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Am. J. Trop. Med. Hyg., 70(4), 2004, pp. 351-356
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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DEVELOPMENT OF A QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ASSAY SPECIFIC FOR ORIENTIA TSUTSUGAMUSHI

JU JIANG, TEIK-CHYE CHAN, JOSEPH J. TEMENAK, GREGORY A. DASCH, WEI-MEI CHING, AND ALLEN L. RICHARDS
Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Division of Vaccines and Related Products Applications, Food and Drug Administration, Rockville, Maryland; Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, Maryland


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5,552 copies/µL of mouse blood, 14,448-86,012 copies/µL of mouse liver/spleen homogenate, and 3-21 copies/µL of monkey blood.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scrub typhus is a mild to fatal disease depending on both the Orientia tsutsugamushi strain encountered and the genetic background and physical condition of the patient. The disease typically presents with fever, headache, maculopapular rash, eschar (pathognomic lesion), lymphadenopathy, and central nervous system involvement.1,2 Accurate diagnosis is important since without appropriate and effective antibiotic treatment, serious disease and high mortality rates (up to 50%) can occur.1–5 Scrub typhus is caused by infection with O. tsutsugamushi following the bite of an infected trombiculid mite. The mite acts as both the reservoir and vector, but only the parasitic larval stage (chigger) feeds on humans and rodents. Geographic distribution of the disease occurs within an area of about 13 million km2 including Afghanistan and Pakistan to the west; Russia to the north; Korea and Japan to the northeast; Indonesia, Papua New Guinea, and northern Australia to the south; and some smaller islands in the western Pacific.1,5–7 Scrub typhus has been contracted in undisturbed rain forests, secondary vegetative growth areas, plantations, rice paddies, riverbanks, semiarid deserts, and urban areas.2,7–10

Historically, laboratory diagnosis of scrub typhus was most often performed by recognition of serum reactivity to Proteus mirabilis Kingsbury strain antigen (OXK) in the Weil-Felix test. Although the test is easy to perform and inexpensive, its lack of specificity and sensitivity (less than 50% of cases during the second week of illness are reactive) has led to the use of serologic assays that use O. tsutsugamushi antigens.11,12 These assays include an indirect immunofluorescent antibody test,13 an indirect immunoperoxidase assay,12,14 an enzyme-linked immunosorbent assay (ELISA),15 and a dot-blot immunoassay.16 Recently, diagnostic assays using the immunodominant 56-kD outer membrane protein antigens17–21 have eliminated the need to grow and purify O. tsutsugamushi in a biosafety level (BSL)-3 containment laboratory. Increased use of antigen-specific ELISAs and rapid flow through assays are expected due to an enhanced stability and consistency of the antigen preparation as well as a reduction in cost, transport, hazard, and reproducibility problems presently associated with whole-cell antigen preparations.22 However, these serologic assays are still limited in clinical value because development of a detectable antibody response to O. tsutsugamushi usually occurs in the second week of illness,23 there are no minimal titers established for diagnostic tests, and paired sera showing a rise in antibody titer are recommended for laboratory diagnosis of scrub typhus.24

Agent detection can be accomplished earlier after onset of disease than antibody detection and therefore decreases the time required for specific diagnosis. The most definitive means of agent detection is by culture of O. tsutsugamushi in laboratory animals, yolk sacs of embryonated chicken eggs, or tissue culture.25 However, the procedures are slower and less efficient than serologic assays and require a sophisticated BSL-3 laboratory that most diagnostic laboratories do not have.

Due to the very low peak concentration of O. tsutsugamushi in patients’ blood (approximately 5–10 organisms/µL), direct detection of the agent-specific antigens by antigen capture methods has been insensitive and requires preconcentration of the antigen.26,27 However, with the advent of the polymerase chain reaction (PCR), where agent-specific nucleic acid can be amplified a million-fold, O. tsutsugamushi DNA can be readily detected with great sensitivity in clinical specimens such as blood and tissue samples,28–30 as well as in arthropod vectors.31,32 This sensitivity has been enhanced by the development of the nested PCR assays that are performed using two separate amplification steps.32 In addition to detection, the amplicon can be used for restriction fragment length polymorphism (RFLP) typing or sequence analysis.30–32

