Differentiation leptospira species and serovars by pcr. Our data suggest that r typhirickettsia spp, o tsutsugamushi, and leptospira spp infections are important causes of cns infections in laos. Pcr tests developed by other researchers also provide fast testing platforms, but the primers used can only differentiate between pathogenic and nonpathogenic leptospira and cannot identify individual species smythe et al. Apr 25, 2019 as an efficient, costeffective method for leptospira detection, we performed a single sequencing run on a pooled library of the described leptospiral and bacterial pcr products by using the miseq. Pcr and real time pcr, looking for dna and 16s rrna of microorganisms, have been used as a useful tool to detect microorganisms in a variety of biological samples. B0051 ultrasensitive qualitative detection of leptospira bacteria by real time polymerase chain reaction. Leptospirosis is a bacterial disease that affects humans and animals. The causal agent of the disease is the bacterium leptospira interrogans. Lungworms dictyocaulus viviparus are nematodes that cause parasitic bronchitis husk or hoose in cattle. Lept pcr detection kit insert pi446003 norgen biotek. A proportion of patients who are positive by pcr are negative by culture. Pdf polymerase chain reaction for detection of leptospira.
Nov, 20 the aim of the present study was to consider the wide usage of urinary pcr as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. According to the available information to date, real time pcr technique have been used for a number of tests to identify the pathogenic and saprophytic strain of leptospira, which. These relatively high infection rates could represent a hazard to public health. Pcr based genetic marker for the detection of leptospira. A nested polymerase chain reaction pcr using primers from the lipl32 sequence of leptospira spp. Pcr and culture identification of pathogenic leptospira spp. Leptospira, real time pcr, saprophyte, leptospirosis 1. In our hands, these assays can detect between 102 and.
It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. Some infected persons, however, may have no symptoms at all. These severely neglected infections represent a potentially large proportion of. Pcr on postmortem tissues, such as liver or kidney, and abortion tissues, such as placenta, fetal kidney or fetal stomach contents, are. A 331bp sequence from the leptospira interrogans serovar canicola rrs 16s gene was amplified, and the pcr products. The leptospirosis diagnostic serology service is located within the department of microbiology at monash university, melbourne and performs testing for human leptospirosis for victoria, australia. Conventional pcr assays have been developed, but all have limitations which have restricted their widespread use. Oligonucleotide sequences of lep f, lep r, lau01, and lau02 used for the mpcr amplification of the leptospira species are listed in table 1.
Multiplex real time pcr is carried out to detect pathogenic leptospira pp. Direct detection and differentiation of pathogenic. The differential diagnosis of leptospirosis is difficult due to the varied and often flu like symptoms which may result in a missed or delayed diagnosis. Molecular detection of pcr is rapid, highly sensitive and specific. Bacterial dna can be extracted from various sample types as blood, plasma or urine. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. Molecular methods such as polymerase chain reaction pcr are considered quick, and better in terms of sensitivity and specificity. Leptospirosis is a disease of worldwide significance that infects both animals and humans. Serological testing for leptospira is useful during the convalescent phase of the disease and for serovar identification. A quantitative pcr taqman assay for pathogenic leptospira spp. Among pinniped populations, periodic epizootics of leptospirosis are responsible for significant dieoffs. The aim of the present study was to compare the use of mat and pcr for the diagnosis of leptospirosis in cattle. Nov 12, 2019 molecular methods such as polymerase chain reaction pcr are considered quick, and better in terms of sensitivity and specificity. Background bacteria of the genus leptospira, the causative agents of leptospirosis, are categorized into pathogenic and nonpathogenic species.
The aim of this study was to identify the 1 and lipl32 genes in ompl leptospira strains isolated from urine samples of cattle. Leptospirosis, diagnosis, pcr, 16s rrna, lesptospira. Nov 28, 2011 recently, a variety of molecular techniques, including conventional and realtime polymerase chain reaction pcr, have been developed for the specific detection of pathogenic bacteria from the genus leptospira. In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction pcr to 26 serovars of leptospira interrogans, l. Imspcr was able to detect 10 2 leptospira cells per ml of human sera and urine, corresponding to 25. Evaluation of boxpcr and ericpcr as molecular typing tools. Leptospira interrogans endpoint pcr kit product insert. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test mat which relies on.
