Development and Validation of a Quantitative Real-Time Polymerase Chain Reaction Assay Specific for the Detection of Rickettsia felis and Not Rickettsia felis -Like Organisms

Abstract

Human infections with Rickettsia felis have been reported worldwide. Recent studies have revealed the presence of many closely related but unique rickettsiae, referred to as Rickettsia felis–like organisms (RFLO), identified in various arthropods. Due to the recent discovery of the lack of specificity of earlier R. felis–specific assays, there has become a need to develop a new generation of R. felis–specific molecular assays that will differentiate R. felis not only from other rickettsiae but more importantly from other members of the R. felis genogroup that may not be pathogenic to humans. This new generation of assays is essential for determining the true risk for flea-borne spotted fever (FBSF) by surveying arthropod vectors/hosts. Because of the lack of specificity of previous assays developed to detect R. felis infections, prior surveys may have overestimated the prevalence of R. felis in arthropod vectors and thus the perceived risk of FBSF. We have developed a specific quantitative real-time polymerase chain reaction (qPCR) assay to detect R. felis (RfelB). Specificity of the assay was determined by testing it with a panel of 17 related Rickettsia species and 12 nonrickettsial bacterial DNA preparations. The RfelB qPCR assay was positive for R. felis DNA and negative for all of the 17 related Rickettsia species and 12 nonrickettsia bacterial DNA preparations. The limit of detection of the RfelB qPCR assay was determined to be two copies (two genoequivalents) per microliter of R. felis target ompB fragment–containing plasmid. Validation of the RfelB qPCR assay was accomplished by testing 83 previously sequence-confirmed R. felis and RFLOs containing DNA preparations from human and flea samples collected from different geographical locations around the world. This assay will be useful for rapid detection, identification, and enumeration of R. felis, an emerging human pathogen of worldwide importance, from both clinical and environmental samples.

Introduction

Rickettsia felis is an obligate intracellular gram-negative bacteria and etiologic agent of flea-borne spotted fever (FBSF) (Adams et al. 1990, Raoult and Roux 1997). FBSF is characterized by signs and symptoms common to other rickettsioses, including fever, headache, myalgia, and rash. The presence of R. felis has been reported worldwide both in humans and arthropods (Parola 2011). It is transmitted to humans by the cat flea (Ctenocephalides felis), which is considered to be the primary vector and reservoir. R. felis has also been detected in other flea species, namely Ctenocephalides canis, Xenopsylla cheopis, Pulex irritans, Tunga penetrans, Echidnophaga gallinacea, ticks, and other nonbiting insects (Hun and Troyo 2012). Complicating the determination of the true prevalence of R. felis in arthropods during the last decade has been an increasing number of reports of Rickettsia felis–like organisms (RFLOs) identified in various arthropods around the world.

In Asia, Rickettsia sp. RF2125 and Rickettsia sp. RF31 were detected in Ctenocephalides species near the Thailand–Myanmar border (Parola et al. 2003) and in Malaysia (Mokhtar and Tay 2011). Other RFLOs reported in cat and dog fleas in Asia include the Rickettsia sp. cfl and cf5 and Rickettsia sp. SE313 in Bangkok (Foongladda et al. 2011) and a Rickettsia sp. detected in Pediobius rotundatus in Iran (Weinert et al. 2009).

In Africa, Rickettsia sp. SE313 and RF2125 genotypes were reported in stick tight flea (E. gallinacea), rat mite (Ornithonyssus bacoti) in Egypt (Loftis et al. 2006, Reeves et al. 2007), and RF2125 in hedgehog fleas (Archaeopsylla erinacei) in Algeria (Bitam et al. 2006). Recent studies have identified the two R. felis genotypes (RF2125, SGL01) in a rare flea (Synosternus pallidus), tsetse flies (Glossina morsitans), and mosquitoes (Anopheles gambiae) in Senegal (Mediannikov et al. 2012, Roucher et al. 2012, Socolovshi et al. 2012). Similar agents were detected in A. gambiae in Gabon (Socolovshi et al. 2012) and in fecal samples of chimpanzee, gorilla, and bonobos in Cameroon and Democratic Republic of Congo (Keita et al. 2013). In Kenya, Candidatus Rickettsia asemboensis (Rickettsia sp. F30), a RFLO with 99% homology to RF2125 was detected in C. felis and C. canis fleas (Jiang et al. 2013).

In North America, Rickettsia sp. RF2125 and RF31 were detected in C. felis in South Carolina (Reeves et al. 2005, Nelder et al. 2009). In South America, Rickettsia spp. TwKM03, Hf187, and RF2125 have been reported in C. canis and C. felis from Uruguay and Peru (Venzal et al. 2006, Forshey et al. 2010).