Real-time quantitative PCR assays are as sensitive as nested PCR assays but have the additional advantages of 1) faster results, 2) potential for automation for high throughput, 3) multiplexing, and 4) quantitative information useful for clinical monitoring of appropriate response to treatment and prognosis, and for experimental studies. In this report, we describe the development of a real-time quantitative PCR (qPCR) procedure with an agent-specific fluorescent probe able to detect O. tsutsugamushi nucleic acid at a lower limit of detection of 3-10 target sequence copies/µL. Furthermore, we have demonstrated the successful application of this qPCR methodology by quantitating O. tsutsugamushi nucleic acid in cynomolgus monkey and Swiss CD-1 mouse tissue samples.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primers and probes. Sequences of primers and probe for O. tsutsugamushi were selected from the 47-kD outer membrane protein gene based on the sequences of the Karp, Kato, Gilliam, Boryong (L31934, L11697, L31933, and L319335 GenBank numbers, respectively), and TH1817 (Dasch GA, unpublished data) strains using Primer Express version 1.0 software (PE Applied Biosystems, Foster City, CA). The primer set (forward primer OtsuFP630: 5'-AAC TGA TTT TAT TCA AAC TAA TGC TGC T-3' and degenerate reverse primer OtsuRP747: 5'-TAT GCC TGA GTA AGA TAC RTG AAT RGA ATT-3') was capable of producing an amplicon of 118 basepairs. The 31-basepair fluorescent Taq-Man probe OtsuPR665 (5'-6FAM-TGG GTA GCT TTG GTG GAC CGA TGT TTA ATC T-TAMRA-3') was labeled at the 5'-end with 6-carboxyfluorescein (FAM) reporter dye and at the 3'-end with 6-carboxytetramethylrhodamine (TAMRA) quencher dye. The oligonucleotide primers were synthesized by Sigma Genosys (The Woodlands, TX), and the probe was synthesized at PE Applied Biosystems.

Sequences of primers and probe for the Rickettsia-specific qPCR assay were selected from a 17-kD gene consensus sequence derived from 21 species of Rickettsia. The degenerate forward primer R17K135F: 5'-ATG AAT AAA CAA GGK ACN GGH ACA C-3', the degenerate reverse primer R17K249R: 5'-AAG TAA TGC RCC TAC ACC TAC TC-3' and probe R17Kbprobe: 5'-6FAM-CGC GAC CCG AAT TGA GAA CCA AGT AAT GCG TCG CG-Black Hole Quencher (BHQ)-1-3' were synthesized by Sigma Genosys. Degenerate positions contained equal molar base concentrations of adenine, guanine, cytosine, and thymine (N); guanine and thymine (K); adenine and guanine (R); and adenine, thymine, and cytosine (H). The presence of a target sequence of the small subunit ribosomal RNA (16S rRNA) gene was detected by a PCR assay using conserved eubacterial primers (forward: 5'-GTT CGG AAT TAC TGG GCG TA-3' and reverse: 5'-AAT TAA ACC GCA TGC TCC AC-3') as previously described.33

Nucleic acids. Three panels of nucleic acids used to assess the specificity of the PCR assays included 26 O. tsutsugamushi DNAs, 17 Rickettsia DNAs and 18 other bacterial DNAs (Table 1Go). The DNAs were extracted from the bacterial cultures34–39 (Taye AB and others, unpublished data) by an automated extraction method and a nucleic acid extractor (Model 340A; Applied Biosystems) as previously described.31 The bacteria were grown either in L929 cells (O. tsutsugamushi, R. africae, R. sharonii, R. parkeri, and R. rhicpicephali), Vero cells (other spotted fever group Rickettsia and R. bellii), P388D1 (Neorickettsia), and DH82 (Ehrlichia), chicken embryonated yolk sac (R. prowazekii, R. typhi, and Francisella persica), or on standard bacterial media (all other bacteria). Cat fleas, obtained from FleaData, Inc. (Freeville, NY), were constitutively infected with R. felis (> 95%)40 and used to extract nucleic acid containing R. felis DNA with the DNeasy tissue kit (Qiagen, Valencia, CA). For the qPCR, the plasmid pWMC-Kt47, which contains the entire open reading frame of the 47-kD antigen gene of O. tsutsugamushi Kato strain in pVR1012 (Vical, San Diego, CA), was used in the amount of 107–100 copies/µL.