Leptospirosis is caused by various species of leptospira, a spirochete in the family leptospiraceae, order spirochaetales. Detection of dna in clinical samples is increasingly used, and pcr assays have been developed for leptospirosis. Amplicons 497 bp were obtained from 21 pathogenic reference serovars belonging to four species l. A 331bp sequence from the leptospira interrogans serovar canicola rrs 16s gene was amplified, and the pcr products were analyzed by dnadna hybridization by using a 289bp fragment internal to the amplified dna. Background leptospirosis is a zoonotic disease scattered throughout the world 1,2. Lungworm outbreaks in cattle can result in production losses from reduced weight gain, lowered milk yield and death in young calves. The national leptospirosis service nls is the national laboratory reference centre for the diagnosis of leptospira infections. A quantitative pcr taqman assay for pathogenic leptospira. Rapid distinction between leptospira interrogans and leptospira biflexa by pcr amplification of 23s ribosomal dna. Detection of pathogenic leptospires in urine from naturally. In this study, we present the development of a realtime pcr rtpcr for the detection of pathogenic leptospira the pathogenic rtpcr, and we. Lssppcr analysis was performed to detect dna polymorphisms in a 285 bp. In humans, it can cause a wide range of symptoms, some of which may be mistaken for other diseases. From table 2, it was summarized that box pcr clustered the isolates into 4, 6, and 6 clusters, with discriminatory indices 0.
The increasing number of reports from species of pathogenic leptospira spp. Sensitive realtime pcr detection of pathogenic leptospira. Using leptospira interrogans serovar icterohaemorrhagiae dna, the lower limit of detection was found to be 20 genomic equivalentsreaction with a 95% cutoff value. From table 2, it was summarized that boxpcr clustered the isolates into 4, 6, and 6 clusters, with discriminatory indices 0. Whole blood and urine are tested simultaneously to allow for diagnosis of sick animals in the early stages of infection and for the detection of urinary shedding in sick animals. Molecular taxonomic analysis of a 1,200bp segment of the leptospiral 16s ribosomal rna gene was used to identify leptospira to the species level. Pcr is increasingly being reported as a rapid and definitive diagnostic tool in many species, such as buffaloes marianelli and others 2007, small ruminants lilenbaum and others 2009 and cattle hernandezrodriguez and others 2011. Pdf rapid distinction between leptospira interrogans and.
In this study, pcr followed by restriction endonu clease analysis of the amplified target pcr rea. Spirochetes of the genus leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. The polymerase chain reaction pcr has the potential to make a dramatic impact in diagnosing leptospirosis. Frontiers a validated multiplex realtime pcr assay for the. Human leptospirosis is an acute febrile illness that presents with many manifestations and is found worldwide. Comparison of realtime pcr assays for detection of. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test mat. There are over 230 known serovars in the genus leptospira.
The lau01 and lau02 primer pair was designed by aligning the lipl32 gene accession number. Identification of ompl1 and lipl32 genes to diagnosis of. In order to overcome these limitations, a realtime pcr assay was developed using a 423 bp target on the lipl32 gene, which is conserved among. Detection of leptospires in urine by polymerase chain reaction. Pdf a quantitative pcr taqman assay for pathogenic. From 1 april 2015, a new combined clinical diagnostic and reference. Pcr and culture identification of pathogenic leptospira. To assess the specificity of the primers, dna samples of reference leptospiral strains belonging to 7 pathogenic species and 5 nonpathogenic or saprophytic species were subjected to both pcr tests. Leptospirosis is a blood infection caused by the bacteria leptospira. Thus an alternative method is needed to provide a quick diagnosis of the disease at this stage. We tested a biobank of paired samples of serum and urine from the. All pathogenic leptospires were positive when tested with both assays.
Rga and bim strain 1051 enabled the amplification by pcr of target dna fragments. Without treatment, leptospirosis can lead to kidney damage. Serum pcr was found to be less sensitive than pcr performed on other fractions for detection of leptospira dna 11, 22. A 331bp sequence from the leptospira interrogans serovar canicola rrs 16s gene was amplified, and the pcr products were analyzed by dnadna hybridization by using a 289bp fragment internal to the. Leptospirosis is a zoonosis caused by pathogenic leptospira spp. Illness may be self limiting or cause hepatorenal failure weil syndrome.
In this study, we present the development of a realtime pcr rtpcr for the detection of pathogenic leptospira the. Its clinical signs resemble that of other diseases like dengue and flu, and it is difficult to. On 8 november 20, a storm surge caused by super typhoon. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. Download fulltext pdf polymerase chain reaction for detection of leptospira spp. Pcr is important for early diagnosis during the acute phase of illness before an antibody response is mounted.
Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. In cattle, it causes economy losses with abortions, stillbirths, prema. In our hands, serum was also found not to be the best fraction for detecting leptospires, whatever the kit of dna extraction used. Primers for polymerase chain reaction pcr were synthesized from clones derived from a leptospira hardjo. In this work we describe a novel and simple taqmanbased multi. The basic unit of leptospira taxonomy is the serovar. Antibiotics, such as tetracyclines, needed for the treatment of murine typhus and scrub typhus, are not routinely advised for empirical treatment of cns infections. Keeping in view the importance of pcr in disease diagnosis and pathogen detection the present study is aimed at pcr based detection of leptospira interrogans using lipl 32 gene specific primers. Diagnosing and managing canine leptospirosis diagnostic update.
Leptospirosis is a disease of worldwide significance that infects many. An immunomagnetic separationpcr method for detection of. Leptospira microagglutination testing cornell university. Detection of pathogenic leptospira bacteria in pinniped. Rapid detection of pathogenic leptospires by pcr molecular. Thirtyfive leptospira serovars from the species leptospira interrogans, leptospira borgpetersenii, leptospira santarosai, leptospira kirschneri, leptospira weilii, leptospira biflexa and leptospira meyeri were characterized by the lowstringency single specific primer pcr lssppcr technique. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where leptospira spp. Novel taqman pcr for detection of leptospira species in urine. Leptospirosis is considered an underdiagnosed disease. Leptospira was first observed in 1907 in kidney tissue slices of a leptospirosis victim. Pcr detection of leptospira dna in rodents and insecti vores. Detection of seven species of pathogenic leptospires by pcr using.
Rapid detection of pathogenic leptospires by pcr molecular methods soni muhsinin1, rahma ziska1 1bandung school of pharmacy, bandung, west java, indonesia abstract leptospirosis is an animalborne disease zoonosis caused by bacterial infection of the leptospira genus. The rate of leptospira detection by pcr per animal species was 6% for mastomys spp. Historically, several pairs of primers for pcr detection of leptospires have been published, some based on specific gene targets rrna genes, repetitive elements or sequences derived from genomic libraries. Sixteen strains were obtained from urine samples and, dna was isolated to perform two polymerase chain reaction pcr.
This is an improvement over other pcr tests available to test for leptospira. Direct detection and differentiation of pathogenic leptospira. Molecular typing of pathogenic leptospira isolates using the boxa1r primer generated 4 to 11 bands ranging from 200 to 20,000 bp figure 1. Lowstringency single specific primer pcr for identification. Orientia, rickettsia, and leptospira pathogens as causes. There are a limited number of realtime pcr qpcr assays for detecting leptospira and not all of these assays are able to distinguish pathogenic from nonpathogenic species. B0051 ultrasensitive qualitative detection of leptospira by real time polymerase chain reaction. Leptospira greek leptos, fine, thin and latin spira, coil is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species. Development and validation of a realtime pcr for detection of. Although several pcrbased methods are currently in use, there is little information on their comparability. Lep f and lep r primer amplified a 330 bp sequence from the 16s rrna gene. A total of 684 samples submitted for the molecular diagnosis of leptospira spp.
Evaluation of boxpcr and ericpcr as molecular typing. Signs and symptoms can range from none to mild headaches, muscle pains, and fevers to severe bleeding in the lungs or meningitis. A quantitative realtime pcr assay was used to compare levels of leptospira in urban and rural environmental surface waters in sites in the peruvian amazon region of iquitos. Development of speciesspecific pcr primer sets for the. Frontiers a validated multiplex realtime pcr assay for. Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of leptospira. The samples included urine, blood, kidney, placenta, liver, and fetal tissues supplementary table s1 obtained from.
For analysis of the pcr data, the pcr reaction is loaded on an agarose dna gel along with the provided dna ladder for qualitative analysis. Currently, direct detection of leptospira can be done in clinical laboratories by conventional and by realtime pcr qrtpcr. Along the west coast of north america, the most recent leptospirosis epizootic occurred in 2004, during which samples were collected. Leptospirosis, caused by the spirochete leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. Simultaneous detection of pathogenic and saprophyte. Like gramnegative bacteria, leptospira have an outer membrane studded with lipopolysaccharide lps on the surface, an inner membrane and a layer of peptidoglycan cell wall. Leptospira detection pcr test for viral infections dr. Pcr is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms.
