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 ...
Introduction Secondary traumatic stress is highly prevalent among nurses, especially among nurses working within the emergency department (ED). Reducing healthcare worker secondary traumatic stress is important for ensuring the delivery of high quality, safe patient care. This paper reports on the development and implementation of a secondary traumatic stress reduction program. Methods We used an adaption of a 5-week intervention based on the Accelerated Recovery Program to test whether there would be a reduction in secondary traumatic stress in a pilot sample of nine ED nurses. Outcomes were assessed using the Secondary Traumatic Stress Scale (STSS), Somatic Symptoms Scale (SSS), and Compassion Satisfaction subscale (CSS) measures. Results Eight of nine nurses were able to complete at least three of the five sessions. Results indicate significant change in STSS (F[5,23] = 4.22, p = .007) and SSS (F[3,15] = 4.42, p = .02) scores, but not CSS (F[5,23] = 0.83, p = .54) scores. Pairwise comparisons revealed that the beneficial effects of the program happened early. For both STSS and SSS, scores at sessions 1 and 2 were generally higher than subsequent sessions. We also found a trend for continued effects on STSS at a four-month follow-up (t23 = 1.95, p = .064). Conclusion Overall, results indicate the 5-week program was associated with a significant reduction in secondary traumatic stress and related somatic symptoms in healthcare workers.
Murine typhus, which is caused by Rickettsia typhi, has a wide range of clinical manifestations. It has a low mortality rate but may result in meningoencephalitis and interstitial pneumonia in severe cases. Comparisons of complete genome sequences of R. typhi isolates from North Carolina, USA (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 single nucleotide polymorphism (SNP) and 7 insertion-deletion (INDEL) sites in these highly syntenic genomes. Assays were developed to further define the distribution of these variant sites among 15 additional isolates of R. typhi with different histories from Asia, the USA, and Africa. Mismatch amplification mutation assays (MAMA) were validated for 22 SNP sites, while the 7 INDEL sites were analyzed directly on agarose gels. Six SNP types, 9 INDEL types, 11 total types were identified among these 18 isolates. Replicate DNA samples as well as comparisons of isolates with different passage and source histories gave consistent genetic typing profiles. Comparison of the SNP and INDEL markers to R. typhi’s nearest neighbor Rickettsia prowazekii demonstrated that the majority of the SNPs represent intra-species variation that arose post divergence of these two species while several INDEL sites also exhibited intraspecies variability among the R. prowazekii genomes that have been completely sequenced. The assays for the presence of these SNP and INDEL sites, particularly the latter, comprise a low technology gel method for consistently distinguishing R. typhi and R. prowazekii as well as for differentiating genetic types of R. typhi.
Clinical and laboratory diagnosis of rickettsial diseases is challenging because of the undifferentiated symptoms (commonly fever, headache, and malaise) and low bacteremia (< 100 genomic copies [gc]/mL) during the early acute stage of illness. Early treatment with doxycycline is critical for a positive outcome, especially in Rickettsia rickettsii (Rocky Mountain spotted fever) infections where cases may be fatal within 5 to 10 days from symptom onset, emphasizing the need for more sensitive diagnostics. A real-time reverse transcriptase polymerase chain reaction (PCR) assay, RCKr, was developed and validated for Rickettsia spp. nucleic acid detection in human clinical samples. The limit of detection for RCKr was determined to be 20 gc/mL, compared with our 2013 (Kato et al.) laboratory developed test, PanR8 at 1,800 to 2,000 gc/mL. Inclusivity, exclusivity, accuracy, and precision results correlated as expected. From an evaluation of 49 banked clinical samples, RCKr detected 35 previously positive samples, as well as two specimens that were PanR8 real-time PCR negative yet clinically diagnosed as possible rickettsiosis. Ct values from RCKr clinical sample testing show a 100-fold increase relative to PanR8. Additional testing is needed to understand the clinical sensitivity of RCKr; however, this study demonstrates RCKr to have high analytical specificity and sensitivity for Rickettsia detection.
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