Widespread Use of Realtime PCR for Rickettsial Diagnosis

SHORT COMMUNICATION Widespread use of real-time PCR for rickettsial diagnosis Aure´ lie Renvoise´ , Jean-Marc Rolain, Cristina Socolovschi & Didier Raoult

Unite´ de Recherche en Maladies Infectieuses et Tropicales Emergentes CNRS-IRD UMR6236-198, Faculte´ de Me´ decine, Universite´ de la Me´ diterrane´ e, Marseille, France

Correspondence: Didier Raoult, Unite´ de Abstract Recherche en Maladies Infectieuses et Tropicales Emergentes CNRS-IRD UMR6236- We report 2 years of experience with rickettsial molecular diagnosis using real- 198, Universite´ de la Me´ diterrane´ e, Faculte´ time PCR at the French National Reference Center. All Rickettsia genomes avail- de Me´ decine, 27 bd Jean Moulin, 13385 able were compared to discover specific sequences to design new sets of primers Marseille Cedex 5, France. Tel.: and probes. The specificity was verified in silico and against a panel of 30 rick- +33491324375; fax: +33491387772; ettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, e-mail: [email protected] primers and probes that were both specific and sensitive were routinely used

Received 6 July 2011; revised 6 October for the diagnosis of rickettsial infections from clinical specimens. We retained 2011; accepted 31 October 2011. sets of primers and probes to detect spotted fever group Rickettsia, typhus Final version published online 8 December group Rickettsia, Rickettsia conorii, Rickettsia slovaca, Rickettsia africae and Rick- 2011. ettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, DOI: 10.1111/j.1574-695X.2011.00899.x nine R. conorii, ﬁve Rickettsia sibirica mongolitimonae, four R. slovaca, two R. australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi Editor: J. Stephen Dumler and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive Keywords Rickettsia; real-time PCR; molecular and reduces delay in the diagnosis of rickettsial infections. These real-time PCR diagnosis; genome; point-of-care strategy. assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.

Members of the genus Rickettsia may be classified into rapid and sensitive (Stenos et al., 2005; Henry et al., 2007; spotted fever group (SFG) Rickettsia, typhus group (TG) Kidd et al., 2008). Genomic approaches have recently Rickettsia, Rickettsia bellii group and Rickettsia canadensis increased our knowledge of Rickettsia sp., and massive group (Merhej & Raoult, 2010). Rickettsiae can be trans- amounts of genomic data have become available (Ogata mitted to humans by blood-sucking arthropods and are et al., 2001; Fournier et al., 2007; Merhej & Raoult, 2010). associated with specific diseases termed rickettsioses. For We used these sequence data to develop specific qPCR example, Rickettsia conorii is associated with Mediterra- methods to improve the diagnosis of rickettsial infections. nean spotted fever (MSF) (Parola et al., 2005), Rickettsia Here, we report 2 years of experience with rickettsial africae with African tick-bite fever (ATBF) (Jensenius molecular diagnosis using qPCR at the French National et al., 2003), Rickettsia sibirica ssp. mongolitimonae with Reference Center (FNRC). lymphangitis-associated rickettsiosis (LAR) (Fournier All rickettsial genomes available were compared to dis- et al., 2005), Rickettsia slovaca with ‘scalp eschar and neck cover sequences that are specific for either SFG or TG or lymphadenopathy after tick bite’ (SENLAT) (Angelakis for the identification of Rickettsia spp. at the species level. et al., 2010), Rickettsia australis with Queensland tick Specific primers and probes which were selected by gen- typhus (QTT) (Parola et al., 2005), Rickettsia typhi with ome comparison were designed based on these specific murine typhus (Civen & Ngo, 2008) and Rickettsia honei sequences (Supporting information, Table S1). Specificity with Flinders Island spotted fever (FISF) (Parola et al., was verified in silico using blastN analysis on GenBank

IMMUNOLOGY & MEDICAL MICROBIOLOGY 2005). When a rickettsiosis is clinically suspected, biologi- database. Specificity was also verified in vitro using a local cal diagnosis can be obtained using serology, cell culture collection panel of 30 rickettsial strains. Sensitivity was and/or molecular tools (Parola et al., 2005); among the determined using 10-fold serial dilutions. Finally, primers molecular tools, real-time quantitative PCR (qPCR) is and probes that were both specific and sensitive were

ª 2011 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 64 (2012) 126–129 Published by Blackwell Publishing Ltd. All rights reserved Widespread use of real-time PCR for rickettsial diagnosis 127 routinely used for the diagnosis of rickettsial infections a non-plasmidic gene. Consequently, the set ‘RAF’ was from clinical specimens. used to detect R. africae in clinical samples from January As an FNRC for rickettsioses, we routinely receive clin- 2010 to December 2010. ical samples from patients with suspected rickettsiosis. We retrospectively collected data for the molecular These samples are obtained from both locally hospitalized diagnosis of rickettsioses from January 2009 to December patients and from outpatients throughout France and the 2010 to assess the usefulness of this strategy. rest of the world. Total DNA was extracted from the sam- Except for the ‘SFG’ set, which had been previously ples using a QIAmp DNA Mini kit (Qiagen, Hilden, Ger- described (Socolovsch et al., 2010), the sets were found to many) as described in the manufacturer’s instructions. be specific for the corresponding rickettsial species both Master mixtures were prepared with a QuantiTect Probe in silico and in vitro, when tested against a panel of 30 PCR kit (Qiagen) following the manufacturer’s instruc- rickettsial strains (Fig. 1a). Sensitivity was also evaluated tions. Sterile human biopsies were used as negative con- using 10-fold serial dilutions (Fig. 1b). trols; DNA extracted from the cell culture supernatant of A total of 643 clinical specimens corresponding to 465 Rickettsia montanensis served as a positive control when different patients were received at the FNRC from Janu- using the primer and probe set targeting SFG Rickettsia; ary 2009 to December 2010. Among these, 204 originated DNA extracted from the cell-culture supernatant of each from locally hospitalized patients, 218 from other French Rickettsia species served as a positive control for the cor- hospitals and 43 from international hospitals. Forty-five responding primer and probe set. Appropriate handling positive qPCRs were obtained: 31/150 cutaneous biopsies, and DNA extraction were controlled using qPCR target- 8/42 cutaneous swab specimens, 2/223 total blood sam- ing the gene encoding b-actin (Socolovsch et al., 2010). ples and 4/94 serum samples. The first molecular screen- qPCR assays were performed in a LightCycler 3.5 instru- ing of SFG Rickettsia using the set labelled ‘SFG’ was ment (Roche Diagnostics, Mannheim, Germany). The positive for 44 samples; the 45th sample was positive PCR mixture included a final volume of 20 lL with using the set labelled ‘TG’, which detects TG Rickettsia. À 10 lL of the Master mixture, 0.5 lL (20 pmol lL 1)of Among 45 positive results, 11 were obtained from locally À each primer, 2 lL (2 pmol lL 1) of probe, 2 lL of dis- hospitalized patients, 32 from other French hospitals and tilled water and 5 lL of extracted DNA. The amplification two from international hospitals. conditions were as follows: an initial denaturation step at A final diagnosis of R. africae was obtained for 15 sam- 95 °C for 15 min, followed by 40 cycles of denaturation ples (13 cutaneous biopsies, two eschar swabs) corre- at 95 °C, annealing and elongation at 60 °C for 60 s, with sponding to 15 different patients with a diagnosis of fluorescence acquisition in single mode. ATBF; five samples were positive for the sets ‘SFG’ and The first molecular screening was systematically per- ‘RAF-plasmid’, and 10 samples were positive for the formed with a set of primers and a probe targeting SFG Rick- sets ‘SFG’ and ‘RAF’. A final diagnosis of R. conorii was ettsia; if clinically and epidemiologically suspected a obtained for nine samples corresponding to nine different screening was performed to target TG Rickettsia. Based on patients with a diagnosis of MSF; eight samples (cutane- clinical and epidemiological investigations and on serologi- ous biopsies) were positive for the sets ‘SFG’ and ‘RCO’. cal results, if first screening was positive, a second directed One remaining sample (serum) was positive for the set step of molecular screening was performed to target Rickett- ‘SFG’ and negative for ‘RCO’; a final diagnosis of R. co- sia spp. at the species level using various sets of primers and norii was obtained using conventional PCR followed by probes. When diagnosis at the species level could not be sequencing. A final diagnosis of R. honei was obtained for obtained using specific qPCR, conventional PCR followed one sample (serum) corresponding to a patient whose by sequencing of the gltA, ompA and/or ompB genes was per- final diagnosis was FISF (Murphy et al., 2011); it was formed (Parola et al., 2005). For the ‘SFG’ set, a mean cycle positive for the set ‘SFG’, and a final diagnosis of R. honei threshold (Ct) value below 35 indicates the sample is posi- was obtained by sequencing because no specific primer tive, and a Ct value above 35 indicates the sample is positive and probe set was available in our laboratory (Murphy if another set is positive and/or a sequence is obtained and/ et al., 2011). A final diagnosis of R. sibirica ssp. mongoli- or serology is positive. Thus, samples are run in duplicate timonae was obtained for five samples corresponding to using sets targeting two different genes. four different patients with a diagnosis of LAR, including From January 2009 to December 2009, the set ‘RAF- a person returning from Egypt (Socolovschi et al., 2010). plasmid’ was used to detect R. africae; its target gene is The samples (three cutaneous biopsies, two eschar swabs) located on a plasmid of the species. Following recent were positive for the set ‘SFG’. A final diagnosis of R. sib- R. africae genome sequencing, it was reported that this irica ssp. mongolitimonae was obtained using conventional plasmid might be unstable. To avoid false-negative PCR followed by sequencing because no specific primer results, we designed a new primer and probe set targeting set was available in our laboratory. A final diagnosis of

