, microorganisms characterized by prominent cross-reactivity. Mediterranean spotted fever (MSF), also known as boutonneuse fever, is a spotted fever group (SFG) rickettsiosis caused and transmitted by Rickettsia conorii and Rhipicephalus species (dog ticks), respectively. It is prevalent in Mediterranean countries including France, Spain, Portugal, Italy, and Tunisia. Furthermore, the geographical distribution extends even to sub-Saharan Africa, the Middle East, and the Indian subcontinent. However, cases contracted in southern Africa, especially in South Africa and Zimbabwe, tend to show somewhat distinctive clinical features from those of classic MSF cases; that is, milder clinical course, multiple eschars, regional lymphadenopathy, but often no rash.1 These cases were regarded as a rural form of MSF caused by a different SFG rickettsia.

In 1992, a new SFG rickettsia was identified in a patient showing the above milder disease and was named Rickettsia africae.2 This has led to a new SFG rickettsiosis entity, African tick-bite fever (ATBF), which is transmitted by Amblyomma species, ticks found on cattle and wild game. Because R. conorii cross-reacts with R. africae in immunofluorescence antibody assay (IFA), some or even many of the previously reported MSF cases might have been ATBF. Since then, differentiation between these two rickettsioses has been encouraged to gain insights into the epidemiology, clinical features, and appropriate therapies of the two diseases among natives as well as travelers. Herein, we report a Japanese traveler returning from Kenya with fever, a prominent rash, but no eschar. The patient was first suspected of having an African SFG rickettsiosis by IFA and was subsequently confirmed as MSF by Western immunoblotting combined with cross-adsorption.

CASE REPORT

In October 2002, a 52-year-old Japanese male presented at our (H.Y.) clinic with a 4-day history of chills, shivering, fever (40°C), general fatigue, generalized maculopapular rash, and muscle weakness. His initial symptom of chills started 5 days after a 9-day travel to Kenya to visit lakes as well as for a safari drive in national parks as part of an organized tour. He had used a repellent spray but had noticed neither insect bites on his body nor dogs around him. His maculopapular rash first appeared on the forearms and extended to the whole body but spared palms and soles (Figure 1). Besides the generalized rash, physical examination did not reveal any eschars, lymph node enlargement, or hepatosplenomegaly. Laboratory analyses showed the following values: hemoglobin, 13.9 g/dL; leukocyte count, 11,300/μL with predominant neutrophils; platelet count, 189 × 103/μL; aspartate aminotransferase (AST), 78 IU/L; alanine aminotransferase, 41 IU/L; lactate dehydrogenase (LDH), 653 IU/L (normal, 232–427); creatine kinase (CK), 1,851 IU/L (normal, 48–280); blood urea nitrogen, 22.1 mg/dL; serum creatinine, 1.1 mg/dL; C-reactive protein, 17.4 mg/dL. Two days after admission, the platelet count decreased to 62 × 103/μL, and the AST and LDH levels increased to 260 and 895, respectively.

Based on a presumptive diagnosis of rickettsiosis, and because he was severely prostrated and seemed deteriorating, he was begun on minocycline, 100 mg b.i.d., by intravenous route. Two days after initiation of the antibiotic therapy, clinical conditions, including the fever and rash as well as the AST level, started to ameliorate. The antibiotic treatment was continued for 17 days, and eventually the patient was discharged free of any subjective and objective abnormalities after a 3-week hospitalization.

SPECIFIC LABORATORY TESTS

First, laboratory tests were performed at the National Institute of Infectious Diseases, Tokyo, Japan. The PCR detection of the citrate synthase gene (gltA) of SFG rickettsiae was performed with whole blood and sera taken 5, 7 and 11 days after disease onset, and all the results were negative. Then, using the last two sera, IFA was performed as described previously.3 R. conorii 7 strain (VR-613), R. japonica YH strain (VR-1363), and Orientia tsutsugamushi Karp and Gilliam strains were propagated in L-929 (mouse fibroblasts) or Vero (green monkey kidney) cells, and these infected cells, as well as purified R. typhi (Panbio Limited, Brisbane, Australia), were used as antigens. Sera were diluted at 1:20 to 1:640 for IFA. Both IgM and IgG antibodies against O. tsutsugamushi were negative (< 1:20) in either of the two sera. However, IgM antibodies seroconverted from < 1:20 to 1:80 against R. typhi and to ≥ 1:640 against R. japonica. IgM antibodies against R. conorii were already positive at 1:20 in the first serum and increased to ≥ 1:640 in the second serum. IgG antibodies seroconverted from < 1:20 to ≥ 1:640 against either of R. conorii and R. japonica. IgG antibodies against R. typhi were already positive at 1:20 in the first serum and increased to 1:160 in the second serum. These results clearly showed that the patient had a SFG rickettsiosis.

