A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi

Paris, Daniel; Aukkanit, Nuntipa; Jenjaroen, Kemajittra; Blacksell, Stuart D.; Day, Nicholas P. J.

doi:10.1111/j.1469-0691.2008.02671.x

Cited by 73 publications

(65 citation statements)

References 23 publications

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“…The higher sensitivity of our assay probably relates to a more accurate classification of the patients and differences in the strains of causative O. tsutsugamushi organisms. In addition, we also used a larger number of clinical samples than previous studies (11,19,25,26). A substantial proportion of patients (38 cases) showed equivocal IFA results, including persistently high titers of both IgG and IgM without an apparent 4-fold increase.…”

Section: Discussionmentioning

confidence: 99%

“…Taking the data together, this multiplex real-time PCR with an internal control is sensitive, specific, and practical for scrub typhus diagnosis. Although several real-time PCR assays for scrub typhus diagnosis have been reported in previous studies (9)(10)(11), the use of multiplex real-time PCR with an internal control has never been published. The level of genetic polymorphism found in O. tsutsugamushi is quite high; consequently, assays based on a single gene sequence can yield false negatives due to mismatches between the template and the primers or probes.…”

Section: Discussionmentioning

confidence: 99%

“…The 47-kDa gene is unique in O. tsutsugamushi and is absent in members of the genus Rickettsia (9). Although the groEL gene is present in other bacteria as well, there are some regions that are highly conserved among O. tsutsugamushi strains but differ from those in members of the genus Rickettsia (11). DNA sequence alignments of these two genes are shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…We obtained previously published DNA sequences of the 47-kDa gene and the groEL gene of several O. tsutsugamushi strains (11,29) from GenBank, aligned the sequences using the BioEdit program, and manually designed the primers and probes from the most conserved regions. The 47-kDa gene is unique in O. tsutsugamushi and is absent in members of the genus Rickettsia (9).…”

Section: Methodsmentioning

confidence: 99%

“…The use of real-time PCR assays targeting the 47-kDa gene or 16S rRNA gene (using hydrolysis probes) or the 60-kDa heat shock groEL gene (using SYBR green) has been reported previously (9)(10)(11). However, multiplex real-time PCR using several probes labeled with different fluorochromes has never been used to diagnose scrub typhus.…”

mentioning

confidence: 99%

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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%

Section: Discussionmentioning

confidence: 99%