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TABLE 1
Orientia tsutsugamushi–specific sPCR and qPCR*
 
Swiss CD-1 mouse blood and tissue (liver/spleen) samples were collected 7 and 10 days after infection with O. tsutsugamushi Karp and Gilliam strains, respectively. The DNA was extracted from 150 µL of blood and 25 µL of homogenized liver/spleen samples using The Neasy tissue kit and eluted in 100 µL and 200 µL of the kit AE buffer (Qiagen), respectively.

Cynomolgus monkeys were infected with different amounts of O. tsutsugamushi Karp strain, bacteria and blood samples were collected every other day post-infection. The DNA was extracted from 100 µL of blood from each monkey using the DNeasy tissue kit and eluted in 50 µL of the kit AE buffer (Qiagen).

The maintenance and care of experimental animals complied with the Animal Welfare Act and the National Institutes of Health guidelines for the humane use of laboratory animals. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 1996.

Standard PCR (sPCR) and Real-time qPCR. The sPCR was conducted in 50-µL reaction volumes containing 1 µL of DNA template, 5 µL of 10x PCR buffer with 15 mM MgCl2 (Perkin Elmer, Foster City, CA), 5 µL of 2 mM dNTPs (Idaho Technology, Salt Lake City, UT), 2.5 µL of each 10 µM primer, and 0.25 µL (5 units/µL) of AmpliTaq Gold DNA polymerase (Perkin Elmer). The reaction mixtures were incubated at 95°C for 10 minutes followed by 45 cycles of three-step amplification at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for seven minutes using a GeneAmp PCR system 9700 (Perkin Elmer). The PCR amplicons were visualized and compared to molecular weight standards (100-basepair ladder; Invitrogen, Carlsbad, CA) after electrophoresis on 2% aga-rose gels at 150 volts for approximately 40 minutes in a Horizon 58 gel electrophoresis system (Gibco-BRL Life Technologies, Inc., Gaithersburg, MD) and staining with ethidium bromide (Gibco-BRL Life Technologies, Inc.).

The real-time qPCRs for the O. tsutsugamushi 47-kD and the Rickettsia 17-kD gene assays were conducted in a total volume of 25 µL of qPCR mixture consisting of 1 µL of DNA template, 2.5 µL of 10x PCR buffer with 50 mM MgCl2, 2.5 µL of 2 mM dNTPs (Idaho Technology), 0.25 µL (5 units/µL) of platinum Taq DNA polymerase (Invitrogen), 0.25 µL (10 µM) of each primer, and 0.5 µL (10 µM) of probe for the O. tsutsugamushi 47-kD assay or 0.75 µL (10 µM) of primer and 1.0 µL (10 µM) of probe for the Rickettsia 17-kD assay. Both qPCRs were incubated at 94°C for five minutes followed by 45–50 cycles of two-step amplification at 94°C for five seconds and 60°C for 30 seconds on a Cepheid (Sunnyvale, CA) SmartCycler System. Fluorescence was monitored during every thermal cycle at annealing step and data were analyzed with SmartCycler software (version 1.2b). Initial sensitivity and specificity determinations for the O. tsutsugamushi assay were conducted with the ABI Prism7700 Sequence Detection System (Applied Biosystems) and results were similar to those obtained with the Cepheid SmartCycler. Three no template controls were consistently negative for each reaction. Plasmid pWMC-Kt47 DNA (105 copies/µL) was used as a positive control and gave consistent threshold (Ct) values between 24 and 25 cycles for the O. tsutsugamushi 47-kD assay.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The O. tsutsugamushi 47-kD sPCR assay was first optimized with DNA extracted from the Karp strain of O. tsutsugamushi, and its specificity was evaluated with nucleic acid preparations from 19 strains of O. tsutsugamushi, 13 species of Rickettsia and 16 species of other bacteria (Table 1Go), and several eukaryotic cell DNAs (mouse, dog, flea, monkey, and chicken). The appropriate size amplicon (118 kb) was produced from all 19 strains of O. tsutsugamushi, but not from Rickettsia or other bacteria nucleic acid samples assessed, or the host DNAs in which the obligate intracellular bacteria were cultivated, indicating that the sPCR assay was specific for O. tsutsugamushi.

