Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

Dittrich, Sabine; Castonguay-Vanier, Josée; Moore, Catrin E.; Thongyoo, Narongchai; Newton, Paul N.; Paris, Daniel

doi:10.1128/jcm.02786-13

Cited by 37 publications

(25 citation statements)

References 44 publications

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“…Reduced sensitivity may also be attributed to low bacterial load in real-time PCR-positive but LAMP-negative patient specimens, a phenomenon that has been described previously (35). This idea is supported by results showing that 96% of LAMP false negatives have a real-time C T value of Ն30.…”

Section: Discussionsupporting

confidence: 74%

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

et al. 2015

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Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resourcepoor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n ‫؍‬ 252) and unextracted respiratory specimens (n ‫؍‬ 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community. Mycoplasma pneumoniae is a human pathogen that accounts for an estimated 15% to 20% of cases of community-acquired pneumonia (CAP) (1, 2). An atypical bacterial pneumonia, M. pneumoniae infection can result in both pulmonary and extrapulmonary manifestations, including pharyngitis, encephalitis, Stevens-Johnson syndrome, septic arthritis, and pericarditis, among others (3). Ongoing endemic M. pneumoniae infection is overlaid with cyclical epidemics of disease, typically occurring every 3 to 5 years (1). Although symptoms of M. pneumoniae infection are generally mild or subclinical, the organism accounts for up to 33% of hospitalizations among children and adults with bacterial pneumonia (4). In the absence of a diagnostic result, empirical therapy can be misdirected, resulting in inappropriate use of antibiotics and continued spread of M. pneumoniae in the community. This may promote the evolution of antibiotic resistance and leads to negative economic impact due to health care costs and missed school and work days.Traditional diagnostics for M. pneumoniae infections exist but are underused due to their cost, time-intensive nature, or requirement for specialized expertise and equipment. M. pneumoniae is fastidious and slow-growing, with isolation from liquid culture requiring up to 8 weeks and often resulting in low yield (5, 6). Appropriate interpretation of serological data requires paired acute-phase and convalescent-phase serum samples, constraining their use to retrosp...

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Section: Discussionsupporting

confidence: 74%

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

et al. 2015

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“…This difference in reactivity toward TGR and SFGR suggests that the assay is highly specific. While the detection limit using NHP spiked with cultured R. typhi was about 2000 copies/ml (Table 4), it is higher than the median of 210 DNA copies/mL in blood of confirmed murine typhus patients [44], suggesting that the assay may not be sensitive enough to be clinical useful. Since no evaluation of 17-RPA was done using clinical samples, it is warranted that additional evaluations should be done to further characterize the clinical sensitivity and specificity of the 17-RPA.…”

Section: Discussionmentioning

confidence: 99%

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

et al. 2015

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Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.

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“…Several methods for the rapid molecular diagnosis of rickettsiosis based on genomic sequence data have been reported (3,(6)(7)(8)(9). However, a method that can simultaneously detect the DNA of several Rickettsia spp.…”

Section: Discussionmentioning

confidence: 99%

“…For the detection of Rickettsia spp. other than R. japonica, qPCR (7) and LAMP assays for Rickettsia causing spotted fever (8) and murine typhus (9), respectively, have also been reported. However, simple LAMP assay suitable for the simultaneous detection of all the Rickettsia spp.…”

Section: Introductionmentioning

confidence: 99%

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Rickettsia</i> spp.

et al. 2017

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SUMMARY:We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 639 C. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

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Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

Cited by 37 publications

References 44 publications

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Rickettsia</i> spp.

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