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

Koukeo Phommasone1, Daniel H. Paris1,2,3, Tippawan Anantatat3, Jose´e Castonguay-Vanier1, Sommay Keomany4, Phoutthalavanh Souvannasing4, Stuart D. Blacksell1,2,3, Mayfong Mayxay1,2,5, Paul N. Newton1,2* 1 Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, 2 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, 3 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 4 Salavan Provincial Hospital, Salavan, Salavan Province, Lao PDR, 5 Faculty of Postgraduate Studies, University of Health Sciences, Vientiane, Lao PDR

Scrub typhus, , and and amoxicillin for seven days The buffy coat was positive for the O. group all occur in and recovered fully. Ethical approval was tsutsugamushi 47-kDa and groEL target the Lao PDR (Laos) [1,2]. Scrub typhus granted by the Lao National Ethics genes as well as the Rickettsia genus 17- and murine typhus account for ,16% and Committee for Health Research and the kDa and R. typhi ompB target genes by the 10%, respectively, of acute undifferentiat- Oxford Tropical Research Ethics Com- diagnostic real-time PCR assays, indicat- ed fever in blood culture–negative adults mittee, United Kingdom, and the patient ing potential dual positivity for O. tsutsuga- admitted to hospital in the capital city, provided written consent to publication of mushi and Rickettsia spp. The copy numbers Vientiane [1]. However, typhus-like ill- clinical details. determined for both pathogens were nesses are significant diagnostic challenges; Subsequently, the patient’s acute serum within the range normally seen at our patients with , dengue, ty- sample was assayed for immunoglobulin laboratory (56/59 and 75/130 copies/ml phoid, and malaria are also common and (Ig)M and IgG antibody titres against for the 47-kDa and ompB real-time assays, can present with similar symptoms and reference O. tsutsugamushi antigens (pooled respectively). That samples were processed signs. Although these pathogens are com- Karp, Kato, and Gilliam) and R. typhi in separate pre- and post-PCR work areas, mon and mixed (or concurrent) infections antigen (Wilmington strain) by indirect the evidence of multigene PCR positivity, are expected, the laboratory diagnosis of immunofluorescent assay [4]. The admis- and that no other dual positive samples mixed infection is a vexed subject. Reports sion serum had titres of scrub typhus were found makes contamination extreme- of mixed infections often use only serolog- IgM,400 and IgG = 1,600 and murine ly unlikely. Further characterisation was ical criteria. The problems of antibody typhus IgM,400 and IgG,400. Conva- performed (Table 1), including a panel of persistence and interspecies cross-reaction lescent serum was not available. DNA conventional nested PCR assays targeting raise uncertainty as to whether these from admission EDTA anticoagulated the 17-kDa (product size 524 bp), 56-kDa results represent true mixed infections, buffy coat was extracted and used as (product size 620 bp), and 47-kDa (prod- sequential infections, or cross-reactions. template for the O. tsutsugamushi 47-kDa- uct size 785 bp) target genes. All three We report a patient with concurrent scrub gene-based real-time PCR assay, the R. assays provided positive PCR amplicons typhus and murine typhus, demonstrated typhi ompB-gene-based real-time PCR as- and the products were purified and by dual PCR positivity, and discuss say, the Rickettsia genus 17-kDa-gene-based sequenced by Macrogen (Korea). Among evidence for identifying mixed infections. real-time PCR assay, and the O. tsutsuga- the candidates with the same BLAST mushi groEL-gene-based real-time PCR. score results for the 17-kDa PCR ampli- Patient Each run contained duplicate low-positive con (367 bp sequence), the geographically dilutions of linearized pGEM plasmids, closest related strain found was R. typhi As part of a study investigating the ranging from 104 to a single copy/ml, as strain TH1526 (max. score 640, max. aetiology of fever among patients with external controls (Table 1). identity 99%, query coverage 97%, E- negative malaria tests, we recruited pa- tients at Salavan Provincial Hospital, Citation: Phommasone K, Paris DH, Anantatat T, Castonguay-Vanier J, Keomany S, et al. (2013) Concurrent Salavan Province, southern Laos [3]. A Infection with Murine Typhus and Scrub Typhus in Southern Laos—the Mixed and the Unmixed. PLoS Negl 20-year-old female rice farmer from Trop Dis 7(8): e2163. doi:10.1371/journal.pntd.0002163 Naxay Village (15u62937.060N; Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of America 106u33942.130E), Salavan District, whose Published August 29, 2013 house was surrounded by vegetable gar- Copyright: ß 2013 Phommasone et al. This is an open-access article distributed under the terms of the dens, presented at Salavan Provincial Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any Hospital in July 2009 with 14 days of medium, provided the original author and source are credited. headache associated with three days of Funding: Funded by the Wellcome Trust of Great Britain (Grant number 089275/Z/09/Z, www.wellcome.ac.uk), fever, , and , having taken the World Health Organization Regional Office for the Western Pacific (www.wpro.who.int), with grants from the Australian Agency for International Development, the Ministry of Foreign Affairs of Japan and the United five days of oral cephalexin. She was States Agency for International Development and by the Foundation for Innovative New Diagnostics (www. febrile (38.5uC), but physical examination finddiagnostics.org) through a grant from the UK Department for International Development. The funders had was otherwise normal without rash or no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. eschar. She was suspected to have scrub Competing Interests: The authors have declared that no competing interests exist. typhus and was prescribed empirical * E-mail: [email protected]