To decrease the time required to obtain a result and to accurately and sensitively quantitate the number of positive templates in the original samples, we developed a qPCR assay using a probe specific for a 31-basepair sequence within the O. tsutsugamushi 118-basepair amplicon. The qPCR assay detected all 26 strains of O. tsutsugamushi evaluated, but did not detect 17 Rickettsia or 18 other bacteria DNA preparations, or the host cell DNA extracted concurrently with the obligate intracellular bacteria (Table 1Go). The integrity of the Rickettsia and bacterial DNAs were confirmed with a qPCR assay based upon the conserved Rickettsia 17-kD antigen gene and a sPCR assay for the 16S rRNA gene (Table 1Go). The 26 strains of O. tsutsugamushi originated from seven different countries (Papua New Guinea, Japan, Burma, Thailand, China, Malaysia, and Australia) and were genetically quite diverse. Fourteen of the 26 strains were assessed in another study in which the 56-kD outer membrane protein gene sequences of 25 strains were determined and compared to each other and to 63 published strain sequences. The 14 strains from this study were well distributed with in the phylogenitc tree developed from the 88 total strains and had sequence homologies with the other 74 strains analyzed that ranged from a low of 70% to a high of 100% (Taye AB and others, unpublished data). In addition, Dasch and others determined that with the use of PCR-RFLP analysis, the19 strains included in this study also varied genetically among themselves and among 92 other strains evaluated.36

To determine the sensitivity of the real-time PCR assay for O. tsutsugamushi, we used plasmid pWMC-Kt47, which contains the open reading frame sequence of the Kato 47-kD gene that was ligated into the plasmid VR1012 (Vical). The concentration of the plasmid was determined by an optical density reading at 260 nm.41 Serial ten-fold dilutions of pWMC-Kt47 in molecular biology-grade water (Sigma Chemical Company, St. Louis, MO) were performed, resulting in final target concentrations of 107–100 copies/µL. This assay consistently detected between 3 and 10 copies of the target sequence per reaction.

To evaluate the utility of the qPCR assay with laboratory-derived animal tissue samples, mouse and monkey blood and mouse liver/spleen homogenates infected with O. tsutsugamushi were tested. Blood collected from Swiss CD-1 outbred mice after inoculation with 1,000 50% murine lethal dose (MuLD)50 produced 27-5,552 copies/µL of blood collected 7 and 10 days post-infection (Table 2Go). Homogenized liver/ spleen samples from CD-1 mice inoculated with 1,000 MuLD50 of either the Karp or Gilliam strain on days 7 and 10, respectively, were found to be positive (14,448–86,012 copies/µL) by the qPCR assay for the O. tsutsugamushi 47-kD gene (Table 2Go). Blood samples collected from six cynomolgus monkeys infected with 106-100 MuLD50 produced 3–21 copies/µL of blood on days 8–18, depending upon the titer of the inoculum (Table 2Go). Enzyme-linked immunosorbent assays performed on these same blood samples indicated that Orientia-specific antibodies developed 4–8 days after detection of Orientia-specific DNA (Chattopadhyay S and others, unpublished data). These results are similar to those of earlier studies in which the presence of Orientiae in the blood of scrub typhus patients was shown to precede the humoral response of the host by approximately one week.26,42


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TABLE 2
Real-time quantitative polymerase chain reaction (qPCR) detection and quantitation of Orientia tsutsugamushi in mouse and monkey tissues*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The 47-kD gene sequences of O. tsutsugamushi strains Karp, Kato, Gilliam and Boryong are similar to those of the genes of the high-temperature requirement (HtrA) family of stress response proteins that have both chaperone and endoprotease activities (Chao C-C and others, unpublished data).43 The HtrA gene is induced by different environmental stress conditions (e.g., elevated temperature) in a variety of bacteria (Escherichia coli originally as DegP, R. prowazekii, R. typhi, R. conorii, Haemophilus influenzae, Brucella abortus, Yersinia entercolitca, Salmonella enterica, etc.) and have been shown to contribute to the envelope protein management and potentially play a role in the pathogenesis for some of these species.43 The HtrA family of proteins has also been found in eukaryotic organisms including humans (hHtrA1, hHtraA2, hHtrA3, and hHtrA4) where their activities appear to be more diverse, including roles in cell protein regulation, tumor suppression, and apoptosis.44 Because of the immunogenicity and conservation of these genes and gene products, they have a potential role as reagents for diagnostic assays when target-specific domains can be identified.

