scholarly journals Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Megan E. Reller ◽  
J. Stephen Dumler
Keyword(s):  
Quantitative Pcr ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Epidemiologic Studies ◽  
Qpcr Assay ◽  
Spotted Fever Group ◽  
Analytical Sensitivity ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

scholarly journals Searching and Finding the Hidden Treasure: A Retrospective Analysis of Rickettsial Disease Among Dutch International Travelers

2020 ◽  
Author(s):  
Sophia G de Vries ◽  
Louise E van Eekeren ◽  
Hans van der Linden ◽  
Benjamin J Visser ◽  
Martin P Grobusch ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
Medical Records ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Empiric Antibiotic Therapy ◽  
Spotted Fever Group ◽  
Orientia Tsutsugamushi ◽  
Rickettsial Disease ◽  
Empiric Antibiotic

Abstract Background Rickettsial disease (RD) is a prevalent and underestimated cause of febrile illness worldwide, especially in the absence of an inoculation eschar. We attempted to quantify this underestimation at our clinic, by investigating past cases of febrile illness in travelers who had tested negative for leptospirosis, a disease that can initially present similarly to non-eschar RD, and which we routinely consider when other important causes of unspecified febrile illness have tested negative. Methods We performed a retrospective analysis in febrile returned travelers from Asia, Africa, or the Americas between 2010 and 2017, who had tested negative for leptospirosis. Serologic immunofluorescence assays were performed for Orientia tsutsugamushi (scrub typhus), typhus group, and spotted fever group RD. We performed a medical records review of all patients who tested positive. In case of a fitting medical history, cases were deemed either confirmed (based on convalescent serology) or suspected (based on single serology). Results Among 97 patients, convalescent serology was available in 16 (16.5%) patients, and a single serology in 81 (83.5%) patients. RD was the likely diagnosis in 8 of 16 (50.0%) patients with convalescent serology, and in 8 of 81 (9.9%) with single serology. Of the 16 confirmed/suspected cases, 11 (69%) had been missed and 7 (44%) had not received adequate empiric antibiotic therapy. Conclusions This study shows that non-eschar RD is an important and poorly recognized cause of illness in travelers, even in a specialized travel clinic. A lower threshold to test and treat for RD is warranted in returning travelers with febrile illness.

scholarly journals A Novel TaqMan Assay for Detection of Rickettsia 364D, the Etiologic Agent of Pacific Coast Tick Fever

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Sandor E. Karpathy ◽  
Alex Espinosa ◽  
Melissa H. Yoshimizu ◽  
Jill K. Hacker ◽  
Kerry A. Padgett ◽  
...  
Keyword(s):  
Pacific Coast ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Etiologic Agent ◽  
Taqman Assay ◽  
Spotted Fever Group ◽  
Content Type ◽  
Clinical Sensitivity ◽  
Detection And Identification ◽  
Diagnostic Applications

ABSTRACT Pacific Coast tick fever is a febrile illness associated with the bite of Dermacentor occidentalis and results from an infection due to the intracellular pathogen Rickettsia 364D (also known by the proposed name “Rickettsia philipii”). Current molecular methods for the detection of this pathogen rely on the amplification of a conserved spotted fever group rickettsial gene (ompA) followed by DNA sequencing of the amplicon to identify the species. This work describes the development of a Rickettsia 364D-specific TaqMan assay to simplify and accelerate the detection and identification processes. The assay demonstrated a sensitivity of 1 genomic copy per 4-μl sample and is highly specific for Rickettsia 364D. The utility of this assay for ecological and diagnostic samples was evaluated using banked specimens collected in a single-blind manner and yielded a clinical sensitivity and specificity of 100%. In conclusion, we describe the development and evaluation of a novel TaqMan real-time PCR assay for the detection and identification of Rickettsia 364D suitable for ecological and diagnostic applications.

scholarly journals Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

2015 ◽  
Vol 53 (7) ◽  
pp. 2095-2102 ◽  
Author(s):  
Samson S. Y. Wong ◽  
Rosana W. S. Poon ◽  
Sandy Chau ◽  
Sally C. Y. Wong ◽  
Kelvin K. W. To ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Copy Number ◽  
Mitochondrial Gene ◽  
Qpcr Assay ◽  
Dna Copy Number ◽  
Predictive Values ◽  
Content Type ◽  
Lesional Skin ◽  
Real Time Quantitative Pcr

Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration ofSarcoptes scabieimites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochromecoxidase subunit 1 (cox1) gene ofS. scabiei. Thecox1gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. TheS. scabieiDNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (medianS. scabieiDNA copy number, 3.604 versus 2.457 log10copies per reaction;P= 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated.

scholarly journals Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Pricila da Silva Cunha ◽  
Heloisa B. Pena ◽  
Carla Sustek D’Angelo ◽  
Celia P. Koiffmann ◽  
Jill A. Rosenfeld ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Test ◽  
Quantitative Pcr ◽  
Target Genes ◽  
Cost Effective ◽  
Qpcr Assay ◽  
Deletion Syndrome ◽  
Comparative Genomic ◽  
Real Time Quantitative Pcr ◽  
Subtelomeric Deletion

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36:PRKCZandSKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescentin situhybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR ofPRKCZandSKIis a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

scholarly journals Epidemiology of Spotted Fever Group Rickettsioses and Acute Undifferentiated Febrile Illness in Villeta, Colombia