PLOS Neglected Tropical Diseases | www.plosntds.org 1 August 2013 | Volume 7 | Issue 8 | e2163 Table 1. Overview of PCR-based and DNA sequencing results.

PCR positivity criteria

Scrub typhus Murine typhus Strength of evidence Technique [reference]

47-kDa real-time PCR (26 pos.) 17-kDa real-time PCR (26 pos.) Strong* Jiang et al., 2004 [20] groEL real-time PCR (26 pos.) ompB real-time PCR (26 pos.) Strong* Henry et al., 2007 [21] Paris et al., 2009 [22] 56-kDa nested PCR (620 bp) 17-kDa nested PCR (524 bp) Very strong. Product size Horinouchi et al., 1996 [23] 47-kDa nested PCR (785 bp) confirmation via gel electrophoresis Jiang et al., 2012 [24] DNA sequences for 47-kDa1 and DNA sequence for 17-kDa Extremely strong. BLAST result Altschul et al., 1990 [25] 56-kDa2 nested PCR amplicons nested PCR amplicon3 with 97–100% coverage for amplicon similarities

*positivity criteria in analogy to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [19]. GenBank accession numbers: 1BankIt1587796 Seq2 KC283067. 2BankIt1587796 Seq3 KC283068. 3BankIt1587796 Seq1 KC283066. doi:10.1371/journal.pntd.0002163.t001 value 3e-180), from a patient with murine demonstrating mixed infection with very We suggest that reports of mixed typhus from Chiang Rai, N. Thailand. high specificity, this approach will suffer infections include an explicit discussion of The 47-kDa amplicon (744 bp) matched from low sensitivity, as significant pro- the likely specificity and sensitivity of the O. tsutsugamushi Ikeda strain (max. score portions of patients with good evidence diagnostic assays used and the likelihood 1314, query coverage 100%, E-value 0.0) of mono-infection (with fourfold rises in that the observations represent true con- and the nested 56-kDa amplicon (523 bp) specific IgM) are PCR negative for both current mixed infections (or coinfections), matched O. tsutsugamushi T1125175_KH scrub typhus [11] and murine typhus or sequential infections due to persistence 56-kDa type-specific antigen (max. (unpublished data). Moreover, there are of antibody or false positives due to assay score = 640, query coverage = 99%, E- cross-reactions between IgM against O. cross-reactions (‘‘dual positivity’’). A grad- value 0.0). tsutsugamushi and R. typhi [12] and very ing system of evidence, analogous to the The infecting O. tsutsugamushi strain is few objective data on serological respons- GRADE guidelines and Infectious Diseas- very similar to the human-pathogenic es in confirmed mixed infections. West- es Society of America guidelines [17,18], Cambodian isolate T1125175_KH and ern blotting has been used to distinguish may be helpful. For example, grade I the animal-derived (Rattus rajah) Thai serological responses [13]. In Vientiane (culture or molecular detection of both strain TA763, making this the first Lao City, 4% of well adults had IgG anti- pathogens or direct observation such as in scrub typhus patient with a strain similar bodies against both scrub typhus and a malaria film), grade II (serological to another nonhuman vertebrate strain murine typhus [2], suggesting the possi- diagnosis with either seroconversion or [5,6]. Similarly, human pathogenicity of a bility