Sera obtained 26 and 45 days after disease onset were then sent to Unité des Rickettsias, Marseille, France, mainly for differentiation between MSF and ATBF. First, microimmunofluorescence (MIF) assay was performed using whole-cell antigens of O. tsutsugamushi serotypes Karp, Kato, Gilliam, and Kawasaki,4 as well as with a large panel of other antigens including Bartonella henselae, Francisella tularensis, Coxiella burnetii, Anaplasma phagocytophilum, R. conorii, R. africae, R. felis, R. japonica, R. slovaca, R. helvetica, Indian tick typhus rickettsia, and R. honeï. This MIF assay was conducted as previously described for other rickettsiae.5 An MIF test was considered positive if 1) a single serum showed antibody titers of ≥ 1:64 for IgM and/or ≥ 1:128 for IgG antibodies, acute and convalescent sera showed 2) a seroconversion or 3) a fourfold or greater increase in titers.6,7 Both of our sera showed positive IgM and IgG antibodies (≥ 1:1,024) against all of the above last eight SFG rickettsial antigens, but negative results for O. tsutsugamushi, F. tularensis, C. burnetii, and Anaplasma phagocytophilum, and a weakly positive result for B. henselae.

Western immunoblotting was performed before and after cross-absorption with relevant antigens. Briefly, the serum was diluted at 1:1,000 or 1:2,000 (according to the serological titers) with R. conorii and R. africae antigen suspensions that were adjusted to contain 2 mg of protein/mL in a buffered saline solution (TBS; 20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.1% merthiolate). The mixtures were shaken for 24 hours at 4°C and centrifuged for 15 minutes at 10,000 × g. Supernatants were then diluted at 1:2 in TBS-0.5% nonfat dry-milk and applied to blots that were prepared as described previously.8 Figure 2 clearly shows that the patient’s antibodies were produced by R. conorii but not R. africae infection.

COMMENTS

Increasing attention is focused on African SFG rickettsioses among European and American travelers to Africa, be it MSF or ATBF. Thus, by IFA using R. conorii, a German group identified 78 cases of “tick typhus” in their university clinic during 1992–1998, 71.8% of which were contracted in southern Africa.9 Similarly, 31 U.S. travelers to Africa were diagnosed as having SFG rickettsioses during 1999–2002 by IFA with R. conorii and R. rickettsii.10 From 1983 through March 2003, 388 cases of SFG rickettsioses have been reported among travelers to sub-Saharan Africa or the French West Indies.1 One of the authors (M.K.) also reported two Japanese cases of African SFG rickettsioses after travel to southern Africa.11 With increasing physicians’ awareness, considerably more cases could be recognized as African SFG rickettsioses among travelers.

Many of the previously reported African SFG rickettsiosis cases might be ATBF but not MSF. Thus, using Western immunoblotting with or without cross-adsorption, one of the authors (D.R.) showed that the majority of tick-bite–related disease cases among European travelers visiting sub-Saharan Africa were ATBF (58%) but not MSF (1%).12 In addition, the above literature survey disclosed that 64% of those cases were either microbiologically confirmed as or clinically suspected of ATBF.1 Despite this recent wide recognition of ATBF, literature is scarce about MSF cases among travelers to sub-Saharan countries, such as Kenya where the disease is known to be distributed as Kenyan tick typhus. Thus, our case report could contribute to detecting more MSF cases and investigating the epidemiologic and clinical features, especially among travelers.