(a) Panel of rickettsial strains used to evaluate specificity of primers and SFG probes (specificity was evaluated using a dilution of 10–3) (b) 45 TG T Rickettsia aeschlimannii MC16 40 RAF T Rickettsia africae ESF-5 RCO T 35 Rickettsia akari MK RSLO Rickettsia amblyommii Darkwater 30 RAUS Rickettsia asiatica IO-1 T 25 T Rickettsia australis Phillips 20 Rickettsiabellii 369L42-1T T 15

Rickettsia canadensis 2678 Cycle thresholds Rickettsia conorii subsp. caspia A-167 T 10 10–10 10–9 10–8 10–7 10–6 10–5 10–4 10–3 10–1 1 Rickettsia conorii subsp. conorii Malish 7 T Rickettsia conorii subsp. israelensis ISTTCDC1 T Ten-fold serial dilutions Rickettsiafelis URRWXCal2T Rickettsia heilongjiangensis 054T (c) 45 RAF Rickettsia helvetica C9P9 T RSLO T Rickettsia honei RB 35 Rickettsia hulinii HL-93 T Rickettsia japonica YM 25 Rickettsia massiliae Mtu1 T

Rickettsia montanensis M/5-6 T Cycle thresholds 15 Rickettsia parkeri Maculatum20 T 4 01 0 Rickettsia prowazekii ME + 00 + 02 + 03 + E + .E .E .E .E + 05 Rickettsia raoultii DnS14 1.E 1. 1 1 1 1 Ten-fold serial dilutions Rickettsia raoultii strain RpA4 (bacteria mL–1) Rickettsia rhipicephali 3-7-6 T Rickettsia rickettsii Rickettsia sibirica subsp. mongolitimonae HA-91 T Rickettsia sibirica subsp. sibirica 246T Rickettsia slovaca 13-B T Rickettsia tamurae strain AT1 Rickettsia typhi Wilmington T Orientia tsutsugamushi Kato

Fig. 1. Sensitivity and speciﬁcity of the newly designed primers and probes. (a) Panel of 30 rickettsial species used in this study to evaluate speciﬁcity. (b) Evaluation of the sensitivity of the different sets of primers and probes used in this study: for the set detecting SFG Rickettsia, the evaluation of sensitivity presented here was performed using Rickettsia africae DNA; for TG Rickettsia, Rickettsia typhi DNA was used; for R. africae, R. africae DNA was used; for Rickettsia conorii, R. conorii DNA was used; for Rickettsia slovaca, R. slovaca DNA was used; for Rickettsia australis, R. australis DNA was used. (c) Determination of the sensitivity for the sets of primers and probes targeting R. africae and R. slovaca with a known inoculum. They present a limit of detection at 103 bacteria mLÀ1 for the set ‘RAF’ and 1 bacterium mLÀ1 for the set ‘RSLO’.

