A multiplex PCR‐based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia

Wang, Yajuan; Kong, Fanrong; Yang, Yonghong; Gilbert, Gwendolyn L.

doi:10.1002/ppul.20749

Cited by 39 publications

(23 citation statements)

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“…This involves development of assays that are symptom based and can detect multiple respiratory pathogens, including B. pertussis (55)(56)(57)(58). A disadvantage of the latter approach can be that multiple sets of primers may reduce the sensitivity of detection.…”

Section: Pcr Assaysmentioning

confidence: 99%

Laboratory Diagnosis of Pertussis

2015

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SUMMARYThe introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

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Section: Pcr Assaysmentioning

confidence: 99%

Laboratory Diagnosis of Pertussis

2015

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“…We previously developed a simple method to rapidly detect and identify bacterial and fungal pathogens using PCR followed by hybridization with species-specific oligonucleotides in a reverse line blot (RLB) assay (20,32,34). This approach has the capacity for the simultaneous analysis of a large number (ϳ40) of strains against multiple probes and can be adapted to include different gene targets within the same assay (14).…”

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confidence: 99%

Identification of Pathogenic Nocardia Species by Reverse Line Blot Hybridization Targeting the 16S rRNA and 16S-23S rRNA Gene Spacer Regions

et al. 2010

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Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 speciesspecific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n ‫؍‬ 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacerdirected probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

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“…17 Tampoco se evaluó sistemáticamente la presencia de Mycoplasma pneumoniae y el cultivo fue la única forma de pesquisa bacteriana, a pesar de que los métodos moleculares podrían incrementar la identificación de estos agentes patógenos. 18 Sin embargo, los méto-dos empleados son exactamente iguales que los utilizados por Moreno y cols., 6 al estudiar su escala simplificada y que los de la descripción original BPS, 7 por lo que es muy poco probable que las diferencias observadas no se relacionen con lo antes mencionado. Además, fuera de lo considerado para el virus influenza, no hay evidencia de que se haya modificado sustancialmente el patrón epidemiológico de las neumonías en el escaso tiempo transcurrido entre ambos estudios (2 años), ni desde la descripción original del BPS (6 años).…”

Section: Discussionunclassified

Validación de una regla de predicción simplificada para la presunción de etiología en niños con neumonía

López

Torres²,

Davenport³

et al. 2011

Arch Argent Pediat

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Arch Argent Pediatr 2011;109(6):499-503 / 499 RESUMEN Introducción. Identificar inicialmente los niños con neumonía bacteriana puede reducir el uso inadecuado de antibióticos. El puntaje BPS (Bacterial Pneumonia Score) es un modelo de predicción clínica que alcanzó adecuada precisión diagnós-tica en la predicción de niños con neumonía bacteriana. Sin embargo, como la interpretación de la radiografía de tórax que incluye este modelo es algo compleja, se desarrolló una versión simplificada, cuya validación en otra población aún no se ha realizado. Objetivo. Validar una regla de predicción clíni-ca simplificada para identificar niños con riesgo de presentar neumonía bacteriana. Métodos. Se estudiaron niños <5 años internados por neumonía con etiología confirmada (viral o bacteriana), y se registraron componentes de la regla de predicción clínica simplificada (temperatura axilar, edad, neutrófilos totales, neutrófi-los inmaduros y radiografía de tórax). Resultados. Se incorporaron 168 pacientes (23 presentaron neumonía bacteriana y 145 viral). Aquellos con neumonía bacteriana presentaron un valor de la escala mayor que los de etiología viral (5,3 ± 2,5 contra 2,6 ± 2,02; p <0,001). Un puntaje ≥3 fue identificado como el mejor punto de corte para predecir neumonía bacteriana (área bajo la curva de predicción relativa [aucROC]= 0,79; IC95%: 0,68-0,90), y fue significativamente más frecuente en pacientes con neumonía bacteriana que viral (19/23 contra 72/145, p= 0,003; OR= 4,8; IC95%: 1,4-17,6). La predicción de neumonía bacteriana mostró una sensibilidad de 82,6%, especificidad 50,3%, valor predictivo positivo 20,9%, valor predictivo negativo 94,8%, razón de verosimilitud positiva 1,66 y razón de verosimilitud negativa 0,35. Conclusiones. La regla de predicción clínica simplificada evaluada mostró una limitada capacidad diagnóstica para identificar a niños con neumonía bacteriana y resultó inferior al BPS. Palabras clave: neumonía, infecciones del sistema respiratorio, diagnóstico diferencial.

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A multiplex PCR‐based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia

Abstract: The mPCR/RLB assay is a sensitive tool for identification of respiratory pathogens, including mixed infections and bacteria requiring special culture methods.

Cited by 39 publications

References 28 publications

Laboratory Diagnosis of Pertussis

Laboratory Diagnosis of Pertussis

Identification of Pathogenic Nocardia Species by Reverse Line Blot Hybridization Targeting the 16S rRNA and 16S-23S rRNA Gene Spacer Regions

Validación de una regla de predicción simplificada para la presunción de etiología en niños con neumonía

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