Comparison of Conventional, Nested, and Real-Time Quantitative PCR for Diagnosis of Scrub Typhus

Kim, Dong-Min; Park, Geon; Kim, Hyong Sun; Lee, Joo Young; Neupane, Ganesh Prasad; Graves, Stephen; Stenos, John

doi:10.1128/jcm.01216-09

Cited by 68 publications

(61 citation statements)

References 23 publications

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“…Here, we intentionally designed long probes (33 and 29 bp), which are known to be more tolerant of probe-template mismatches than short probes (18). In addition, a real-time PCR result with a quantification cycle (C q ) value of over 37 to 38 cycles may be considered to represent a negative or a false-positive result (19); therefore, detection of two genes increases the accuracy of the results. For example, samples positive for the two genes can be interpreted as true positives, regardless of the C q values, whereas nested or repeated real-time PCRs would be required to confirm samples positive for only one gene.…”

Section: Discussionmentioning

confidence: 99%

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

et al. 2017

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Scrub typhus, caused by Orientia 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 targeting O. tsutsugamushi 47-kDa, groEL, and human interferon beta (IFN-␤ 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.KEYWORDS Orientia tsutsugamushi, real-time PCR, scrub typhus S crub typhus is a mite-borne infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. A characteristic feature of the disease is patients presenting nonspecific symptoms, including fever, headache, myalgia, cough, and abdominal pain, which cannot be differentiated from symptoms of other systemic infections. The presence of eschar can help diagnose this illness; however, it is found only in some patients (1). Although the clinical course of scrub typhus is usually mild and self-limiting, delaying the treatment in severe cases can lead to complications such as renal failure, myocarditis, meningoencephalitis, and death (2). Since scrub typhus is one of the most common causes of acute undifferentiated febrile illness (AUFI) in areas of endemicity (3, 4), an early, definite diagnosis is essential for providing appropriate treatment and gathering accurate epidemiological data.Scrub typhus diagnosis mainly relies on serologic tests, particularly the indirect immunofluorescence assay (IFA), whereby the illness is identified by a 4-fold increase in antibody titers in paired sera (5, 6) and/or a positive IgM titer in a single serum sample (7,8). However, these serologic tests require paired serum samples and good technician expertise, and even then they often return false negatives during the early phase of disease. In addition, reinfection by different O. tsutsugamushi strains is not uncommon in areas of endemicity and reinfected patients may sometimes

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Section: Discussionmentioning

confidence: 99%

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

et al. 2017

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“…A comparative analysis was done by Dong min Kim et al, to study the sensitivity and specificity of conventional PCR (C-PCR), nested PCR (N-PCR) and real time quantitative PCR (Q-PCR) by targeting the Orienta tsutsugamushi specific 47kDa gene with blood samples obtained from patients within 4 weeks of onset of fever [7] . He concluded that RT-PCR had 100% sensitivity and specificity and was considered as one of the preferred assays for diagnosis of scrub typhus.…”

Section: Discussionmentioning

confidence: 99%

“…Orienta tsutsugamushi expresses type specific proteins coded by 56KDa and 47KDa genes. The 56KDa gene is unique not expressed by other Rickettsia, so used for vaccine development [7] . The 47KDa gene which is also known as HtrA encodes for the outer membrane protein, is more sensitive by real time quantitative PCR rather than conventional PCR, as the amplicon containment is easier to achieve [7] .…”

Section: Introductionmentioning

confidence: 99%

“…The 56KDa gene is unique not expressed by other Rickettsia, so used for vaccine development [7] . The 47KDa gene which is also known as HtrA encodes for the outer membrane protein, is more sensitive by real time quantitative PCR rather than conventional PCR, as the amplicon containment is easier to achieve [7] . This 47KDa gene of Karp strain is highly conserve product, which is both group reactive and species specific, also recognised by both antibodies and 'T' cells, they play a significant role in molecular diagnosis.…”

Section: Introductionmentioning

confidence: 99%

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Screening of Scrub Typhus among Patients Presenting with Pyrexia of Unknown Origin in Tertiary Care Hospital

Vasanthi¹

2018

jmscr

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“…Kompleks mikrobiyal popülasyonlara ait hedef dizilerin spesifik bir şekilde amplifikasyonu klasik PCR yöntemleriyle bazen mümkün olmadığı görülmekte ve oluşan non-spesifik fragmentlerin amplifikasyonu sonucu yanlış pozitiflikler ortaya çıkabilmektedir. Nested PCR ile söz konusu bu olumsuzluk giderilmekte, aynı zamanda düşük DNA'ya sahip örneklerde ikinci kez PCR uygulaması ile teşhisteki sensitivite artırılabilmektedir [13,18] . Son yıllarda ruminant ve vektör kenelerde babesiosis'in spesifik teşhisinde nested PCR yönteminin sıklıkla uygulandığı görülmektedir [13,19,20] .…”

Section: Rlbunclassified

Sığırlarda Babesia bovis ve Babesia bigemina’nın Reverse Line Blotting, Nested PCR ve Real Time PCR Teknikleri İle Karşılaştırmalı Tanısı

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et al. 2013

Kafkas Univ Vet Fak Derg

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SummaryThis study was carried out to compare Reverse Line Blotting (RLB), Nested PCR and Real Time PCR techniques in the molecular diagnosis of Babesia bovis and Babesia bigemina in cattle. Genomic DNA extractions were performed on 400 blood samples which were previously collected from cattle in various provinces of Turkey and stored in the laboratory with respect to use in different project studies. The concentrations of the DNAs were measured in NanoDrop spectrophotometer and analyzed by RLB, Nested PCR and Real Time PCR techniques after preparing the suitable concentrations. Totally 18 (4.50%), 59 (14.75%), 16 (4.00%) and 2 (0.50%) of examined samples were found to be infected with B. bovis, B. bigemina, Babesia spp. and B. bovis + B. bigemina mix, respectively by RLB. 23 (5.75%), 71 (17.75%), 7 (1.75%) and 23 (5.75%), 75 (18.75%), 9 (2.00%) of the examined samples were found to be infected with B. bovis, B. bigemina and B. bovis + B. bigemina mix by Nested PCR and Real Time PCR, respectively. When comparing the Nested PCR and RLB results with Real Time PCR assay, 94.4% and 88.8% sensitivity and both 100.0% specificity were determined, respectively. 5, 9 and 2 out of the total 16 Babesia spp. positivity's in RLB test were determined as B. bigemina, B. bovis, B. bovis + B. bigemina mix, respectively by both Real Time and Nested PCR. In conclusion, Real Time PCR was found to be more sensitive than Nested PCR and RLB in the investigation of B. bovis and B. bigemina in cattle with this study.

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Comparison of Conventional, Nested, and Real-Time Quantitative PCR for Diagnosis of Scrub Typhus

Cited by 68 publications

References 23 publications

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

Screening of Scrub Typhus among Patients Presenting with Pyrexia of Unknown Origin in Tertiary Care Hospital

Sığırlarda Babesia bovis ve Babesia bigemina’nın Reverse Line Blotting, Nested PCR ve Real Time PCR Teknikleri İle Karşılaştırmalı Tanısı

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