Article

Detection of pathogenic Leptospira spp. through TaqMan Polymerase Chain Reaction targeting the LipL32 Gene

National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Diagnostic microbiology and infectious disease (Impact Factor: 2.46). 05/2009; 64(3):247-55. DOI: 10.1016/j.diagmicrobio.2009.03.014
Source: PubMed

ABSTRACT

Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In this study, we developed a real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp. Using Leptospira interrogans serovar Icterohaemorrhagiae DNA, the lower limit of detection was found to be 20 genomic equivalents/reaction with a 95% cutoff value. The assay detected pathogenic Leptospira strains, but not intermediately pathogenic or nonpathogenic strains. When testing the assay on spiked clinical specimens, whole blood and plasma were better specimens for detecting the same initial number of leptospires compared with serum from clotted and centrifuged blood. Leptospira spiked at the same concentration was better detected in centrifuged urine. This real-time PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis.

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    • "Además, poseen más de 250 serovariedades patógenas, que por su similitud antigénica están agrupadas en 25 serogrupos. Protocolos actuales de PCR no definen el serovar/serogrupo infectante, a pesar de distinguir entre especies patógenas y saprófitas (Stoddard et al., 2009; Picardeau, 2013). Así, la prueba de Microaglutinación (MAT) es la herramienta diagnóstica de referencia internacional con mejor capacidad de distinguir y cuantificar anticuerpos contra determinado serogrupo (patógeno y saprófito). "
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    ABSTRACT: La leptospirosis es una zoonosis bacteriana de gran impacto. Los perros y otros animales pueden infectarse, constituyendo un factor importante en la diseminación de la bacteria al humano. La infección es causada por cualquiera de los serovares patógenos de los 25 serogrupos de Leptospira spp. El estudio tuvo como objetivo identificar serogrupos de Leptospira spp presentes en perros con diagnóstico clínico presuntivo de leptospirosis de la ciudad de Lima. Se obtuvieron 305 muestras de suero sanguíneo de perros de 31 distritos de Lima Metropolitana. Se realizó la prueba de microaglutinación (MAT), estableciéndose títulos ≥1/100 de serorreactividad como seropositivos. Se utilizaron cepas de referencia de los 25 serogrupos, incluyendo al nuevo serogrupo Iquitos, serovar varillal, cepa VAR10, aislado y reportado en casos humanos en Perú. Se detectaron 177 seropositivos (58.0%), 31 (10.2%) de los cuales fueron coaglutinaciones. Los serogrupos reaccionaron contra 18 serogrupos siendo los de mayor frecuencia: Iquitos (15.1%), Tarassovi (12.1%), Canicola (12.1%), Australis (4.6%), Icterohaemorrhagiae (4.3%), Pomona (3.9%), Mini (3.3%) y Ballum (2.6%). No se identificaron serorreactores en los serugrupos Bataviae, Celledoni, Hebdomadis, Lousiana, Panama, Ranarum y Sarmin.
    Preview · Article · Dec 2015 · Revista de Investigaciones Veterinarias del Peru
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    • "Some authors agree that serum PCR is less sensitive than PCR performed on other types of sample, e.g. whole blood for detection of Leptospira DNA (Stoddard et al., 2009; Bourhy et al., 2011). Besides low DNA concentration , the low percentage of typing samples might be due to the use of storage at − 70 °C for several years, and the associated freezing and thawing, which can cause DNA damage (Visvikis et al., 1998; Ross et al., 1990). "
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    ABSTRACT: Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6L. interrogans, one L. wolffii and one L. broomii) and 58 out of 85 (68.2%) clinical samples (55L. interrogans, 2L. meyeri, and one L. kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.
    Full-text · Article · Nov 2015 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
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    • "LipL32_45F – 5 0 AAG CAT TAC TTG CGC TGG TG 3 0 and LipL32_286R – 5 0 TTT CAG CCA GAA CTC CGA TT 3 0 ), which generate a 242 bp fragment (Stoddard et al., 2009 "
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    ABSTRACT: A strain of Leptospira kirschneri (serogroup Grippotyphosa) was cultured from urine of a mare post-abortion in Brazil and characterized by serogrouping, multiple-locus variable-number tandem repeat analysis, PGFE, and sequencing of genes rrs and secY. Strains of L. kirschneri have apparently never been recovered from horses in tropical area, only in Europe and USA. Knowledge of local epidemiology is important to interpret genetic profiles of leptospires circulating in an area.
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