A Highly Sensitive and Specific Real-Time PCR Assay for the Detection of Spotted Fever and Typhus Group Rickettsiae

Stenos, John; Graves, Stephen; Unsworth, Nathan B.

doi:10.4269/ajtmh.2005.73.1083

Cited by 251 publications

(175 citation statements)

References 24 publications

Supporting

Mentioning

173

Contrasting

Unclassified

Order By: Relevance

“…This fi nding is likely to be an underestimate of the actual extent of rickettsial disease because of the small volume of blood tested in each PCR and possible sample deterioration during transport and storage (between sample collection and testing). Although PCR has yet to be extensively evaluated for the diagnosis of murine typhus (5-8), a sizeable body of evidence supports the high sensitivity and specifi city of PCR for the diagnosis of rickettsial diseases (2,4,9,10). The real-time PCR used in our study has a high analytical sensitivity and specifi city for R. typhi (5), and sequencing of amplicons from our patients further supports the specifi city of the assay.…”

Section: Discussionmentioning

confidence: 69%

Murine Typhus and Febrile Illness, Nepal

Zimmerman¹,

Murdoch²,

Rozmajzl³

et al. 2008

Emerg. Infect. Dis.

View full text Add to dashboard Cite

Murine typhus was diagnosed by PCR in 50 (7%) of 756 adults with febrile illness seeking treatment at Patan Hospital in Kathmandu, Nepal. Of patients with murine typhus, 64% were women, 86% were residents of Kathmandu, and 90% were unwell during the winter. No characteristics clearly distinguished typhus patients from those with blood culture-positive enteric fever.I n 2001, we found Salmonella enterica serotype Typhi and S. enterica serotype Paratyphi A to be the most common causes of bloodstream infections among adults with febrile illness who sought treatment at Patan Hospital in Kathmandu, Nepal (1). Another important fi nding was the relatively high percentage of patients (11%) who had immunoglobulin (Ig) M antibodies against Rickettsia typhi in peripheral blood. Because most testing was performed on unpaired acute-phase sera, and a high percentage of seropositive results were found in a group of healthy study participants, we were uncertain whether these participants had acute murine typhus or more distant past infection.Recent studies have shown the value of PCR for diagnosing scrub typhus (2-4), and a real-time assay for R. typhi has recently been validated (5). In our study, we tested archived blood samples from our febrile adult cohort (1) with this R. typhi PCR to better characterize the incidence of murine typhus and to determine whether clinical features could help distinguish murine typhus from enteric (typhoid) fever. The StudyWe studied consecutive adult patients with fever (axillary temperature >38°C; >13 years of age) who sought treatment at Patan Hospital from January 15 through March 15, 2001 (winter) and July 2 through August 10, 2001 (summer), as detailed elsewhere (1). The study was approved by the Nepal Health Research Council and the Institutional Review Board of the Centers for Disease Control and Prevention. Blood from each patient was injected into blood culture bottles and serum samples were tested for R. typhi IgM antibodies (INDX Multi-Test Dip-S-Ticks SDLST; Integrated Diagnostics, Inc., Baltimore, MD, USA). In addition, whole blood samples (stored at −80ºC) were tested by real-time PCR for R. typhi at the Naval Medical Research Center, Silver Spring, MD (NMRC) and at Canterbury Health Laboratories, Christchurch, New Zealand (CHL). Details of the assay have been described elsewhere (5). We extracted DNA from 200 μL of whole blood and used primers and probes specifi c to a portion of the outer membrane protein B (ompB) unique to R. typhi to amplify and detect the target sequence in a SmartCycler (Cepheid, Sunnyvale, CA, USA) at NMRC and in a LightCycler (Roche Diagnostics, Mannheim, Germany) thermocycler at CHL. Thermocycling parameters included an initial denaturation step (2 min at 9°C) followed by 45 cycles of denaturation (94°C for 5 s) and annealing/ elongation (60°C for 30 s) steps. Positive samples were defi ned as those that demonstrated fl uorescence above background levels. Template-free controls assayed at the same time and under the same conditions as the experimental a...

show abstract

Section: Discussionmentioning

confidence: 69%

Murine Typhus and Febrile Illness, Nepal

Zimmerman¹,

Murdoch²,

Rozmajzl³

et al. 2008

Emerg. Infect. Dis.

