Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospira-specific IgM enzyme-linked immunosorbent assay in a clinical environment

Slack, Andrew T.; Symonds, Meegan L.; Dohnt, Michael F.; Harris, Christina; Brookes, Dianne L.; Smythe, Lee D.

doi:10.1016/j.diagmicrobio.2006.10.004

Cited by 71 publications

(75 citation statements)

References 16 publications

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“…Additionally, unlike MAT, ELISA is a genus specific tests that does not enable the assessment of presumptive infecting serogroups, and as such lacks an important epidemiological application. In The Netherlands, MAT is always [23,24]. These PCRs have a high diagnostic accuracy notably in this early acute phase, hence perfectly complementing serodiagnostic testing.…”

Section: Discussionmentioning

confidence: 99%

» Indexing and Archiving | More Bacteriology & Parasitology Journals Journal of Bacteriology & Parasitology Download Journal Flier JBP Editorial Board Scientific Associations Ann Albihn 2010- Governmental commission on climate change and infectious diseases 2009 Commission on climate change and animal inf. diseases to the governmental investigation on the handling of animal inf. diseases(Djursmittoutredningen), Agricult. Ministry 2007- 2010 The trend surveillance and analysis Group (omvärldsråd) at

Goris¹,

Leeflang²

2012

J Bacteriol Parasitol

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Laboratory case definition of leptospirosis is scarcely defined by a solid evaluation that determines cut-off values in the tests that are applied. This study describes the process of determining optimal cut-off titers of laboratory tests for leptospirosis for a valid case definition of leptospirosis. In this case the tests are the microscopic agglutination test (MAT) and an in-house IgM enzyme-linked immunosorbent assay (ELISA) both on single serum and paired samples using a positive culture as the reference test in the Dutch population. The specificity was assessed using panels of sera from healthy donors, cases with known other diseases and non-leptospirosis cases with symptoms compatible with leptospirosis. Cases were divided into three periods corroborating the acute phase (1-10 days post onset of illness (DPO)), the early convalescent (11-20 DPO) and the late convalescent phase (>20 DPO). Cut-off titers for MAT and IgM ELISA were determined as 1:160 and 1:80 respectively for all three periods. These cut-off titers combined 100% specificity with a sensitivity that changed according to the stage of disease for both tests. The low sensitivities in the early acute phase are consistent with the dynamics of the humoral immune response. IgM ELISA yielded higher sensitivities compared to MAT in the acute and early convalescent stages. Moreover, the optimal sensitivity of MAT, the gold standard was < 82%, implying that a significant part of global cases is missed by this recommended test. MAT and IgM ELISA manifested partly complementary, resulting in a higher sensitivity when combining the results of these two tests. The availability of paired samples and of adequate clinical and epidemiological data are other parameters that will significantly increase the sensitivity of laboratory confirmation. This study enables fine-tuning of the current laboratory definition towards an improved case finding and implies that solid validation of laboratory parameters for case definition will improve both the diagnosis for individual patient care and for estimating the disease burden at a worldwide scale.

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Section: Discussionmentioning

confidence: 99%

» Indexing and Archiving | More Bacteriology & Parasitology Journals Journal of Bacteriology & Parasitology Download Journal Flier JBP Editorial Board Scientific Associations Ann Albihn 2010- Governmental commission on climate change and infectious diseases 2009 Commission on climate change and animal inf. diseases to the governmental investigation on the handling of animal inf. diseases(Djursmittoutredningen), Agricult. Ministry 2007- 2010 The trend surveillance and analysis Group (omvärldsråd) at

Goris¹,

Leeflang²

2012

J Bacteriol Parasitol

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“…DNA was eluted in a final volume of 200 ml. Real-time PCR was performed, in version 1 of the LightCycler thermocycler (Roche Scientific), using the modified TaqMan method described by Slack et al (2007). Test samples giving cycle-to-threshold (Ct) values of 5-40, 41-45 and .45 cycles were considered positive, 'equivocal' and negative, respectively.…”

Section: Silver Stainingmentioning

confidence: 99%

The role of fruit bats in the transmission of pathogenic leptospires in Australia