In Europe, an agent identical to Rickettsia sp. TwKM03 was identified in Ctenophalides spp. from northern Spain (Blanco et al. 2006). Rickettsia sp. RF2125 was found in P. irritans (human flea) in Hungary (Hornok et al. 2010). Rickettsia sp. Cf1 and Cf5 and Rickettsia sp. RF2125 were detected in A. erinacei (hedgehog fleas) in Germany (Gilles et al. 2009).

Even though prevalence of these RFLOs is high in arthropods, their pathogenicity is currently unknown (Mokhtar et al. 2011). Further studies are needed to focus on the pathogenicity of these organisms as well as determining more precisely their identification, prevalence, and distribution. Misdiagnosis, underestimation, or overestimation of FBSF due to R. felis infection could be as a result of a lack of specific molecular methods in most resource constrained countries (Hun and Troyo 2012). Identification of R. felis has previously been determined by using quantitative real-time PCR (qPCR) assays (Hun and Troyo 2012), PCR restriction fragment length polymorphisms (RFLP), or sequencing of one or more gene fragments (Schriefer et al. 1994). Indirect fluorescence antibody (IFA) test is regarded as the gold standard for diagnosis of rickettsioses even though it is complicated, laborious, expensive, and cannot differentiate between species of Rickettsia (La Scola and Raoult 1997). Early detection of R. felis is imperative for patient management of FBSF. The previous assays for detecting R. felis (e.g., Henry et al. 2007) have been shown to detect RFLOs (Jiang et al. 2013), necessitating development of a new diagnostic assay specific for R. felis. The objective of this study was to develop and validate a qPCR assay specific for detecting R. felis using R. felis and RFLOs obtained from human and arthropod samples.

Materials and Methods

Primer and probe design

The RfelB qPCR assay was developed targeting a fragment specific to the R. felis outer membrane protein B gene (ompB) (GenBank no. CP000053) to design primers and a fluorescent (FAM- [6-carboxyfluorescein] labeled) Taqman probe. Comparison of the published R. felis ompB sequence and other Rickettsia species was performed using Mega 5 software. A unique 20-base-pair sequence of the R. felis ompB was identified to be the probe for the qPCR assay. The probe sequence was evaluated with Beacon Designer 7.9 software (PREMIER Biosoft, Palo Alto, CA), and the primer sequences that were suitable for working with the probe were then identified. The primers amplified a 97-base-pair fragment of ompB (Tables 1 and 2). The oligonucleotide primers and probe were synthesized by Eurofins MWG Operon (Huntville, AL).

Table 1.

RfelB-Specific Primers and Probe

Primer	Size (bp)	Oligonucleotide Sequence 5′ to 3′	Tm (°C)	Gene location
RfelB forward	22	5′-TAATTTTAACGGAACAGACGGT-3′	57.1	789–810
RfelB reverse	25	5′- GCCTAAACTTCCTGTAACATTAAAG-3′	59.7	885–860
RfelB probe	20	5′- FAM- TGCTGCTGGTGGCGGTGCTA-BHQ]-3′	66.6	837–857

Table 2.

Alignment Table of OmpB Fragment Sequence of Rickettsia felis, Rickettsia felis–Like Organisms and Other Rickettsial Species, GenBank Accession Numbers, and the Sites of the RfelB qPCR Primer and Probe Sequences

qPCR assay optimization

The RfelB qPCR assay was optimized and validated using the StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA). The final master mix (20 μL) comprised 10 μL of 2× Supermix-UDG (Invitrogen, Foster City, CA), primers, probe, water, magnesium chloride, and 2 μL of template. Optimal conditions for the RfelB qPCR assay were achieved by varying the concentration of the primers (0.2–0.8 μM) and probe (0.1–0.5 μM) by 0.1-μM increments. The concentration of MgCl₂ was varied from 3 mM to 7 mM in increments of 1 mM. Annealing temperatures were varied from 56°C to 61°C. The final thermocycling conditions consisted of first hold at 50°C for 2 min, second hold 95°C for 2 min, and then 45 cycles of denaturation at 95°C for 15 s and annealing/elongation at 59°C for 30 s.

Specificity of the RfelB qPCR assay

Specificity of the assay was determined using a genomic DNA panel of 17 closely related rickettsiae and 12 nonrickettsial bacteria, as described previously (Jiang et al. 2012).

Limit of detection

Limit of detection (LOD) was performed using a synthetic plasmid containing the target sequence of R. felis ompB fragment. The qPCR was performed in triplicate using 10-fold serially diluted plasmid concentrations ranging from 10⁸ to 10⁻¹ copies/μL.