of previous exposures to both fourfold antibody responses to both path- Kato-related TA716-like O. tsutsugamushi organisms and/or serological cross-reac- ogens, without evidence of cross-reactions, strain originally described from the In- tions. or using Western blotting), and grade III dochinese ground squirrel (Menetes berd- Mixed O. tsutsugamushi and Leptospira (serological diagnosis based on admission morei) has been recently reported from spp. infections have been reported, but serology without exclusion of cross-reac- Thailand [7]. none of these included positive PCR or tions or antibody persistence or culture, culture for both pathogens (Table 2). Such molecular, or admission serological detec- Mixed Infections infections are especially important as tion). Grades I to III would have decreas- leptospirosis would be expected to respond ing specificity but increasing sensitivity in We present a patient with clear molec- to penicillins or cephalosporins while scrub diagnosing true mixed infections. Sero- ular diagnostic evidence of concurrent typhus would not [14]. Mixed and conversion could also be regarded as grade mixed infection with scrub typhus and scrub typhus infections have been reported I evidence if documented with a diagnostic murine typhus. Such infections may go in Taiwan but only using serological test providing highly specific evidence for unrecognized. Although clinically similar, assays. Mixed infections of Plasmodium seroconversion. The relative importance the diseases have markedly different path- falciparum with both scrub typhus and of sensitivity and specificity will depend on ophysiology [8]. Although both pathogens murine typhus diagnosed by PCR and/ the question being asked and the clinical would be expected to respond to doxycy- or dynamic serology was documented use of the data. When different grades of cline, O. tsutsugamushi generally causes the among febrile pregnant women on the evidence are used for different pathogens more severe disease and would not be Thai–Burmese border (Table 2). Interpre- in a ‘‘mixed’’ infection, we suggest that the expected to respond to fluoroquinolones, tation would be more intricate if either (or grade with the highest number (least which have been used for murine typhus both) pathogen(s) caused chronic infec- specificity) is used. [9]. Mixed infection with these two tions. This has not been demonstrated for For patients with grade I evidence, pathogens was demonstrated using PCR R. typhi (although we can find no evidence further care is required as molecular and IFA among three patients in Yunnan that it has been expressly looked for), but methods have different specificities for Province, China [10]. there have been suggestions that O. pathogen diagnosis. Real-time PCR spec- Although culture or molecular detec- tsutsugamushi may cause long-term infec- ificity is higher if type-specific genes are tion should be the gold standard for tions [15,16]. used (e.g., 56-kDa and 47-kDa genes for O.

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

Number of patients Country Rickettsial pathogen and diagnosis Additional pathogen and diagnosis Evidence grade Reference

