Dataset Information

Clinical usefulness of 16S ribosomal RNA real-time PCR for the diagnosis of scrub typhus.

ABSTRACT: Scrub typhus is a major acute febrile disease in the Asia-Pacific region. The purpose of the present study is to investigate the clinical usefulness of real-time PCR (Q-PCR) of 16S rRNA for the diagnosis of scrub typhus. We examined blood specimens from 148 adult patients who were confirmed to have scrub typhus from September 2008 to December 2009. Among the 148 scrub typhus patients, 36 patients were treated with antibiotics before admission. To evaluate the clinical usefulness of 16S rRNA Q-PCR, we compared its diagnostic accuracy to the accuracy of the following methods: nested PCR (N-PCR) targeting the gene encoding the 56-kDa protein, Q-PCR targeting the gene encoding the 47-kDa protein, and conventional PCR (C-PCR), targeting the 16S rRNA gene. According to 16S rRNA Q-PCR and 47-kDa Q-PCR, the mild group had copy numbers of 234.4 ± 261.9 and 130.5 ± 128.3, whereas the severe group had copy numbers of 584.4 ± 911.4 and 244.7 ± 210.9, respectively. In both tests, the mean copy numbers were significantly greater in the severe group (P = 0.037 and P = 0.035). 16S rRNA Q-PCR detected Orientia tsutsugamushi infections with a sensitivity of 91.9% (95% CI 86.3-95.7), and 56-kDa N-PCR, 47-kDa Q-PCR, and 16S rRNA C-PCR exhibited lower sensitivities of 81.1% (95% CI 73.8-87.0), 74.3% (95% CI 66.5-81.1), and 87.8% (95% CI 81.5-92.6), respectively, for all 148 patients. In addition, 16S rRNA Q-PCR exhibited a sensitivity of 99.1% (95% CI 95.1-100.0) in the 112 patients who were not treated with antibiotics before admission. 16S rRNA Q-PCR is clinically useful for the rapid diagnosis of scrub typhus and is more accurate than the 56-kDa N-PCR, 47-kDa Q-PCR, and 16S C-PCR methods.

SUBMITTER: Yun NR

PROVIDER: S-EPMC8275794 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Orientia tsutsugamushi is the causative agent of scrub typhus. For the diagnosis of scrub typhus, we investigated the performances of conventional PCR (C-PCR), nested PCR (N-PCR), and real-time quantitative PCR (Q-PCR) targeting the O. tsutsugamushi-specific 47-kDa gene. To compare the detection sensitivities of the three techniques, we used two template systems that used plasmid DNA (plasmid detection sensitivity), including a partial region of the 47-kDa gene, and genomic DNA (genomic detection sensitivity) from a buffy coat sample of a single patient. The plasmid detection sensitivities of C-PCR, N-PCR, and Q-PCR were 5 × 10(4) copies/μl, 5 copies/μl, and 50 copies/μl, respectively. The results of C-PCR, N-PCR, and Q-PCR performed with undiluted genomic DNA were negative, positive, and positive, respectively. The genomic detection sensitivities of N-PCR and Q-PCR were 64-fold and 16-fold (crossing point [Cp], 37.7; 426 copies/μl), respectively. For relative quantification of O. tsutsugamushi bacteria per volume of whole blood, we performed real-time DNA PCR analysis of the human GAPDH gene, along with the O. tsutsugamushi 47-kDa gene. At a 16-fold dilution, the copy number and genomic equivalent (GE) of GAPDH were 1.1 × 10(5) copies/μl (Cp, 22.64) and 5.5 × 10(4) GEs/μl, respectively. Therefore, the relative concentration of O. tsutsugamushi at a 16-fold dilution was 0.0078 organism/one white blood cell (WBC) and 117 organisms/μl of whole blood, because the WBC count of the patient was 1.5 × 10(4) cells/μl of whole blood. The sensitivities of C-PCR, N-PCR, and Q-PCR performed with blood samples taken from patients within 4 weeks of onset of fever were 7.3% (95% confidence interval [CI], 1.6 to 19.9), 85.4% (95% CI, 70.8 to 94.4), and 82.9% (95% CI, 67.9 to 92.8), respectively. All evaluated assays were 100% specific for O. tsutsugamushi. In conclusion, given its combined sensitivity, specificity, and speed, Q-PCR is the preferred assay for the diagnosis of scrub typhus.

Project description:Introduction: Scrub typhus is a mite-borne infection widespread in Southeast Asia, with clinical symptoms such as fever, chills, skin rash, eschar at the bite site, and other signs of acute febrile illness. The Rickettsia pathogen (Orientia tsutsugamushi) is always difficult to be diagnosed at an early stage by traditional clinical diagnostic methods, especially for patients without typical eschar. This greatly increases the mortality of patients with scrub typhus. A new approach should be introduced to improve its clinical diagnosis. Methods: During May 2018 to March 2021, 13 samples from 10 patients with suspected scrub typhus were collected. Metagenomic next-generation sequencing (mNGS) and other diagnostic methods (including serology using Weil-Felix reaction and indirect immunofluorescence test (IIFT) for scrub typhus and respiratory tract profile IgM as well as culture for routine bacteria) were used to identify the pathogens in this study. Results: The results of mNGS were all positive, with mapped reads of O. tsutsugamushi ranging from 1 to 460. Eight patients (80%) were diagnosed as scrub typhus. The other two were diagnosed as suspected scrub typhus due to the limited number of reads of the pathogen (one and two, respectively). According to clinical evidences, nine of the 10 patients were finally diagnosed as scrub typhus, except for patient 9 (suspected scrub typhus by mNGS with one specific reads of the pathogen) diagnosed as acute exacerbation of chronic obstructive pulmonary disease. For the five scrub typhus patients without typical eschar, mNGS gave all positive results (4-460 specific reads). For other methods, only Weil-Felix reaction of one patient detected the pathogen. In addition, the respiratory tract profile (IgM) detected various pathogens, but all were confirmed to be false positive. Conclusions: mNGS performed better than conventional clinical methods to early diagnose scrub typhus. This approach can be routinely carried out for early and precise diagnosis in clinical infections, especially for those hard to be identified by traditional diagnostic methods.

Dataset Information

Clinical usefulness of 16S ribosomal RNA real-time PCR for the diagnosis of scrub typhus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets