Detection of
            <i>Anaplasma marginale</i>
            and
            <i>A. phagocytophilum</i>
            in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Reinbold, James B.; Coetzee, Johann F.; Sirigireddy, Kamesh R.; Ganta, Roman R.

doi:10.1128/jcm.02405-09

Cited by 36 publications

(30 citation statements)

References 29 publications

(42 reference statements)

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“…chaffeensis TRP120 gene. The detection limit was 100 copies of the target DNA per reaction, which is similar to some previous reports [32,33]. High speciﬁcity (100%) was observed when we performed the duplex real-time PCR assay on 27 members of the order Rickettsiales, 3 related bacteria and 13 common clinically pathogenic bacteria.…”

Section: Discussionsupporting

confidence: 86%

Detection of A. phagocytophilum and E. chaffeensis in Patient and Mouse Blood and Ticks by a Duplex Real-Time PCR Assay

Dong

Zhang

2013

PLoS ONE

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Human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellus undulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D . silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.

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Section: Discussionsupporting

confidence: 86%

Detection of A. phagocytophilum and E. chaffeensis in Patient and Mouse Blood and Ticks by a Duplex Real-Time PCR Assay

Dong

Zhang

2013

PLoS ONE

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“…The one‐way ROC curve determined the optimal negative cut‐off value to be 20%I, which resulted in an Se of 73% and an Sp of 91.2%. The two‐way ROC curve determined the optimal cut‐off value to be 15.3%I and resulted in a 74.2% Se and 81.2% Sp (Reinbold et al., 2010a). In another study where duplicate testing was performed for each of 659 samples from calves entering two commercial feedlots in Iowa, substantial but not perfect agreement (κ statistic of 0.74 ± 0.036 at 30%I) between the duplicate cELISA tests was reported (Coetzee et al., 2010).…”

Section: Diagnosismentioning

confidence: 99%

A Review of Bovine Anaplasmosis

Aubry

Geale

2010

Transboundary and Emerging Diseases

342

236

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Bovine anaplasmosis, caused by Anaplasma marginale, is an infectious but non-contagious disease. It is spread through tick bites or by the mechanical transfer of fresh blood from infected to susceptible cattle from biting flies or by blood-contaminated fomites including needles, ear tagging, dehorning and castration equipment. Transplacental transmission of A. marginale may contribute to the epidemiology of bovine anaplasmosis in some regions. Bovine anaplasmosis occurs in tropical and subtropical regions worldwide. Cattle of all ages are susceptible to infection with A. marginale, but the severity of disease increases with age. Once cattle of any age become infected with A. marginale, they remain persistently infected carriers for life. Diagnosis of bovine anaplasmosis can be made by demonstration of A. marginale on stained blood smears from clinically infected animals during the acute phase of the disease, but it is not reliable for detecting infection in pre-symptomatic or carrier animals. In these instances, the infection is generally diagnosed by serologic demonstration of antibodies with confirmation by molecular detection methods. The susceptibility of wild ruminants to infection by A. marginale and the role of wild ruminants in the epidemiology of bovine anaplasmosis are incompletely known owing to lack of published research, lack of validation of diagnostic tests for these species and cross-reaction of Anaplasma spp. antibodies in serologic tests. Control measures for bovine anaplasmosis vary with geographical location and include maintenance of Anaplasma-free herds, vector control, administration of antibiotics and vaccination.

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“…Immunosorbent Assay Relative to Quantitative Reverse Transcriptase PCR for Detection of Anaplasma marginale and A. phagocytophilum R einbold et al (2) reported performance results from a study comparing a commercial competitive enzyme-linked immunosorbent assay (cELISA) against a duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) developed for the detection of Anaplasma marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples.…”

Section: Sensitivity and Specificity Of A Competitive Enzyme-linkedmentioning

confidence: 99%

Sensitivity and Specificity of a Competitive Enzyme-Linked Immunosorbent Assay Relative to Quantitative Reverse Transcriptase PCR for Detection of Anaplasma marginale and A. phagocytophilum

Aubry

Paré

2012

J Clin Microbiol

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R einbold et al. (2) reported performance results from a study comparing a commercial competitive enzyme-linked immunosorbent assay (cELISA) against a duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) developed for the detection of Anaplasma marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples.Although estimates of sensitivity and specificity (the ability of a test to accurately detect diseased [or infected] and nondiseased [noninfected] animals, respectively) of the cELISA relative to the qRT-PCR are properly reported in the abstract, they seem to have been confused in the Results and Discussion. For example, in the receiver operating characteristic (ROC) curve analysis in Fig. 2a, the x axis (and corresponding legend) is indicated to represent the true-negative rate (i.e., the specificity), while it should rather be the false-positive rate or 1 Ϫ specificity. In addition, the legend to Fig. 2b incorrectly identifies the two curves: the sensitivity plot is the one represented by diamonds (not squares), while the specificity plot is the one represented by squares (not diamonds).In ROC curve analysis, the optimal cutoff is the one that maximizes the sum of sensitivity and specificity (1). The 15.3% inhibition negative cutoff proposed by the authors was determined by the intersection of the plots of sensitivity and specificity, but it does not optimize sensitivity and specificity (as can be seen in Fig. 2b, using a 20% cutoff yields a much higher specificity while only slightly decreasing sensitivity). The use of an ROC statistic such as Youden's J, would have been appropriate. The sensitivity and specificity reported in Results for the 15.3% cutoff (74.2 and 81.2%) do not agree with Fig. 2b. Since they were estimated by the intersection of the two curves, the sensitivity and specificity values should have been equal (approximately 75%).It also appears that sensitivity and specificity were inverted in the Discussion. The authors compared their results to those of the licensure study for the cELISA, where a sensitivity of 95% was reported with a negative cutoff value set at 28% (3). The authors indicate that "In order to achieve similar sensitivity results, the percentage of inhibition used as the negative cutoff value in our study would have needed to be set at 40%." Without the actual data on hand, we are not in a position to determine exactly what the negative cutoff value should have been to obtain a sensitivity of 95%, but by looking at Fig. 2b, it appears the cutoff should have been in the 2 to 3% range instead. Also in the Discussion, at the 40% inhibition cutoff, it is indicated that sensitivity and specificity estimates are 100% and 60%, respectively. Again, it appears the two terms were confused, since it is the specificity that would be 100% and the sensitivity 60% at this cutoff.The consequence of this confusion is that for various cutoff values, the estimates of sensitivity and specificity reported are erroneous. REFERENCES1. Dohoo I, Martin W, Stry...

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Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Cited by 36 publications

References 29 publications

Detection of A. phagocytophilum and E. chaffeensis in Patient and Mouse Blood and Ticks by a Duplex Real-Time PCR Assay

Detection of A. phagocytophilum and E. chaffeensis in Patient and Mouse Blood and Ticks by a Duplex Real-Time PCR Assay

A Review of Bovine Anaplasmosis

Sensitivity and Specificity of a Competitive Enzyme-Linked Immunosorbent Assay Relative to Quantitative Reverse Transcriptase PCR for Detection of Anaplasma marginale and A. phagocytophilum

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