Advertisement
Symposium

Symposium In Symposium articles, authors discuss a real-world problem, from the clinic, laboratory, or community, and how best it can be tackled.

See all article types »

Concurrent Infection with Murine Typhus and Scrub Typhus in Southern Laos—the Mixed and the Unmixed

  • Koukeo Phommasone,

    Affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR

    X
  • Daniel H. Paris,

    Affiliations: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

    X
  • Tippawan Anantatat,

    Affiliation: Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

    X
  • Josée Castonguay-Vanier,

    Affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR

    X
  • Sommay Keomany,

    Affiliation: Salavan Provincial Hospital, Salavan, Salavan Province, Lao PDR

    X
  • Phoutthalavanh Souvannasing,

    Affiliation: Salavan Provincial Hospital, Salavan, Salavan Province, Lao PDR

    X
  • Stuart D. Blacksell,

    Affiliations: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

    X
  • Mayfong Mayxay,

    Affiliations: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Faculty of Postgraduate Studies, University of Health Sciences, Vientiane, Lao PDR

    X
  • Paul N. Newton mail

    paul@tropmedres.ac

    Affiliations: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

    X
  • Published: August 29, 2013
  • DOI: 10.1371/journal.pntd.0002163

Scrub typhus, murine typhus, and spotted fever group rickettsia all occur in the Lao PDR (Laos) [1], [2]. Scrub typhus and murine typhus account for ~16% and 10%, respectively, of acute undifferentiated fever in blood culture–negative adults admitted to hospital in the capital city, Vientiane [1]. However, typhus-like illnesses are significant diagnostic challenges; patients with leptospirosis, dengue, typhoid, and malaria are also common and can present with similar symptoms and signs. Although these pathogens are common and mixed (or concurrent) infections are expected, the laboratory diagnosis of mixed infection is a vexed subject. Reports of mixed infections often use only serological criteria. The problems of antibody persistence and interspecies cross-reaction raise uncertainty as to whether these results represent true mixed infections, sequential infections, or cross-reactions. We report a patient with concurrent scrub typhus and murine typhus, demonstrated by dual PCR positivity, and discuss evidence for identifying mixed infections.

Patient

As part of a study investigating the aetiology of fever among patients with negative malaria tests, we recruited patients at Salavan Provincial Hospital, Salavan Province, southern Laos [3]. A 20-year-old female rice farmer from Naxay Village (15°62′37.06″N; 106°33′42.13″E), Salavan District, whose house was surrounded by vegetable gardens, presented at Salavan Provincial Hospital in July 2009 with 14 days of headache associated with three days of fever, myalgia, and vomiting, having taken five days of oral cephalexin. She was febrile (38.5°C), but physical examination was otherwise normal without rash or eschar. She was suspected to have scrub typhus and was prescribed empirical doxycycline and amoxicillin for seven days and recovered fully. Ethical approval was granted by the Lao National Ethics Committee for Health Research and the Oxford Tropical Research Ethics Committee, United Kingdom, and the patient provided written consent to publication of clinical details.

Subsequently, the patient's acute serum sample was assayed for immunoglobulin (Ig)M and IgG antibody titres against reference O. tsutsugamushi antigens (pooled Karp, Kato, and Gilliam) and R. typhi antigen (Wilmington strain) by indirect immunofluorescent assay [4]. The admission serum had titres of scrub typhus IgM<400 and IgG = 1,600 and murine typhus IgM<400 and IgG<400. Convalescent serum was not available. DNA from admission EDTA anticoagulated buffy coat was extracted and used as template for the O. tsutsugamushi 47-kDa-gene-based real-time PCR assay, the R. typhi ompB-gene-based real-time PCR assay, the Rickettsia genus 17-kDa-gene-based real-time PCR assay, and the O. tsutsugamushi groEL-gene-based real-time PCR. Each run contained duplicate low-positive dilutions of linearized pGEM plasmids, ranging from 104 to a single copy/µl, as external controls (Table 1).

