Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens

Briese, Thomas; Palacios, Gustavo; Kokoris, Mark S.; Jabado, Omar; Liu, Zhiqiang; Renwick, Neil; Kapoor, Vishal; Casas, Inmaculada; Pozo, Francisco; Limberger, Ron; Pérez-Breña, Pilar; Ju, Jingyue; Lipkin, W. Ian

doi:10.3201/eid1102.040492

Cited by 151 publications

(150 citation statements)

References 14 publications

Supporting

Mentioning

145

Contrasting

Unclassified

Order By: Relevance

“…(Table 1). 22,23,29 Of template DNA, 2 μL was used in all reactions. Reaction conditions were 94°C for 15 minutes; one cycle at 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds, followed by 11 cycles with annealing temperature decreased by 1°C in each cycle.…”

Section: Methodsmentioning

confidence: 99%

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

Tadin¹,

Tokarz²,

Markotić³

et al. 2016

The American Society of Tropical Medicine and Hygiene

Self Cite

View full text Add to dashboard Cite

Abstract. Croatia is a focus for many rodent-borne zoonosis. Here, we report a survey of 242 rodents and small mammals, including 43 Myodes glareolus, 131 Apodemus flavicollis, 53 Apodemus agrarius, three Apodemus sylvaticus, six Sorex araneus, four Microtus arvalis, one Microtus agrestis, and one Muscardinus avellanarius, collected at eight sites in Croatia over an 8-year period. Multiplex MassTag polymerase chain reaction (PCR) was used for detection of Borrelia, Rickettsia, Bartonella, Babesia, Ehrlichia, Anaplasma, Francisella tularensis, and Coxiella burnetii. Individual PCR assays were used for detection of Leptospira, lymphocytic choriomeningitis virus, orthopoxviruses, flaviviruses, hantaviruses, and Toxoplasma gondii. Of the rodents, 52 (21.5%) were infected with Leptospira, 9 (3.7%) with Borrelia miyamotoi, 5 (2%) with Borrelia afzelii, 29 (12.0%) with Bartonella, 8 (3.3%) with Babesia microti, 2 (0.8%) with Ehrlichia, 4 (1.7%) with Anaplasma, 2 (0.8%) with F. tularensis, 43 (17.8%) with hantaviruses, and 1 (0.4%) with an orthopoxvirus. Other agents were not detected. Multiple infections were found in 32 rodents (13.2%): dual infections in 26 rodents (10.7%), triple infections in four rodents (2.9%), and quadruple infections in two rodents (0.8%). Our findings indicate that rodents in Croatia harbor a wide range of bacteria and viruses that are pathogenic to humans.

show abstract

Section: Methodsmentioning

confidence: 99%

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

Tadin¹,

Tokarz²,

Markotić³

et al. 2016

The American Society of Tropical Medicine and Hygiene

Self Cite

View full text Add to dashboard Cite

show abstract

“…Assay limitations have been described previously. 2 Definitive identification of pathogen(s) detected by the multiplex PCR assay was achieved through subsequent specific single-plex PCR amplification and sequencing of the product.…”

Section: Methods Development Of Multiplex Pcr Assay For Vrismentioning

confidence: 99%

“…PCR primers were designed as previously described 2 and optimized using targeted sequences cloned into pCR2.1-TOPO (Invitrogen) or samples with 10 8 live organisms per milliliter of lysis buffer (NucliSENS, bioMérieux) obtained from the Clinical Microbiology Service. Samples were subjected to PCR amplification with a multiplex PCR kit (Qiagen), primers at 0.5 mM each in a final reaction volume of 20 ml, and the following cycling protocol: an annealing step with a temperature reduction in 1°C increments from 65°C to 58°C during the first 8 cycles and then continuing with a cycling profile of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds for 34 cycles in an MJ PTC200 thermal cycler (MJ Research).…”

Section: Methods Development Of Multiplex Pcr Assay For Vrismentioning

confidence: 99%

Evaluation of a multiplex polymerase chain reaction for early diagnosis of ventriculostomy-related infections

Gordon

Tokarz

Briese

et al. 2015

JNS

Self Cite

View full text Add to dashboard Cite

V entriculostomy devices are used in the management of acute hydrocephalus by providing therapeutic drainage of CSF and intracranial pressure monitoring. However, ventriculostomy-related infections (VRIs) frequently occur, causing significant morbidity and mortality.6 Diagnosis of ventriculomeningitis is difficult because similar clinical and CSF parameters are often present after intraventricular hemorrhage and neurosurgery. 5 To reduce the risk of VRIs, many institutions use prolonged antibiotic "prophylaxis" during the use of ventriculostomy devices despite limited evidence of efficacy. 7Definitive diagnostic culture results can take several days and are often negative because of antibiotic inhibition. 8,10 Given the inability to obtain a rapid, sensitive diagnosis and the potential for serious neurological sequelae associated with delayed treatment, most clinicians treat em- obJect Diagnosis of ventriculostomy-related infections (VRIs) is challenging due to the lack of rapid, sensitive assays for pathogen detection. The authors report the development of a multiplex polymerase chain reaction (PCR) assay for differential diagnosis of common VRI pathogens. methods MassTag PCR was used to develop a multiplex assay for detection of 11 VRI pathogens. The assay was established and optimized using cloned template standards and spiked samples and was then evaluated on CSF specimens from ventricular drains. Subjects were grouped into definite VRI, possible VRI, or no VRI based on conventional microbiology, CSF evaluation, and clinical parameters. results CSF specimens were obtained from 45 subjects (median age 49 years, interquartile range 32-63 years; 51% were male). The assay detected 10-100 genome copies. It detected a pathogen in 100% (6 of 6) of definite VRI cases in which a pathogen targeted by the assay was present; these represented 67% of all definite VRIs (6 of 9). Among subjects with a possible VRI, the assay detected a pathogen in 29% (5 of 17). In subjects without overt infection the presence of a pathogen was detected in 32% of subjects (6 of 19), albeit with lower signal compared with the VRI group. coNclusioNs MassTag PCR enabled parallel testing of CSF specimens for 11 pathogens of VRI. The high sensitivity of PCR combined with possible device colonization, specimen contamination, and concurrent antibiotic treatments limit the clinical value of the assay, similar to other current diagnostic approaches. With further optimization, multiplex PCR may provide timely identification of multiple possible VRI pathogens and guide management, complementing classic culture approaches.

show abstract

“…Si bien los virus son los agentes que con mayor frecuencia producen IRA a todas las edades, comparativamente son más comunes en niños que en adultos y causan cuadros más graves en grupos de riesgo, como menores de 2 años, ancianos, inmunosuprimidos y pacientes con comorbilidades 4,7 . En estos pacientes es fundamental encontrar técnicas diagnósticas rápidas, sensibles y precisas, especialmente en el estudio de infecciones respiratorias bajas 8 .…”

unclassified

Aporte de la biología molecular en el diagnóstico de infecciones respiratorias agudas

et al. 2016

Rev. chil. enferm. respir.

View full text Add to dashboard Cite

show abstract

Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens

Cited by 151 publications

References 14 publications

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

Evaluation of a multiplex polymerase chain reaction for early diagnosis of ventriculostomy-related infections

Aporte de la biología molecular en el diagnóstico de infecciones respiratorias agudas

Contact Info

Product

Resources

About