Validation

The RfelB qPCR assay was validated by using previously characterized specimens containing R. felis, R. typhi, and RFLOs collected from fleas (n=63) and humans (n=20). The total number of specimen tested from Kenya were (n=38), Indonesia (n=2), Thailand (n=21), and United States (n=22) (Jiang et al. 2006, Maina et al. 2012, Jiang et al. 2013, Richards et al. unpubl. data). The 83 specimens were tested with the RfelB qPCR assay using the cycling conditions outlined above. Concordance between the RfelB qPCR assay and sequencing was computed using kappa statistics with GraphPad Software Inc. (2013). Strength of agreement based on kappa was judged according to Altman Kappa benchmark guidelines: <0.2=poor; 0.2 to <0.4=fair; 0.4 to <0.6=moderate; 0.6 to <0.8=good; >0.8=very good (Altman 1991).

Results

Optimization

The optimum conditions were determined to be 0.6 μM of the forward and reverse primers, 0.2 μM of probe, 6 mM of MgCl₂, and 59°C annealing temperature. The ideal conditions were defined as the lowest concentration of R. felis DNA that produced consistent positive results.

Specificity

The RfelB assay demonstrated specificity to R. felis. All of the 17 closely related Rickettsia species and the 12 nonrickettsial bacteria species (Table 3) were consistently negative whereas R. felis DNA was consistently detected in all the runs.

Table 3.

Specificity of RfelB Quantitative Real-Time PCR Assay

			16S				16S
	17 kDa	RfelB	rRNA		17 kDa	RfelB	rRNA
Rickettsia species	qPCR	qPCR	sPCR	Non-Rickettsia spp.	qPCR	qPCR	sPCR
R. conorii	+	−	+	Corynebacterium sp.			+
R. parkeri	+	−	+	Bartonella vinsonii	−	−	+
R. montanensis	+	−	+	Bartonella quintana	−	−	+
R. bellii	+	−	+	Proteus mirabilis	−	−	+
R. africae	+	−	+	Escherichia coli	−	−	+
R. prowazekii	+	−	+	Neorickettsia risticii	−	−	+
R. akari	+	−	+	Wolbachia persica	−	−	+
R. rickettsii	+	−	+	Salmonella enterica	−	−	+
R. slovaka	+	−	+	Staphylococcus aureus	−	−	+
R. honei	+	−	+	Orientia tsutsugamushi	−	−	+
R. canadensis	+	−	+	Neorickettsia sennetsu	−	−	+
R. australis	+	−	+	Legionella bozemanii	−	−	+
R. typhi	+	−	+	Ehrlichia sp.	−	−	+
R. felis	+	+	+
R. sibirica	+	−	+
R. ambylommii	+	−	+
R. japonica	+	−	+
R. rhipicephali	+	−	+

Limit of detection

The LOD was determined based on the logarithmic signal amplification of the serially diluted plasmid where the lowest amount of plasmid DNA was estimated to be two copies of the target R. felis plasmid. The standard curve had a slope of −3.352, y intercept of 39.5, and the R ² value of 0.998. The LOD was determined as the lowest concentration of R. felis DNA that produced consistent positive results in 10 out of 10 runs.

Assay validation

We tested the utility of the RfelB qPCR assay by testing samples containing sequence-confirmed DNA from R. felis, RFLOs, and R. typhi collected from arthropods and humans from Kenya, Indonesia, United States, and Thailand. The no template controls (NTC) were consistently negative for all the reactions whereas the positive control gave a consistent cycle threshold value of 21.50±1.00 for R. felis. A total of 83 previously sequenced specimens identified as R. felis (n=51), RFLOs (n=28), and R. typhi (n=4) were tested with the RfelB qPCR assay. Of the 83 sequenced confirmed specimens, 52 were positive for R. felis by the new RfelB qPCR assay whereas 31 were negative for R. felis (Table 4). The observed agreement was 82/83 (98.80%) with a kappa value of 0.974 (95% confidence interval [CI] 0.925–1.024).

Table 4.

Comparison of Positive and Negative Results Obtained by RfelB qPCR Assay and Sequencing (ompB and/or 17-kD Genes) for Detection of Rickettsia felis

Assay+/–	Sequence positive	Sequence negative	Totals
RfelB qPCR positive	51	1	52
RfelB qPCR negative	0	31	31
Totals	51	32	83

Discussion

FBSF due to infection with R. felis is an emerging human disease. Since its identification in 1990 (Adams et al. 1990), R. felis has been reported in more than 20 countries (Abdad et al. 2011, Parola 2011) in all continents but Antarctica (Hun and Troyo 2012) in both humans and arthropods. The ability of clinicians to suspect and diagnose FBSF remains a challenge due to the nonspecific clinical presentation (Abdad et al. 2011). Accurate enumeration of R. felis infections is also hampered by overlapping distribution and cross-reactivity of R. felis and R. typhi (Civen and Ngo 2008), coupled with the rising number of RFLOs. The notable exponential increase in geographical distribution of FBSF warrants development of a robust, rapid, and reliable assay to diagnose the disease in clinical and research settings.