Although MSF and ATBF have similar clinical features in common, the incidence of these features differs between the two diseases. Thus, consistent with our case, MSF cases present an eschar at a lower rate compared with ATBF, that is, 72% versus 95%,12 with no documented cases showing multiple eschars, while they are commonly seen in ATBF. Conversely, MSF is characterized by a higher rate of skin rash,12 sometimes in a more prominent form as in our patient. Most importantly, MSF cases occasionally follow a deteriorating clinical course, sometimes resulting in death.13,14 An old study conducted by one of the authors (D.R.) on MSF cases in southern France reported that 6% of cases had severe complications such as neurologic, myocardial, and renal disorders and 2.5% died.13 In contrast, such life-threatening complications have not been documented in ATBF.1 An elevated CK level in MSF cases, as observed in our patient, was mentioned elsewhere; however, its relation to muscle symptoms was not studied.15

For laboratory confirmation, biopsy specimens from inoculation eschars provide the best materials for isolation and the genomic demonstration of the causative rickettsia. The whole blood for PCR should ideally be obtained at acute stages, that is, before initiation of antibiotic treatment and before antibodies become detectable. Our first whole blood for PCR was obtained as early as 5 days after disease onset and before initiation of minocycline treatment; however, the result was negative. Conceivably, the causative agent of MSF could be present in the blood only transiently even at acute stages. The commonly used IFA with R. conorii is associated with diverse cross-reactions with other SFG rickettsiae and is thus unable to identify the causative species. Recently, Western immunoblotting was developed using species-specific high-molecular-mass protein antigens and was also shown to be advantageous over IFA in detecting antibodies in early stages of the disease.16 In our case, the Western immunoblotting was combined with cross-adsorption to ensure specificity of the test and proved conclusive.

Antibiotic treatment is indicated in suspected cases of African SFG rickettsioses even without laboratory confirmation. The first-line antibiotics for both MSF and ATBF are tetracyclines, especially doxycycline because this agent is most frequently used. Although our patient responded well to minocycline, the relative efficacy of this agent for MSF needs further study.

In summary, clinicians should be alert to the possibility of African SFG rickettsiosis in any febrile traveler returning from Africa, even in the absence of an eschar or rash. In addition, because of the need to further characterize the epidemiologic and clinical features of the two African SFG rickettsioses, clinicians are encouraged to contact a specialized laboratory when encountering such cases.

Figure 1.
Figure 1.

Prominent maculopapular rash on the patient’s back.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 6; 10.4269/ajtmh.2005.73.1086

Figure 2.
Figure 2.

Western immunoblotting of the patient’s serum before and after cross-adsorption. Molecular markers are shown at the left in kilodaltons (kDa). Untreated serum reacted strongly with the 130 kDa R. conorii antigen but weakly with the 130 kDa R. africae antigen. Subsequently, serum adsorbed with R. conorii antigens did not react with either of the R. conorii and R. africae antigens, whereas that adsorbed with R. africae antigens still retained reactivity with the R. conorii antigen, indicating that the patient’s antibodies were produced by R. conorii but not R. africae infection. (A) Untreated serum tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens. (B) Serum adsorbed with R. conorii antigens and tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens. (C) Serum adsorbed with R. africae antigens and tested with R. conorii (Lane 1) and R. africae (Lane 2) antigens.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 6; 10.4269/ajtmh.2005.73.1086

*

Address correspondence to Mikio Kimura, Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: kimumiki@nih.go.jp

Authors’ addresses: Hiroko Yoshikawa, Department of Infectious Diseases, Niigata City General Hospital, 2-6-1 Schichikuyama, Nii-gata 950-8739, Japan, Telephone: +81-25-241-5151, Fax: +81-25-248-3507. Mikio Kimura, Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, Telephone: +81-3-5285-1111 ext. 2043, Fax: +81-3-5285-1129, E-mail: kimumiki@nih.go.jp. Motohiko Ogawa, Department of Virology 1, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, Telephone: +81-3-5285-1111 ext. 2563, Fax: +81-3-5285-1208. Jean-Marc Rolain and Didier Raoult, Unité des Rickettsies, CNRS UMR6020, Faculté de Médecine, Université de la Méditerranée, 27, boulevard Jean Moulin, 13385 Marseilles Cédex 5, France, Telephone: +33-491-324375, Fax: +33-491-830390.

Financial support: This study was conducted in part as a “Research on Health Sciences Focusing on Drug Innovation” (KH42075) funded by The Japan Health Sciences Foundation.

REFERENCES

  • 1

    Jensenius M, Fournier P-E, Kelly P, Myrvang B, Raoult D, 2003. African tick bite fever. Lancet Infect Dis 3 :557–564.