In developing a PCR diagnostic assay for O. tsutsugamushi, primers were designed based upon sequences of this 47-kD antigen gene that were conserved among five strains of O. tsutsugamushi (Karp, Kato, Gilliam, Boryong, and TH1817), but not found in the HtrA gene of the closely related genus Rickettsia (GenBank Y11782 R. prowazekii, AE008583 R. conorii) or in the human homologs. The sequence for the 47-kD of R. typhi (HtrA homolog) currently listed in Genbank (D78346) and previously reported suggests a sequence very similar to that of O. tsutsugamushi 47-kD antigen gene,43 but it is not found in the newly determined R. typhi genome sequence (Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, http://hgsc.bcm.tmc.edu/microbial/Rtyphi). We did not observe a product produced by our primers in the O. tsutsugamushi 47-kD sPCR assay or a positive response in the O. tsutsugamushi 47-kD qPCR assay with two strains (Wilmington and Museibov) of R. typhi (Table 1Go). However, we did observe a positive reaction for these two R. typhi nucleic acid preparations with real-time PCR assays developed for the Rickettsia 17-kD antigen gene (Table 1Go) and the R. typhi outer membrane protein B gene (Flavin M and others, unpublished data). This suggests that what is currently listed as a sequence for R. typhi 47-kD in GenBank may be in error.

The presently described qPCR assay is clearly useful for the detection and enumeration of O. tsutsugamushi in research samples and laboratory animal specimens. Thus, the assay could be used to monitor scrub typhus vaccine and antibiotic efficacy in animal models. To date, this qPCR assay has been used successfully in our laboratory to evaluate the efficacy of the scrub typhus vaccine candidate, Kp r56, in mice and cynomolgus monkeys challenged by O. tsutsugamushi delivered by needle inoculation (Chattopadhyay S and others, unpublished data). With the development of a chigger challenge model for vaccine efficacy studies, this qPCR assay will be critical in the determination of the challenge dose of the O. tsutsugamushi provided by the infected chigger, a previously recognized fault associated with this method. Currently, the standard procedure for O. tsutsugamushi challenge of vaccinated laboratory animals is by the unnatural needle inoculation method, where knowledge of concentration of the dose is known. This needle challenge method could be supplanted by the chigger challenge procedure with the knowledge of the challenge dose of the chigger provided by the qPCR assay.

In addition, to the use of this assay with experimental animal specimens, it can also be used with human samples. Presently, this qPCR assay has been used to detect and enumerate O. tsutsugamushi Karp in infected human peripheral blood mononuclear cells (Rentas FJ and others, unpublished data). Therefore, this assay should be useful in diagnosing scrub typhus by detecting O. tsutsugamushi in patient samples (blood, eschar, and rash biopsies) shortly after symptoms appear. This early and rapid diagnosis would allow the initiation of agent-specific therapy, offering the best opportunity for quick and complete recovery of the patient.

Received September 24, 2003. Accepted for publication December 14, 2003.

Acknowledgments: We gratefully acknowledge Steve Yevich, Kathryn DeCarlo, Won Suh, and Meaghan Pimsler from the Thomas Jefferson High School for Science and Technology (Alexandria, VA) mentorship program for their invaluable technical support and discussions through the progress of this work.

Financial support: This work was supported by U.S. Department of Defense work unit numbers B998.0000.000.A0074 and B998.0000.000.A0310.

Disclaimer: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.

Authors’ addresses: Ju Jiang, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7249, Fax: 301-319-7460, E-mail: JiangJ{at}nmrc.navy.mil. Teik-Chye Chan, Rickettsial Diseases Department Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7436, Fax: 301-319-7460, E-mail: ChanC{at}nmrc.navy.mil. Joseph J. Temenak, Bacterial Vaccines and Allergenic Products Branch, Division of Vaccines and Related Products Applications, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Department of Health and Human Services, Rockville, MD 20852, Telephone: 301-827-3070, Fax: 301-827-3532, E-mail: Temenak{at}cber.fda.gov. Gregory A. Dasch, Rickettsial Diseases Section, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Centers for Infectious Disease, Centers for Disease Control and Prevention, Mail-stop G-13, 1500 Clifton Road, Atlanta, GA 30033, Telephone: 404-639-4140, Fax: 404-639-4436, E-mail: ged4{at}cdc.gov. Wei-Mei Ching, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7438, Fax: 301-319-7460, E-mail: ChingW{at}nmrc.navy.mil. Allen L. Richards, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7668, Fax: 301-319-7460, E-mail: RichardsA{at}nmrc.navy.mil.

Reprint requests: Allen L. Richards, Rickettsial Diseases Department Naval Medical Research Center 503 Robert Grant Avenue Silver Spring, MD 20910-7500.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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