View full text Add to dashboard Cite

show abstract

“…When tested with O. tsutsugamushi strains Kato, Karp, and Gilliam, the same assay produced a positive result for all three strains, and a band around the 141-bp mark was observed when the products were run on an agarose gel. The assay produced negative results when tested against the DNA of other medically important bacteria previously used (11), and no band was evident around the 141-bp mark when run on an agarose gel.…”

Section: Serologymentioning

confidence: 99%

Isolation of a Novel Orientia Species ( O. chuto sp. nov.) from a Patient Infected in Dubai

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while species-discriminating assays have targeted the 16S rRNA, sca4, ompB, gltA, and ompA genes (18)(19)(20). However, using recently obtained Rickettsia genome sequences to identify new conserved and specific sites suitable for TaqMan assays, we have designed panrickettsia and R. rickettsii-specific assays.…”

mentioning

confidence: 99%

“…Several real-time PCR assays have been developed for rickettsial agent detection, including Rickettsia genus-specific (panrickettsia) and R. rickettsii-specific assays (17)(18)(19)(20). Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while species-discriminating assays have targeted the 16S rRNA, sca4, ompB, gltA, and ompA genes (18)(19)(20).…”

mentioning

confidence: 99%

Assessment of Real-Time PCR Assay for Detection of Rickettsia spp. and Rickettsia rickettsii in Banked Clinical Samples

et al. 2013

View full text Add to dashboard Cite

Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC. Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), is a tick-borne bacterial agent that is a member of the spotted fever group (SFG) rickettsiae. Symptoms of this disease may include, but are not limited to, high fever, headache, and rash, with the potential for a fatal outcome (1). National case fatality rates are Ͻ1% but can approach 7% in some regions to which it is highly endemic; a fatal outcome may be averted with timely administration of doxycycline (2, 3).In the United States, serologic tests are most often used to diagnose RMSF with seroconversion (4-fold titer increase from acute to convalescent phase) by immunofluorescence assay (IFA), the gold standard. However, serologic tests are frequently negative during the acute phase of illness, and currently available molecular tests are not reliable for use in patient management when applied to acute-phase blood samples, which may have very few organisms (3, 4). Clinical management of suspected RMSF cases based on test results is not recommended, partly because of diagnostic assay limitations, and physicians must treat suspected cases empirically (4). While empirical treatment of suspected cases will always be recommended, the development of more sensitive molecular tests that could be applied to patient specimens during the acute phase of illness will enhance the identification of nonfatal cases, improve the timeliness of public health actions following identification of a case, and aid national surveillance efforts by better defining the spectrum and burden of tick-borne rickettsial infections.At the Centers for Disease Control and Prevention (CDC), nested PCR assays have been the standard molecular diagnostic method for testing of blood and fresh tissue specimens. PCR methods for detection of R. rickettsii in clinical samples include nested assays for two SFG target genes, the 17-kDa-protein-encoding gene and the ompA outer membrane protein gene (5-7). The ompA nested amplicon may then be sequenced for species identification (8-15). However, this methodology is time-consuming (1 to 2 days, minimum) and has not proven highly sensitive for the detection of Rickettsia spp. in blood samples during acute disease, except in cases of advanced or fatal illness (4, 16). A 2000-2007 national surveillance summary noted that Ͻ0.5% of reported cases were diagnosed using nested PCR methodology (3).Several real-time PCR assays have been developed for rickettsial agent detection, including Rickettsia genus-specific (panrickettsia) and R. rickettsii-specific assays (17-20). Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while ...

show abstract

A Highly Sensitive and Specific Real-Time PCR Assay for the Detection of Spotted Fever and Typhus Group Rickettsiae

Cited by 251 publications

References 24 publications

Murine Typhus and Febrile Illness, Nepal

Murine Typhus and Febrile Illness, Nepal

Isolation of a Novel Orientia Species ( O. chuto sp. nov.) from a Patient Infected in Dubai

Assessment of Real-Time PCR Assay for Detection of Rickettsia spp. and Rickettsia rickettsii in Banked Clinical Samples

Contact Info

Product

Resources

About