  • 2

    Kelly P, Matthewman L, Beati L, Raoult D, Mason P, Dreary M, Makombe R, 1992. African tick-bite fever: a new spotted fever group rickettsiosis under an old name. Lancet 340 :982–983.

    • Search Google Scholar
    • Export Citation
  • 3

    Furuya Y, Yamamoto S, Otu M, Yoshida Y, Ohashi N, Murata M, Kawabata N, Tamura A, Kawamura A Jr, 1991. Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay. J Clin Microbiol 29 :340–345.

    • Search Google Scholar
    • Export Citation
  • 4

    Amano K, Suzuki N, Fujita M, Nakamura Y, Suto T, 1993. Serological reactivity of sera from scrub typhus patients against Weil-Felix test antigens. Microbiol Immunol 37 :927–933.

    • Search Google Scholar
    • Export Citation
  • 5

    Fournier P-E, Jensenius M, Laferl H, Vene S, Raoult D, 2002. Kinetics of antibody responses in Rickettsia africae and Rickettsia conorii infections. Clin Diagn Lab Immunol 9 :324–328.

    • Search Google Scholar
    • Export Citation
  • 6

    Parola P, Miller RS, McDaniel P, Telford SR III, Rolain J-M, Wongsrichanalai C, Raoult D, 2003. Emerging rickettsioses of the Thai-Myanmar border. Emerg Infect Dis 9 :592–595.

    • Search Google Scholar
    • Export Citation
  • 7

    La Scola B, Raoult D, 1997. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 35 :2715–2727.

    • Search Google Scholar
    • Export Citation
  • 8

    Raoult D, Dasch GA, 1989. Line blot and Western blot immunoassays for diagnosis of Mediterranean spotted fever. J Clin Microbiol 27 :2073–2079.

    • Search Google Scholar
    • Export Citation
  • 9

    Jelinek T, Löscher T, 2001. Clinical features and epidemiology of tick typhus in travelers. J Travel Med 8 :57–59.

  • 10

    McQuiston JH, Paddock CD, Singleton J Jr, Wheeling JT, Zaki SR, Childs JE, 2004. Imported spotted fever rickettsioses in United States travelers returning from Africa: a summary of cases confirmed by laboratory testing at the Centers for Disease Control and Prevention, 1999–2002. Am J Trop Med Hyg 70 :98–101.

    • Search Google Scholar
    • Export Citation
  • 11

    Kimura M, Fujii T, Iwamoto A, 1998. Two cases of spotted fever group rickettsiosis contracted in southern parts of Africa (abstract in English). Kansenshogaku Zasshi 72 :1311–1316.

    • Search Google Scholar
    • Export Citation
  • 12

    Raoult D, Fournier PE, Fenollar F, Jensenius M, Prioe T, de Pina JJ, Caruso G, Jones N, Laferl H, Rosenblatt JE, Marrie TJ, 2001. Rickettsia africae, a tick-borne pathogen in travelers to sub-Saharan Africa. N Engl J Med 344 :1504–1510.

    • Search Google Scholar
    • Export Citation
  • 13

    Raoult D, Weiller PJ, Chagnon A, Chaudet H, Gallais H, Casanova P, 1986. Mediterranean spotted fever: clinical, laboratory and epidemiological features of 199 cases. Am J Trop Med Hyg 35 :845–850.

    • Search Google Scholar
    • Export Citation
  • 14

    Amaro M, Bacellar F, França A, 2003. Report of eight cases of fatal and severe Mediterranean spotted fever in Portugal. Ann N Y Acad Sci 990 :331–343.

    • Search Google Scholar
    • Export Citation
  • 15

    Antón E, Font B, Muñoz T, Sanfeliu I, Segura F, 2003. Clinical and laboratory characteristics of 144 patients with Mediterranean spotted fever. Eur J Clin Microbiol Infect Dis 22 :126–128.

    • Search Google Scholar
    • Export Citation
  • 16

    Teysseire N, Raoult D, 1992. Comparison of Western immunoblotting and microimmunofluorescence for diagnosis of Mediterranean spotted fever. J Clin Microbiol 30 :455–460.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Mikio Kimura, Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, Telephone: +81-3-5285-1111 ext. 2043, Fax: +81-3-5285-1129, E-mail: kimumiki@nih.go.jp.
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