Tulsiani

Cobbold

Graham

et al. 2011

Annals of Tropical Medicine & Parasitology

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Although antileptospiral antibodies and leptospiral DNA have been detected in Australian fruit bats, the role of such bats as infectious hosts for the leptospires found in rodents and humans remains unconfirmed. A cohortdesign, replicated survey was recently conducted in Far North Queensland, Australia, to determine if the abundance and leptospiral status of rodents were affected by association with colonies of fruit bats (Pteropus conspicillatus spp.) via rodent contact with potentially infectious fruit-bat urine. In each of four study areas, a 'colony site' that included a fruit-bat colony and the land within 1500 m of the colony was compared with a 'control site' that held no fruit-bat colonies and was .2000 m from the nearest edge of the colony site. Rodents were surveyed, for a total of 2400 trap-nights, over six sampling sessions between September 2007 and September 2008. A low abundance of rodents but a high carriage of leptospires in the rodents present were found to be associated with proximity to a fruit-bat colony. For example, means of 0.4 and 2.3 fawn-footed melomys (Melomys cervinipes) were collected/100 trap-nights at sites with and without fruit-bat colonies, respectively (P,0.001), but the corresponding prevalences of leptospiral carriage were 100% and 3.6% (P,0.001). Such trends were consistent across all of the sampling sessions but not across all of the sampling sites.Leptospires were not isolated from fruit bats by culture, and the role of such bats in the transmission of leptospires to rodents cannot be confirmed. The data collected do, however, indicate the existence of a potential pathway for transmission of leptospires from fruit bats to rodents, via rodent contact with infectious fruit-bat urine. Fruit bats may possibly be involved in the ecology of leptospires (including emergent serovars), as disseminators of pathogens to rodent populations. Stringent quantitative risk analysis of the present and similar data, to explore their implications in terms of disease prevalence and wildlife population dynamics, is recommended.

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“…Se han desarrollado pruebas de laboratorio mediante PCR para la detección de leptospiras patógenas en el humano (Smythe et al, 2002;Slack et al, 2007;Ahmed et al, 2009;Rojas et al, 2010). En la fase aguda de la enfermedad, las leptospiras están presentes en grandes cantidades en varios fluidos corporales (sangre, líquido cefalorraquídeo, orina), de allí que se puede obtener buenos resultados en los ensayos de PCR antes de iniciar la terapia antimicrobiana (Slack et al, 2007;Ahmed et al, 2009;Villumsen et al, 2010).…”

Section: Introductionunclassified

“…En la fase aguda de la enfermedad, las leptospiras están presentes en grandes cantidades en varios fluidos corporales (sangre, líquido cefalorraquídeo, orina), de allí que se puede obtener buenos resultados en los ensayos de PCR antes de iniciar la terapia antimicrobiana (Slack et al, 2007;Ahmed et al, 2009;Villumsen et al, 2010).…”

Section: Introductionunclassified

Estandarización de una Técnica de PCR en Tiempo Real con Sondas TaqMan para la Detección de Leptospira spp Patógenas en Orina de Canes Domésticos

Sedano

Pinto

Siuce

et al. 2016

Rev. investig. vet. Perú

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El presente estudio tuvo como objetivo estandarizar una técnica de PCR en tiempo real con sondas TaqMan para detectar la presencia de leptospiras patógenas en orina de perro infectada in vitro con cepas patrón de Leptospira sp. Las muestras fueron obtenidas de un perro clínicamente sano y negativo a la prueba de microaglutinación. Se utilizaron los cebadores Lepto R y Lepto F, específicos para Leptospira sp y una sonda TaqMan Lepto probe que amplifican e hibridan, respectivamente, una porción del gen rrs, capaces de diferenciar entre especies patógenas y no patógenas de Leptospira. Se estandarizó el protocolo de PCR en 35 ciclos, con un proceso de desnaturalización inicial a 95 °C por 5 min, seguida por una desnaturalización a 95 °C por 15 s y finalmente el alineamiento y extensión en un solo paso a 60 °C por 1 min. Los valores de ciclo umbral (Ct) determinados durante la estandarización de la PCR estuvieron en un rango de 12.53 a 18.21 para las 25 cepas patógenas de Leptospira sp, en tanto que las cepas saprófitas y otros bacterias no dieron productos específicos. El estándar fue detectado hasta una dilución de 102 con un Ct de 29.98 a una eficiencia de 1.13 y con un coeficiente de correlación (R2) de 0.993. Se detectó ADN en las muestras de orina infectada desde la dilución de 107 leptospiras/ml con un valor de Ct de 17.54 hasta una mínima dilución de 102 leptospiras/ml con un valor de Ct de 29.87.

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Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospira-specific IgM enzyme-linked immunosorbent assay in a clinical environment

Cited by 71 publications

References 16 publications

The role of fruit bats in the transmission of pathogenic leptospires in Australia

Estandarización de una Técnica de PCR en Tiempo Real con Sondas TaqMan para la Detección de Leptospira spp Patógenas en Orina de Canes Domésticos

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