thumbnail

Table 1. Overview of PCR-based and DNA sequencing results.

doi:10.1371/journal.pntd.0002163.t001

The buffy coat was positive for the O. tsutsugamushi 47-kDa and groEL target genes as well as the Rickettsia genus 17-kDa and R. typhi ompB target genes by the diagnostic real-time PCR assays, indicating potential dual positivity for O. tsutsugamushi and Rickettsia spp. The copy numbers determined for both pathogens were within the range normally seen at our laboratory (56/59 and 75/130 copies/µl for the 47-kDa and ompB real-time assays, respectively). That samples were processed in separate pre- and post-PCR work areas, the evidence of multigene PCR positivity, and that no other dual positive samples were found makes contamination extremely unlikely. Further characterisation was performed (Table 1), including a panel of conventional nested PCR assays targeting the 17-kDa (product size 524 bp), 56-kDa (product size 620 bp), and 47-kDa (product size 785 bp) target genes. All three assays provided positive PCR amplicons and the products were purified and sequenced by Macrogen (Korea). Among the candidates with the same BLAST score results for the 17-kDa PCR amplicon (367 bp sequence), the geographically closest related strain found was R. typhi strain TH1526 (max. score 640, max. identity 99%, query coverage 97%, E-value 3e-180), from a patient with murine typhus from Chiang Rai, N. Thailand. The 47-kDa amplicon (744 bp) matched O. tsutsugamushi Ikeda strain (max. score 1314, query coverage 100%, E-value 0.0) and the nested 56-kDa amplicon (523 bp) matched O. tsutsugamushi T1125175_KH 56-kDa type-specific antigen (max. score = 640, query coverage = 99%, E-value 0.0).

The infecting O. tsutsugamushi strain is very similar to the human-pathogenic Cambodian isolate T1125175_KH and the animal-derived (Rattus rajah) Thai strain TA763, making this the first Lao scrub typhus patient with a strain similar to another nonhuman vertebrate strain [5], [6]. Similarly, human pathogenicity of a Kato-related TA716-like O. tsutsugamushi strain originally described from the Indochinese ground squirrel (Menetes berdmorei) has been recently reported from Thailand [7].

Mixed Infections

We present a patient with clear molecular diagnostic evidence of concurrent mixed infection with scrub typhus and murine typhus. Such infections may go unrecognized. Although clinically similar, the diseases have markedly different pathophysiology [8]. Although both pathogens would be expected to respond to doxycycline, O. tsutsugamushi generally causes the more severe disease and would not be expected to respond to fluoroquinolones, which have been used for murine typhus [9]. Mixed infection with these two pathogens was demonstrated using PCR and IFA among three patients in Yunnan Province, China [10].

Although culture or molecular detection should be the gold standard for demonstrating mixed infection with very high specificity, this approach will suffer from low sensitivity, as significant proportions of patients with good evidence of mono-infection (with fourfold rises in specific IgM) are PCR negative for both scrub typhus [11] and murine typhus (unpublished data). Moreover, there are cross-reactions between IgM against O. tsutsugamushi and R. typhi [12] and very few objective data on serological responses in confirmed mixed infections. Western blotting has been used to distinguish serological responses [13]. In Vientiane City, 4% of well adults had IgG antibodies against both scrub typhus and murine typhus [2], suggesting the possibility of previous exposures to both organisms and/or serological cross-reactions.