O. tsutsugamushi Leptospira spp. 9/22 (41%) of patients with leptospirosis had NE Thailand Positive dot blot immunoassay (correlates with MAT 4-fold rise in titre or single titre $1:320 evidence for scrub typhus Watt et al., 2003 [26] IgG titres $1:1,600 or IgM titres $1:400) Grade III O. tsutsugamushi Leptospira spp. 1 patient with cholecystitis, pancreatitis, and Taiwan IgM single titre $1:80 MAT single titre of 1:400 acute renal failure Wang et al., 2003 [27] Grade III O. tsutsugamushi Leptospira spp. 62/540 (12%) of patients with leptospirosis NE Thailand 4-fold rise in specific IgG or IgM titre to $1:200 by Culture, MAT, IFA. 4-fold rise in specific IgG or IgM had evidence for scrub typhus Suputtamongkol et al., 2004 [28] IFA or a single titre of $1:400 titre to $1:200 by IFA or a single titre of $1:400 Grade II O. tsutsugamushi Leptospira sp. 1 patient with had evidence of Taiwan Admission IFA IgM titre 1:80 & IgG 1:40 MAT 4-fold rise in antibody titre and leptospirosis and scrub typhus Lu et al., 2005 [29] Burkholderia pseudomallei by blood culture Grade II (meliodosis+leptospirosis) Grade III (scrub typhus+melioidosis) Grade III (scrub typhus+leptospirosis) O. tsutsugamushi Leptospira spp. 4 patients with leptospirosis had evidence for Taiwan Serology technique not stated Serology technique not stated scrub typhus Ho et al., 2006 [30] Patient 1: PCR positive Patient 1: single titre 1:1,600 Grade II Patient 2: PCR and IgM & IgG positive Patient 2: single titre 1:800 Patient 3: PCR positive, IgM positive, and 4-fold rise in IgG Patient 3: antibody titre increased .4-fold rise Patient 4: PCR positive, IgM positive, and 4-fold rise in IgG Patient 4: antibody titre increased .4-fold rise O. tsutsugamushi Leptospira spp. 1 patient with acute renal failure and pulmonary Taiwan Admission IFA IgM $1:80 plus 4-fold rise in IgG titre on MAT seroconversion to 1:400 haemorrhage Chen et al., 2007 [31] paired sera Grade II O. tsutsugamushi Leptospira spp. 7/87 (8%) of patients with leptospirosis or scrub Taiwan IFA 4-fold rise in titre or a single IgM titre $1:80 MAT 4-fold rise in titre or a single titre $1:320 typhus had evidence for both pathogens Lee et al., 2007 [32] Grade II O. tsutsugamushi & R. typhi Leptospira spp. 11/296 (4%) of patients with leptospirosis had NE Thailand IFA 4-fold rise or a single titre of $1:400 Culture or MAT 4-fold rise or a single titre evidence for infection with scrub typhus or murine Phimda et al., 2007 [33] of $1:400 typhus Grade II O. tsutsugamushi R. typhi 3/8 (38%) of febrile farmers PCR positive for scrub China PCR positive and $4-fold rise in IgG PCR positive and $4-fold rise in IgG typhus or murine typhus were PCR positive for both Zhang et al., 2007 [10] Grade I O. tsutsugamushi & R. typhi 5/144 (3%) of patients with Q fever or typhus Taiwan IFA IgM $1:80 or 4-fold rise in IgG IFA anti-phase II IgG $1:320 or IgM $1:80 (scrub and murine) had evidence for both infections Lai et al., 2009 [34] or a 4-fold rise in IgG titre Grade II O. tsutsugamushi & R. typhi Plasmodium falciparum 5/51 (10%) of pregnant women with malaria had NW Thailand PCR, culture or IFA 4-fold rise in IgM or IgG Giemsa malaria films evidence for murine typhus or scrub typhus McGready et al., 2010 [35] Grade I for scrub typhus-malaria and grade II for murine typhus-malaria O. tsutsugamushi Leptospira spp. MAT seroconversion to 1:1,600 1 patient with shock and respiratory failure Taiwan IFA seroconversion to IgG 1:320 & IgM 1:160 Grade II Wei et al., 2012 [36]

doi:10.1371/journal.pntd.0002163.t002 tsutsugamushi) than if genus-specific genes gene sequences deposited in GenBank Acknowledgments are used (17-kDa genes for Rickettsia spp.), and/or genotyping using SNPs will allow which again are stronger than nonspecific for discrimination at a more subtle level. We thank the staff of the Microbiology conserved ‘‘housekeeping’’ genes (e.g., We suggest that where possible mixed Laboratory, Mahosot Hospital, especially the Director, Dr. Rattanaphone Phetsouvanh, and groEL and 16S rRNA). Sequencing should infections should be confirmed by culture Salavan Provincial Hospital. be attempted if conventional (nested) PCR or detection of specific nucleic acid products are obtained, as BLAST analysis sequences and that the introduction of a will provide high-level confidence with grading system for the strength of evidence confirmation of the amplicon similarity to for mixed infections should be considered.

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