Molecular detection and speciation of pathogenic leptospira. Leptospirosis is transmitted via contact with infected urine, venereal and placental transfer, bite wounds or ingestion. Leptospira species referred to as the pathogenic rtpcr. It is accredited for testing by the national association of testing authorities and by the royal college of pathologists of australasia. In this study, we developed a realtime polymerase chain reaction pcr assay using a taqman probe targeting lipl32, which is present only in pathogenic leptospira spp. Although several pcr based methods are currently in use, there is little information on their comparability. Detection of pathogenic leptospires by realtime quantitative pcr. A severe form, which includes jaundice, kidney failure, and bleeding, is called weil disease.
This test detects but does not differentiate the leptospira serovars that are typically of clinical concern in horses b0051 is included in p0024 equine breeding panel. Leptospira detection kit mylab discovery solutions. Download fulltext pdf download fulltext pdf a quantitative pcr taqman assay for pathogenic leptospira spp article pdf available in bmc infectious diseases 21. The pathogenic rtpcr combines the leptospira primers from the ufi assay with a new hydrolysis probe that targets sequence found in pathogenic leptospira species. In addition, there are a variety of probe technologies and qpcr instruments that are utilized with these assays.
Mylab lifesolutions offers pathodetect leptospira detection kit for detection of pathogenic leptospira spp. Determining risk for severe leptospirosis by molecular. Sixteen strains were obtained from urine samples and, dna was isolated to perform two polymerase chain reaction pcr tests which identified 32 and ompl1 lipl genes. Laboratory studies can confirm the diagnosis and determine the extent of organ involvement. Weils disease, the acute, severe form of leptospirosis, causes the infected individual to become jaundiced skin and eyes become yellow, develop kidney failure, and bleed. Although leptospira infection can be diagnosed by change in antibody titers, titers may be negative during early stages of critical infection. A comparison of three dna extractive procedures with. B0051 is included on p0034 canine breeding panel and p0039 canine anemia panel and p0051 dog show panel.
In addition to mat serology, leptospira realtime pcr testing on blood, urine or ocular fluids may be of diagnostic value in highly suspect clinical cases, especially when the duration of illness is not known. Two sets of primers derived from genomic dna libraries of leptospira serovars icterohaemorrhagiae strain. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. Leptospirosis is caused by spirochaete bacteria that belong to the genus leptospira, which are aerobic, righthanded helical, and 6 20 micrometers long. Pdf leptospirosis diagnosis using nestedpcr researchgate. Pcr on blood will be positive early in infection, usually prior to seroconversion.
B0051 ultrasensitive qualitative detection of leptospira by real time polymerase chain reaction b0051 is included on p0034 canine breeding panel and p0039 canine anemia panel and p0051 dog show panel. Some leptospira are harmless saprophytes that reside in the environment, while others are pathogenic. In this study, four quantitative realtime pcr qpcr assays sybr green and taqman chemistries targeting the secy, lfb1, and lipl32 genes were evaluated as diagnostic assays. Diagnosis of human leptospirosis in a clinical setting. Polymerase chain reaction for detection of leptospira spp. The development of a pcr based typing system for leptospira spp. Pcr polymerase chain reaction pcr tests detect leptospira spp. The imspcr method enhanced detection of leptospira in experimentally contaminated human sera and urine when compared to pcr performed alone. A 330 bp dna fragment from the dna of all leptospira interrogans, leptospira borgpetersenii, leptospira noguchii, leptospira santarosai and leptospira biflexa serovars used in the present study was amplified by pcr.
For identification of individual leptospira species, pcr primers that detect the ompl1 gene sequence for the. The broad spectrum of pcr in the detection of pathogenic and saprophytic leptospires could be explained by the primers employed since the. The detection of leptospira interrogans specific dna is based on endpoint pcr technology, utilizing polymerase chain reaction pcr for the amplification of specific l. This assay was performed as a monoplex reaction, which differs from the multiplex, internallycontrolled design of the ufi assay. Thirtyfive leptospira serovars from the species leptospira interrogans, leptospira borgpetersenii, leptospira santarosai, leptospira kirschneri, leptospira weilii, leptospira biflexa and leptospira meyeri were characterized by the lowstringency single specific primer pcr lssp pcr technique. As an efficient, costeffective method for leptospira detection, we performed a single sequencing run on a pooled library of the described leptospiral and bacterial pcr products by using the miseq. Rapid and simple assays for the identification of individual leptospira species are currently not available.
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