2017 ◽  
Vol 97 (3) ◽  
pp. 782-788 ◽  
Author(s):  
Álvaro A. Faccini-Martínez ◽  
Elkin Valbuena ◽  
Christian Barreto ◽  
Ana M. Palomar ◽  
Luis J. Polo-Terán ◽  
...  
Keyword(s):  
Spotted Fever ◽  
Febrile Illness ◽  
Spotted Fever Group

scholarly journals Spotted Fever GroupRickettsiaInfection and Transmission Dynamics inAmblyomma maculatum

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Chanakan Suwanbongkot ◽  
Ingeborg M. Langohr ◽  
Emma K. Harris ◽  
Wellesley Dittmar ◽  
Rebecca C. Christofferson ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Spotted Fever ◽  
Attachment Site ◽  
Spotted Fever Group ◽  
Tick Feeding ◽  
Tick Saliva ◽  
Content Type ◽  
Feeding Site ◽  
Vertebrate Hosts ◽  
Tick Salivary Glands

ABSTRACTTick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novelRickettsiaspecies or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG)Rickettsiaspecies is transmitted at higher levels during tick feeding. UsingAmblyomma maculatumcohorts infected withRickettsia parkerior “CandidatusRickettsia andeanae,” a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers ofR. parkerithan of “Ca. Rickettsia andeanae” rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localizedR. parkeri, but not “Ca. Rickettsia andeanae,” in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed toR. parkerithan in those exposed to “Ca. Rickettsia andeanae.” The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

Clinical Features of Rickettsial Infection in Children in Tropical Australia—A Report of 15 Cases

2020 ◽  
Vol 66 (6) ◽  
pp. 655-660 ◽  
Author(s):  
Alexandra G A Stewart ◽  
Simon Smith ◽  
Enzo Binotto ◽  
Josh Hanson
Keyword(s):  
Scrub Typhus ◽  
Spotted Fever ◽  
Case Fatality ◽  
Spotted Fever Group ◽  
Tertiary Referral Hospital ◽  
Admission Rates ◽  
Rickettsial Infections ◽  
Retrospective Audit ◽  
Tropical Australia ◽  
The Tropics

Abstract Rickettsial infections are an under-recognized cause of acute, undifferentiated fever in the tropics. In Asia, intensive care unit (ICU) admission rates as high as 21% and case-fatality rates of up to 5% have been reported. This 20-year retrospective audit of children and adults with serologically confirmed scrub typhus or spotted fever group (SFG) infection was performed at a tertiary-referral hospital in tropical Australia. There were 15 paediatric cases during the study period (11 scrub typhus, 3 SFG and 1 undifferentiated). Hypotension [5/15 (33%)], tachycardia [6/15 (40%)] and tachypnoea [6/15 (40%)] were common at presentation. Children were more likely to be hypotensive at admission than adults [5/15 (33%) vs. 5/118 (4%), p = 0.002]. However, no child died or was admitted to ICU, compared with 18/120 (15%) adults who required ICU support during the study period, one of whom died. Paediatric rickettsial infections have a relatively benign clinical course in tropical Australia with serious complications appearing far less frequently than have been reported in the Asian literature.

scholarly journals Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

2017 ◽  
Vol 55 (5) ◽  
pp. 1377-1387 ◽  
Author(s):  
Wiwit Tantibhedhyangkul ◽  
Ekkarat Wongsawat ◽  
Saowaluk Silpasakorn ◽  
Duangdao Waywa ◽  
Nuttawut Saenyasiri ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Asia Pacific ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Serologic Tests

ABSTRACTScrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

scholarly journals Applying Real-Time Quantitative PCR to Fusarium Crown Rot of Wheat

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1021-1028 ◽  
Author(s):  
A. C. Hogg ◽  
R. H. Johnston ◽  
A. T. Dyer
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Field Experiments ◽  
Qpcr Assay ◽  
Crown Rot ◽  
Yield Reduction ◽  
Fusarium Crown Rot ◽  
Persistent Problem ◽  
Real Time Quantitative Pcr ◽  
Severity Scores

Fusarium crown rot (FCR) of wheat is a persistent problem that causes significant losses worldwide. In Montana, FCR is caused primarily by Fusarium culmorum and F. pseudograminearum. Recently, a real-time quantitative PCR (QPCR) assay was developed for FCR using primers and probes specific for a segment of the trichodiene synthase (tri5) gene. The purpose of this study was to determine the utility of QPCR for accessing FCR severity on wheat in field experiments. In 2004 and 2005, plots of spring and durum wheat were inoculated with varying levels of F. pseudograminearum oat inoculum and grown under rain-fed conditions. Two weeks prior to harvest, plants were collected from the plots and assessed for FCR severity and analyzed by QPCR for Fusarium DNA quantities. Disease severity scores (DSS) and Fusarium DNA quantities were positively correlated with each other for all three cultivars in 2004 but for only the durum cultivar in 2005 (P < 0.05). In 2004, grain yields for both spring wheat cultivars were negatively correlated with Fusarium DNA quantities (P > 0.05). When DSS and Fusarium DNA quantities negatively correlated with yield, both measurements were comparable in predicting yield reduction (R = –0.64 and –0.77, respectively). Results indicate that this QPCR assay is effective in measuring FCR severity in wheat.

Sign in / Sign up

Export Citation Format

Share Document