Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine

Gerritsen, M. J.; Olyhoek, T.; Smits, M.A.; Bokhout, B.A.

doi:10.1128/jcm.29.12.2805-2808.1991

Cited by 56 publications

(26 citation statements)

References 18 publications

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“…Detection of leptospires in urine by PCR has been reported for cattle (8,29) and recently for one patient with leptospirosis (18). The use of PCR provides a considerable time gain compared with culturing of leptospires from urine, and PCR can be applied to frozen or formalin-preserved urine samples.…”

mentioning

confidence: 98%

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis

et al. 1994

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We tested urine samples from patients at different stages of current leptospirosis and thereafter to determine whether use of the PCR for detection of leptospires in urine can be a valuable alternative to culturing. The procedure of DNA extraction and subsequent PCR applied to 15 freshly voided urine samples proved to be twice as sensitive as culturing. Overall, we were able to detect leptospires in approximately 90% (26 of 29) of the urine samples. Urine and serum samples were obtained from seven patients, before the eighth day of illness. Although it is generally assumed that leptospiruria starts approximately in the second week of illness, we were able to detect leptospires in all of these early urine samples. In contrast, only two of seven corresponding serum samples gave positive PCR results, which suggests that PCR analysis of urine can be more successful for early diagnosis of leptospirosis than PCR analysis of serum. Urine samples from six patients who had been treated with antibiotics at the time of illness were positive by PCR, implying that the patients were still shedding leptospires in their urine despite treatment. Some of these samples were even taken years after the infection, indicating that shedding of leptospires in urine may last much longer than is generally assumed. We conclude that detection of leptospires in urine with PCR is a promising approach for early diagnosis of leptospirosis and may also be useful in studying long-term shedding.

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confidence: 98%

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis

et al. 1994

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“…Alternative molecular techniques based mainly on PCR have been developed with limited success. PCR methods published to date have either involved amplification of sequences of unknown function (5,6,10,12,17,19,26) or, with a few exceptions (14,23,25), have been used for identification of only a fraction of all possible members of this genus (11,15,16,20,24).…”

mentioning

confidence: 99%

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR

et al. 1997

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Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

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“…Leptospira are very susceptible for acidic environment (pH below 6,8) and after 90 minutes in contact with acid urine, the spirochetes are not detectable with PCR (32,33,34).…”

Section: Materiały I Metodymentioning

confidence: 99%

“…W późniejszej fazie, DNA tych krętków można wykrywać tylko w moczu jednak nieprawidłowe opracowanie próbki po pobraniu skutkuje często wynikiem fałszywie ujemnym. Bakterie z rodzaju Leptospira są wrażliwe na kwaśne środowisko (pH poniżej 6.8) i już po 90 min kontakcie z kwaśnym moczem, nie są wykrywalne metodą PCR (32,33,34). Zgodnie z licznymi opracowaniami naukowymi, pobrany mocz powinien być kilkakrotnie zwirowany, a osad przepłukiwany buforem PBS (chlorek sodu buforowany fosforanami o pH 7,2) oraz sterylną wodą destylowaną.…”

Section: Piśmiennictwounclassified

Leptospirosis in Poland in the years 2014-2017 – characteristics of infections and epidemiological surveillance data

Fiecek¹,

Lewandowska²,

Rogulska³

et al. 2018

Przegl Epidemiol

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The aim of the study was the analysis of current Leptospira spp. infections in Poland on the basis of blood serum samples tests results and clinical data collected from clinicians in the Laboratory NIPH-NIH. METHODS. Clinical materials from 48 patients with clinical symptoms suggesting Leptospira spp suspected of leptospirosis from the years 2014-2017 were included to the study. Blood serum samples collected from patients were tested in Laboratory of Rickettsiae, Chlamydiae and Spirochaetes (currently Laboratory of Vector-borne Diseases) of NIPH-NIH. Levels of specific IgM and IgG antibodies to Leptospira spp. antigens were detected with the enzyme-linked immunosorbent assay (ELISA). RESULTS. Specific antibodies to Leptospira spp. were detected in 18 patients (37.5%). IgM antibodies were found in 6 patients (12.5%) and IgG antibodies were identified in 7 patients (14.6%). Both classes of antibodies of were detected in 5 patients (10.4%). The most samples for study were sent to laboratory from Masovian (13 samples) and Kuyavian-Pomeranian (11 samples) Voivodeships. Not any samples from the Lower Silesia, Lublin, Łódź, Podlaskie and Warmian-Masurian Voivodeships were received. In these patients the most common symptoms of disease were: fever, hepatitis with jaundice and renal failure. CONCLUSIONS. The number of diagnosed human leptospirosis in Poland is low in comparison to the number of cases in other countries, although the Leptospira spp. spirochetes occur in animals in the environment.

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Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine

Cited by 56 publications

References 18 publications

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR

Leptospirosis in Poland in the years 2014-2017 – characteristics of infections and epidemiological surveillance data

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