Multiplex 5′ Nuclease-Quantitative PCR for Diagnosis of Relapsing Fever in a Large Tanzanian Cohort

Reller, Megan E.; Clemens, Emily G.; Schachterle, Steve E.; Mtove, George; Sullivan, David J.; Dumler, J. Stephen

doi:10.1128/jcm.00940-11

Cited by 15 publications

(15 citation statements)

References 26 publications

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“…Also, the multiplex real-time PCR proved highly sensitive, detecting 100 copies, that is more sensitive than the 10 3 –10 5 borreliae/µL reported for microscopy [14], [24]. Previously, borreliae not detectable by microscopy, were detected by using real-time PCR targeting the fla and the glpQ genes [3], [15]. These assays however could not identify borreliae at the species level [3], [15].…”

Section: Discussionmentioning

confidence: 99%

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Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

et al. 2013

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BackgroundIn Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples.Methodology/Principal FindingsWe designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae.Conclusions/SignificanceThe multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae.

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Section: Discussionmentioning

confidence: 99%

“…In particular, real-time PCR targeting the 16S rRNA gene or the glpQ gene improved the sensitivity of the diagnosis when compared to microscopy [14], [15]. Also, we previously showed that PCR-sequencing intergenic spacers could be used for genotyping B. crocidurae , B. duttonii and B. recurrentis [16].…”

Section: Introductionmentioning

confidence: 99%

Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

et al. 2013

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“…In order to estimate the true concentration of pathogen DNA available in whole blood for molecular analysis from patients with bloodstream infections, we analyzed the data available in the literature. Organism-specific quantitative PCR has been used to measure the bacterial DNA present in whole-blood specimens from patients with sepsis, pneumonia, or suspected bloodstream infections (13,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). Each of these studies analyzed whole-blood specimens from patients with suspected or confirmed infections rather than spiked samples for which the genome-to-viable cell ratio is expected to be close to 1:1.…”

Section: Repeated Analytical Testing By Pcr/esi-msmentioning

confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

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“…Several studies have assessed the effect of fastidious bacterial infections in systemic febrile illness, including Rickettsia felis ( 4 – 6 ), Coxiella burnetii ( 7 ), Tropheryma whipplei ( 3 ), and Borrelia spp. ( 1 , 8 ). Tourism, immigration, international business travel, international aid work, and the deployment of troops overseas were documented as contributors to a tremendous increase in international travel during 1996–2004 ( 9 ).…”

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confidence: 99%

Common Epidemiology ofRickettsia felisInfection and Malaria, Africa

Mediannikov¹,

Socolovschi²,

Edouard³

et al. 2013

Emerg. Infect. Dis.

109

121

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This study aimed to compare the epidemiology of Rickettsia felis infection and malaria in France, North Africa, and sub-Saharan Africa and to identify a common vector. Blood specimens from 3,122 febrile patients and from 500 nonfebrile persons were analyzed for R. felis and Plasmodium spp. We observed a significant linear trend (p<0.0001) of increasing risk for R. felis infection. The risks were lowest in France, Tunisia, and Algeria (1%), and highest in rural Senegal (15%). Co-infections with R. felis and Plasmodium spp. and occurrences of R. felis relapses or reinfections were identified. This study demonstrates a correlation between malaria and R. felis infection regarding geographic distribution, seasonality, asymptomatic infections, and a potential vector. R. felis infection should be suspected in these geographical areas where malaria is endemic. Doxycycline chemoprophylaxis against malaria in travelers to sub-Saharan Africa also protects against rickettsioses; thus, empirical treatment strategies for febrile illness for travelers and residents in sub-Saharan Africa may require reevaluation.

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Multiplex 5′ Nuclease-Quantitative PCR for Diagnosis of Relapsing Fever in a Large Tanzanian Cohort

Cited by 15 publications

References 26 publications

Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Common Epidemiology ofRickettsia felisInfection and Malaria, Africa

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