Mixed O. tsutsugamushi and Leptospira spp. infections have been reported, but none of these included positive PCR or culture for both pathogens (Table 2). Such infections are especially important as leptospirosis would be expected to respond to penicillins or cephalosporins while scrub typhus would not [14]. Mixed Q fever and scrub typhus infections have been reported in Taiwan but only using serological assays. Mixed infections of Plasmodium falciparum with both scrub typhus and murine typhus diagnosed by PCR and/or dynamic serology was documented among febrile pregnant women on the Thai–Burmese border (Table 2). Interpretation would be more intricate if either (or both) pathogen(s) caused chronic infections. This has not been demonstrated for R. typhi (although we can find no evidence that it has been expressly looked for), but there have been suggestions that O. tsutsugamushi may cause long-term infections [15], [16].

thumbnail

Table 2. Reports of apparent mixed infections in Asia that included rickettsioses.

doi:10.1371/journal.pntd.0002163.t002

We suggest that reports of mixed infections include an explicit discussion of the likely specificity and sensitivity of the diagnostic assays used and the likelihood that the observations represent true concurrent mixed infections (or coinfections), or sequential infections due to persistence of antibody or false positives due to assay cross-reactions (“dual positivity”). A grading system of evidence, analogous to the GRADE guidelines and Infectious Diseases Society of America guidelines [17], [18], may be helpful. For example, grade I (culture or molecular detection of both pathogens or direct observation such as in a malaria film), grade II (serological diagnosis with either seroconversion or fourfold antibody responses to both pathogens, without evidence of cross-reactions, or using Western blotting), and grade III (serological diagnosis based on admission serology without exclusion of cross-reactions or antibody persistence or culture, molecular, or admission serological detection). Grades I to III would have decreasing specificity but increasing sensitivity in diagnosing true mixed infections. Seroconversion could also be regarded as grade I evidence if documented with a diagnostic test providing highly specific evidence for seroconversion. The relative importance of sensitivity and specificity will depend on the question being asked and the clinical use of the data. When different grades of evidence are used for different pathogens in a “mixed” infection, we suggest that the grade with the highest number (least specificity) is used.

For patients with grade I evidence, further care is required as molecular methods have different specificities for pathogen diagnosis. Real-time PCR specificity is higher if type-specific genes are used (e.g., 56-kDa and 47-kDa genes for O. tsutsugamushi) than if genus-specific genes are used (17-kDa genes for Rickettsia spp.), which again are stronger than nonspecific conserved “housekeeping” genes (e.g., groEL and 16S rRNA). Sequencing should be attempted if conventional (nested) PCR products are obtained, as BLAST analysis will provide high-level confidence with confirmation of the amplicon similarity to gene sequences deposited in GenBank and/or genotyping using SNPs will allow for discrimination at a more subtle level.

We suggest that where possible mixed infections should be confirmed by culture or detection of specific nucleic acid sequences and that the introduction of a grading system for the strength of evidence for mixed infections should be considered.

Acknowledgments

We thank the staff of the Microbiology Laboratory, Mahosot Hospital, especially the Director, Dr. Rattanaphone Phetsouvanh, and Salavan Provincial Hospital.