In this report, we have described a sensitive and specific molecular assay that can discriminate R. felis not only from R. typhi but also from other RFLOs. The results of this study show 98.79% (82/83) concordance of RfelB qPCR assay with sequence results of clinical and arthropod samples containing R. felis, R. typhi, and RFLOs DNA. Kappa analysis indicated a very good agreement between the RfelB qPCR assay and sequencing, with an overall k value of 0.974 (95% CI 0.925–1.024). Out of 83 specimens evaluated, both sequencing and the RfelB qPCR assay determined that 51/83 (61.4%) contained R. felis DNA and 31/83 (37.3%) were negative for R. felis DNA. Of the 31 R. felis–negative samples by the new qPCR assay, 10 (32.3%) were collected from Thailand and were positive for the Candidatus R. asemboensis by Rasemb qPCR assay (Jiang et al. 2013) and were 99% identical to Candidatus R. asemboensis as determined by sequencing. Ten of the 31 (32.3%) R. felis–negative specimens were collected from California, of which 4/10 tested positive for R. typhi and 6/10 sequence positive for the RFLOs: RF2125, RF31, Rickettsia spp. Cf1 and Cf5, and Rickettsia sp. SE313 (Parola et al. 2003, Loftis et al. 2006, Nelder et al. 2009, Foongladda et al. 2011). Candidatus R. asemboensis (Jiang et al. 2013) was detected in 11/31 (35.5%) fleas collected from Kenya.

One sample, from Kenya, that tested positive with the RfelB assay had previously been determined by sequencing and the Candidatus R. asemboensis qPCR (Rasemb) assay to contain Candidatus R. asemboensis DNA (Jiang et al. 2013). This flea DNA preparation came from a pool of 13 fleas. Thus two rickettsial agents were found in this pool of fleas. It is unknown whether they came from the same flea or different fleas. A similar scenario has been reported previously where R. felis and R. typhi were detected in a pool of fleas (Jiang et al. 2006). To unravel this problem of pooled flea specimens, future studies should focus on testing individual fleas to determine if co-infections of R. felis and RFLOs do occur.

The genetic diversity in the R. felis genogroup presents a daunting task for developing a molecular assay that is able to detect R. felis and discriminate it from all other RFLOs. This assay was shown to discriminate R. felis from Rickettsia sp. F30 also known as Candidatus R. asemboensis, Rickettsia spp. Cf1 and Cf5, Rickettsia spp. SE313 (Jiang et al. 2013), RF2125, and RF31 (Parola et al. 2003), and Rickettsia sp. RFLO-18 recently identified from C. felis obtained from Thailand (Richards et al. unpubl. data). Further studies are needed to determine the utility of this assay in discriminating R. felis from other RFLOs not included in the validation of the new assay or whose sequences were not available in GenBank for comparison of probe sequences. These include the Rickettsia sp. SGL01, Candidatus R. dielmo and Rickettsia sp. Hf187 (Venzal et al. 2006, Mediannikov et al. 2012, Roucher et al. 2012). Although we did not get the ompB sequence for Candidatus R. dielmo corresponding with the primer sequence for the RfelB assay, the gltA sequence of Candidatus R. dielmo was found to be 100% identical to RF2125 (Roucher et al. 2012).

This will be a two-stage assay, where a negative result does not necessarily mean that the patient/arthropod sample does not have other rickettsial infections. We recommend that any RfelB-negative sample be retested with a pan-rickettsia assay to rule out the possibility of other rickettsial infections.

The RfelB qPCR assay described herein holds promise as a suitable, specific tool that is affordable, easy to perform, and produces rapid results that can be used for detecting R. felis in clinical settings and surveillance studies. Moreover, the RfelB qPCR assay performed well with human clinical samples and arthropod samples from Kenya, Indonesia, Thailand, and the United States, representing specimens from diverse geographical locations. Ultimately, we anticipate this assay will address the problem of underestimation, overestimation, and misdiagnosis (Hun and Troyo 2012). We hope that other clinicians and researchers will find this test useful for future surveillance studies as well as mapping of R. felis infections to detect disease hotspots.

Footnotes

Acknowledgments

The authors wish to thank the Rickettsial Diseases Research Program laboratory team at the Naval Medical Research Center for their support to undertake this project. We would also like to thank George Washington University (GWU) and American Society for Engineering for Education (ASEE) for their invaluable support. This work is supported by the Global Emerging Infections Surveillance and Response System, a Division of the Armed Forces Health Surveillance Center, work unit number 847705.82000.25GB.A0074.

Author Disclosure Statement

The views expressed in this article are those of the authors and do not necessary reflect the official position of the department of the Navy Department of Defense, or the United States Government. As an employee of the US Government, this work was prepared as part of official duties (ALR) and therefore under Title 17 USC paragraph 105 copyright protection is not available.

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