A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and II

Pérez, Lester J.; Lanka, Saraswathi; DeShambo, Vanessa J.; Fredrickson, Richard; Maddox, Carol W.

doi:10.3389/fmicb.2020.00457

Cited by 14 publications

(8 citation statements)

References 70 publications

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“…The efficacy of real time PCR for the daignosis of leptosprisos has been reported by Fornazari and co-investigators [24]. Recently, Perez and co-workes [25] mentioned that new multiplex qPCR is a dependable tool to diagnose Leptospira infections. In addition, this novel assay can be employed for the detection and differentiation of Leptospira strains from both pathogenic groups I and II.…”

Section: Polymerase Chain Reactionmentioning

confidence: 89%

Immunological and Molecular Diagnostic Techniques for Leptospirosis: An Update

Lema¹,

Atalel²

2022

IJCEMR

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A number of laboratory techniques are employed to establish the diagnosis of microbial diseases that cause significant morbidity as well as mortality in humans and animals throughout the world. Leptospirosis is an emerging and re-emerging enigmatic zoonotic disease of public health and economic importance. Currently, over 600,000 human deaths are attributed due to leptosprirosis annually in the world. However, there is a lack of information on Leptospira strains in remote parts of the world. The diagnosis of leptospirosis is challenging due to non-specific clinical feature. Thus, laboratory tests are necessary to confirm the diagnosis of disease due to its varied symptomatology. The microscopic agglutination test (MAT), which determines agglutinating antibodies in sera for various serovars of Leptospira species is considered the gold standard for the diagnosis of leptospirosis. Enzyme linked immunosorbent assay (ELISA) usually detects only the antibodies reacting with a broadly reactive genus specific antigen and thus gives no indication of the causative serovar or serogroup, which limits its application. Polymerase chain reaction on the other hand is considered sensitive and specific for the rapid detection of Leptospira in clinical samples. It is imperative to employ immunological and molecular techniques in order to make an unequivocal diagnosis of leptospirosis to institute immediate therapy to mitigate the suffering of the patients.

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Section: Polymerase Chain Reactionmentioning

confidence: 89%

Immunological and Molecular Diagnostic Techniques for Leptospirosis: An Update

Lema¹,

Atalel²

2022

IJCEMR

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“…In addition, we coupled this method with a bacterial integrity assay. A triplex PCR (two genes and an internal control) was designed and used to discriminate Leptospira from subclades P1 and P2 in animal samples [ 45 ]. This multiplex qPCR was set up, according the MIQE guidelines [ 46 ], to detect all Leptospira and selectively discriminate between pathogenic and saprophytic strains.…”

Section: Discussionmentioning

confidence: 99%

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay

et al. 2021

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Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.

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“…Serial dilutions of in vitro transcripts in water and plasma were detected by the respective primer/probe sets to determine linearity and efficiency (Figure S4). The limit of quantification (LoQ) was defined using ten-fold serial dilutions in nuclease-free water of quantified transcripts standards (Figure S4) [ 25 ]. Similarly, the analytical sensitivity (ASe) was estimated by evaluating ten-fold serial dilutions of quantified RNA transcripts standards, determined using a Probit regression analysis implemented in the MedCalc Statistical Software version 19.0.7 (MedCalc Software bvba, Ostend, Belgium; 2019).…”

Section: Methodsmentioning

confidence: 99%

Oropouche virus as an emerging cause of acute febrile illness in Colombia

Čiuoderis

Berg

Pérez

et al. 2022

Emerging Microbes & Infections

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Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study ( n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT–PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.

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A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and II

Cited by 14 publications

References 70 publications

Immunological and Molecular Diagnostic Techniques for Leptospirosis: An Update

Immunological and Molecular Diagnostic Techniques for Leptospirosis: An Update

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay

Oropouche virus as an emerging cause of acute febrile illness in Colombia

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