References

  1. 1. Phongmany S, Rolain JM, Phetsouvanh R, Blacksell SD, Soukkhaseum V, et al. (2006) Rickettsial infections and fever, Vientiane, Laos. Emerg Infect Dis 12: 256–262. doi: 10.3201/eid1202.050900
  2. 2. Vallée J, Thaojaikong T, Moore CE, Phetsouvanh R, Richards AL, et al. (2010) Contrasting spatial distribution and risk factors for past infection with scrub typhus and murine typhus in Vientiane City, Lao PDR. PLoS Negl Trop Dis 4: e909 doi:10.1371/journal.pntd.0000909.
  3. 3. Mayxay M, Castonguay-Vanier J, Chansamouth V, Dubot-Pérès A, Paris DH, et al. (2013) The causes of non-malarial fever in Laos – evidence to inform empirical treatment of fever. The Lancet Global Health 1: e46–e54. doi: 10.1016/s2214-109x(13)70008-1
  4. 4. Phetsouvanh R, Blacksell SD, Jenjaroen K, Day NP, Newton PN (2009) Comparison of indirect immunofluorescence assays for diagnosis of scrub typhus and murine typhus using venous blood and finger prick filter paper blood spots. Am J Trop Med Hyg 80: 837–840.
  5. 5. Elisberg BL, Sangkasuvana V, Campbell JM, Bozeman FM, Bodhidatta P (1967) Physiogeographic distribution of scrub typhus in Thailand. Acta Med Biol (Niigata) 15: 61–67.
  6. 6. Duong V, Mai TT, Blasdell K, Lo LV, Morvan C, et al. (2013) Molecular epidemiology of Orientia tsutsugamushi in Cambodia and Central Vietnam reveals a broad region-wide genetic diversity. Infect Genet Evol 15: 35–42. doi: 10.1016/j.meegid.2011.01.004
  7. 7. McGready R, Blacksell SD, Luksameetanasan R, Wuthiekanun V, Jedsadapanpong W, et al. (2010) First report of an Orientia tsutsugamushi type TA716-related scrub typhus infection in Thailand. Vector Borne Zoonotic Dis 10: 191–193. doi: 10.1089/vbz.2008.0199
  8. 8. Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, et al. (2012) Coagulation and inflammation in scrub typhus and murine typhus-a prospective comparative study from Laos. Clin Microbiol Infect 18: 1221–1228. doi: 10.1111/j.1469-0691.2011.03717.x
  9. 9. Tantibhedhyangkul W, Angelakis E, Tongyoo N, Newton PN, Moore CE, et al. (2010) Intrinsic fluoroquinolone resistance in Orientia tsutsugamushi. Int J Antimicrob Agents 35: 338–341. doi: 10.1016/j.ijantimicag.2009.11.019
  10. 10. Zhang LJ, Li XM, Zhang DR, Zhang JS, Di Y, et al. (2007) Molecular epidemic survey on co-prevalence of scrub typhus and murine typhus in Yuxi city, Yunnan province of China. Chin Med J (Engl) 120: 1314–1318.
  11. 11. Sonthayanon P, Chierakul W, Wuthiekanun V, Phimda K, Pukrittayakamee S, et al. (2009) Association of high Orientia tsutsugamushi DNA loads with disease of greater severity in adults with scrub typhus. J Clin Microbiol 47: 430–434. doi: 10.1128/jcm.01927-08
  12. 12. Blacksell SD, Jenjaroen K, Phetsouvanh R, Tanganuchitcharnchai A, Phouminh P, et al. (2010) Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 83: 365–369. doi: 10.4269/ajtmh.2010.09-0534
  13. 13. Rolain JM, Gouriet F, Brouqui P, Larrey D, Janbon F, et al. (2005) Concomitant or consecutive infection with Coxiella burnetii and tickborne diseases. Clin Infect Dis 40: 82–88. doi: 10.1086/426440
  14. 14. Watt G, Jongsakul K, Suttinont C (2003) Possible scrub typhus coinfections in Thai agricultural workers hospitalized with leptospirosis. Am J Trop Med Hyg 68: 89–91.
  15. 15. Smadel JE, Ley HL Jr, Diercks FH, Cameron JA (1952) Persistence of Rickettsia tsutsugamushi in tissues of patients recovered from scrub typhus. Am J Hyg 56: 294–302.
  16. 16. Chung M-H, Lee J-S, Baek J-h, Kim M, Kang J-S (2012) Persistence of Orientia tsutsugamushi in humans. J Korean Med Sci 27: 231–235. doi: 10.3346/jkms.2012.27.3.231
  17. 17. Kish MA (2001) Infectious Diseases Society of America (2001) Guide to development of practice guidelines. Clin Infect Dis 32: 851–854. doi: 10.1086/319366