R. australis was obtained for two samples (cutaneous samples, the Ct obtained using the set ‘SFG’ was signifi- swabs) corresponding to a single patient with a diagnosis cantly higher compared with the positive samples identi- of QTT. The samples were positive for both ‘SFG’ and fied at the species level (36.71/31.95, P = 0.0023). ‘RAUS’. A final diagnosis of R. slovaca was obtained for For one diagnosis of R. honei and five diagnoses of four samples (cutaneous biopsies) corresponding to three LAR, molecular diagnosis was performed by first screen- different patients with a diagnosis of SENLAT. Three ing using the ‘SFG’ set and then sequencing because spe- samples were positive for both the ‘SFG’ and the ‘RSLO’ cific primers and probes were not available. The need to sets. One remaining sample (serum) was positive for the resort to sequencing suggests the genomic databases must set ‘SFG’ and negative for ‘RSLO’; a final diagnosis of be updated regularly to develop new systems of primers R. slovaca was obtained using conventional PCR followed and probes. Increased genomic data for Rickettsia species by sequencing. A diagnosis of TG Rickettsia was obtained will permit the development of accurate qPCR tools. for one sample (serum) using the set ‘TG’; this sample For eight clinical samples, a diagnosis of rickettsiosis corresponded to a patient with a diagnosis of murine was obtained by systematic screening using the ‘SFG’ set. typhus. Diagnosis at the species level was obtained by However, identification at the species level (by different Western blot followed by cross-adsorption. sets of species-specific qPCRs or by conventional PCRs The remaining eight samples (three cutaneous biopsies, targeting gltA and ompA) remained unsuccessful. We two cutaneous swabs, two total blood and one serum) demonstrated that the Ct values for such samples are sig- were positive for the set ‘SFG’, but we could not discrimi- nificantly higher, suggesting that the ‘SFG’ set is more nate at the species level using either molecular or serolog- sensitive than conventional PCR (Angelakis et al., 2009); ical techniques. These samples corresponded to eight however, molecular tools for diagnosis at the species level patients with a diagnosis of rickettsiosis. For these eight are not yet sufficiently sensitive. In such cases, the use of

ª 2011 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 64 (2012) 126–129 Published by Blackwell Publishing Ltd. All rights reserved Widespread use of real-time PCR for rickettsial diagnosis 129 highly sensitive techniques, such as suicide PCR or Wes- Fournier PE, Drancourt M & Raoult D (2007) Bacterial tern blot followed by cross-adsorption, remains necessary genome sequencing and its use in infectious diseases. Lancet (Fournier & Raoult, 2004; Parola et al., 2005). Infect Dis 7: 711–723. Among the positive clinical samples, 68.9% (31/45) Henry KM, Jiang J, Rozmajzl PJ, Azad AF, Macaluso KR & were cutaneous biopsies, 17.8% (8/45) were cutaneous Richards AL (2007) Development of quantitative real-time swabs, 4.4% (2/45) were total blood samples and 8.9% PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted (4/45) were serum samples. The identification of fever. Mol Cell Probes 21:17–23. rickettsial infections using cutaneous swab specimens and Holland CA & Kiechle FL (2005) Point-of-care molecular PCR testing has recently been reported (Bechah et al., diagnostic systems – past, present and future. Curr Opin 2011; Mouffok et al., 2011); based on these preliminary Microbiol 8: 504–509. results, we collected cutaneous swabs from patients rather Jensenius M, Fournier PE, Kelly P, Myrvang B & Raoult D than cutaneous biopsies. Our retrospective analysis recov- (2003) Afican tick bite fever. Lancet Infect Dis 3: 557–564. ered eight positive cutaneous eschar swabs from different Kidd L, Maggi R, Diniz PP, Hegarty B, Tucker M & Breitschwerdt patients, confirming that these provide a rapid and simple E (2008) Evaluation of conventional and real-time PCR assays means method that can be performed easily without the for detection and differentiation of spotted fever group risk of the side effects related to biopsy collection in rickettsia in dog blood. 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Fournier PE & Raoult D (2004) Suicide PCR on skin biopsy online version of this article: specimens for diagnosis of rickettsioses. J Clin Microbiol 42: Table S1. Primers and probes used in this study. 3428–3434. Fournier PE, Gouriet F, Brouqui P, Lucht F & Raoult D Please note: Wiley-Blackwell is not responsible for the (2005) Lymphangitis-associated rickettsiosis, a new content or functionality of any supporting materials sup- rickettsiosis caused by Rickettsia sibirica mongolotimonae: plied by the authors. Any queries (other than missing seven new cases and review of the literature. Clin Infect Dis material) should be directed to the corresponding author 40: 1435–1444. for the article.