  18. 18. Balshem H, Helfand M, Schünemann HJ, Oxman AD, Kunz R, et al. (2011) GRADE guidelines: 3. Rating the quality of evidence. J Clin Epidemiol 64: 401–406. doi: 10.1016/j.jclinepi.2010.07.015
  19. 19. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622. doi: 10.1373/clinchem.2008.112797
  20. 20. Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, et al. (2004) Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70: 351–356.
  21. 21. Henry KM, Jiang J, Rozmajzl PJ, Azad AF, Macaluso KR, et al. (2007) Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever. Mol Cell Probes 21: 17–23. doi: 10.1016/j.mcp.2006.06.002
  22. 22. Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NP (2009) A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect 15: 488–495. doi: 10.1111/j.1469-0691.2008.02671.x
  23. 23. Horinouchi H, Murai K, Okayama A, Nagatomo Y, Tachibana N, et al. (1996) Genotypic identification of Rickettsia tsutsugamushi by restriction fragment length polymorphism analysis of DNA amplified by the polymerase chain reaction. Am J Trop Med Hyg 54: 647–651.
  24. 24. Jiang J, Stromdahl EY, Richards AL (2012) Detection of Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Gulf Coast ticks collected from humans in the United States. Vector Borne Zoonotic Dis 12: 175–182. doi: 10.1089/vbz.2011.0614
  25. 25. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. doi: 10.1016/s0022-2836(05)80360-2
  26. 26. Watt G, Parola P (2003) Scrub typhus and tropical rickettsioses. Curr Opin Infect Dis 16: 429–436. doi: 10.1097/00001432-200310000-00009
  27. 27. Wang NC, Ni YH, Peng MY, Chang FY (2003) Acute acalculous cholecystitis and pancreatitis in a patient with concomitant leptospirosis and scrub typhus. J Microbiol Immunol Infect 36: 285–287.
  28. 28. Suputtamongkol Y, Niwattayakul K, Suttinont C, Losuwanaluk K, Limpaiboon R, et al. (2004) An open, randomized, controlled trial of penicillin, doxycycline, and cefotaxime for patients with severe leptospirosis. Clin Infect Dis 39: 1417–1424. doi: 10.1086/425001
  29. 29. Lu PL, Tseng SH (2005) Fatal septicemic melioidosis in a young military person possibly co-infected with Leptospira interrogans and Orientia tsutsugamushi. Kaohsiung J Med Sci 21: 173–178. doi: 10.1016/s1607-551x(09)70297-9
  30. 30. Ho YH, Chen LK, Tsai PJ, Wang LS (2006) Coinfection with leptospirosis and scrub typhus. Tzu Chi Med J 18: 237–240.
  31. 31. Chen YS, Cheng SL, Wang HC, Yang PC (2007) Successful treatment of pulmonary hemorrhage associated with leptospirosis and scrub typhus coinfection by early plasma exchange. J Formos Med Assoc 106: S1–S6. doi: 10.1016/s0929-6646(09)60344-2
  32. 32. Lee CH, Liu JW (2007) Coinfection with leptospirosis and scrub typhus in Taiwanese patients. Am J Trop Med Hyg 77: 525–527.
  33. 33. Phimda K, Hoontrakul S, Suttinont C, Chareonwat S, Losuwanaluk K, et al. (2007) Doxycycline versus azithromycin for treatment of leptospirosis and scrub typhus. Antimicrob Agents Chemother 51: 3259–3263. doi: 10.1128/aac.00508-07
  34. 34. Lai CH, Chen YH, Lin JN, Chang LL, Chen WF, et al. (2009) Acute Q fever and scrub typhus, southern Taiwan. Emerg Infect Dis 15: 1659–1661. doi: 10.3201/eid1510.090007
  35. 35. McGready R, Blacksell SD, Luksameetanasan R, Wuthiekanun V, Jedsadapanpong W, et al. (2010) First report of an Orientia tsutsugamushi type TA716-related scrub typhus infection in Thailand. Vector Borne Zoonotic Dis 10: 191–193. doi: 10.1089/vbz.2008.0199
  36. 36. Wei YF, Chiu CT, Lai YF, Lai CH, Lin HH (2012) Successful treatment of septic shock and respiratory failure due to leptospirosis and scrub typhus coinfection with penicillin, levofloxacin, and activated protein C. J Microbiol Immunol Infect 45: 251–254. doi: 10.1016/j.jmii.2011.09.015