Cited by (15)
Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR
2012, Saudi Journal of Biological Sciences
Citation Excerpt :
The other serological methods like enzyme-linked immunosorbent assay (ELISA), dipstick ELISA and latex agglutination test (LAT) have already been evaluated but all these techniques suffer from their own limitations (Wagenaar et al., 1994). Southern blot analysis (Pacciarini et al., 1992), Dot-blot, and in situ hybridization analyses (Millar et al., 1987; Terpstra et al., 1987; Zuerner and Bolin, 1988) have overcome some of the limitations of serologic techniques. However, none of these methods can provide definitive diagnosis of leptospirosis in a timely manner to aid clinicians for disease management.
Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7days or 7–30days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.
A simple and rapid nested polymerase chain reaction-restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp.
2009, Diagnostic Microbiology and Infectious Disease
Citation Excerpt :
Although the ELISA method is more sensitive than the MAT technique, it has limitations regarding the standardization of infectious and noninfectious antibody titers, especially in animals (Wagenaar et al., 1994). The introduction of molecular biology assays such as Southern blot analysis (Pacciarini et al., 1992; Perolat et al., 1990; Van Eys et al., 1988; Van Eys et al., 1991; Zuerner and Bolin, 1990), pulsed-field gel electrophoresis (Herrmann et al., 1991, 1992), dot-blot, and in situ hybridization analyses (Millar et al., 1987; Terpstra et al., 1987; Zuerner and Bolin, 1988) have overcome some of the limitations of serologic techniques; however, these techniques cannot provide an early diagnosis and are also not readily applicable to routine work in diagnostic laboratories. Therefore, to overcome this limitation, polymerase chain reaction (PCR) has been used, which is considered as a sensitive and specific assay for the rapid detection of Leptospira infection.
A rapid and specific nested polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) has been developed to detect and differentiate pathogenic and nonpathogenic Leptospira spp. Leptospiral genomic DNA was extracted from suspected human sera using an improved method of standard phenol–chloroform, and specific primers have been used to amplify 16S ribosomal RNA from all pathogenic and nonpathogenic Leptospira spp. The PCR products of all nonpathogenic species were digested with ApoI enzyme, but not pathogenic. To evaluate this assay, we analyzed 283 serum samples collected from suspected patients with leptospirosis. Nested PCR assay confirmed that 42 (14.8%) of 283 samples harbored Leptospira infection, and RFLP assay confirmed 38 (90.5%) of 42 and 4 (9.5%) of 42 positive cases had pathogenic and nonpathogenic Leptospira spp., respectively. Based on sequencing results, Leptospira interrogans, Leptospira kirschneri, and Leptospira wolffii and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected among analyzed samples.
The nested PCR–RFLP assay developed in this study fulfills this requirement in the early stage of infection.
Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine
1998, Research in Veterinary Science
A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 103 to 102 leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.
Magnetic immuno capture PCR assay (MIPA): Detection of Leptospira borgpetersenii serovar hardjo
1997, Veterinary Microbiology
Magnetic immuno PCR assay (MIPA) was developed for the rapid detection of leptospires excreted in urine samples (n = 59) collected from 35 experimentally infected cattle. The immunomagnetic separation of leptospires from inhibitors in frozen formalin fixed bovine urine prior to PCR detection resulted in a marked improvement on previous detection methods. MIPA is a rapid 5 step protocol requiring 70 mins preparation time prior to amplification, which consistently detects 101 organisms. MIPA detected 76% (38/50) of culture positive urines and in addition three urines that were culture negative were shown to be positive by this method of detection. Consequently we conclude that whilst MIPA is an improvement on previously published PCR detection methods, the culture of the organism is still the standard against which other detection methods have to be compared.
Effect of streptomycin treatment on the shedding of and the serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis in experimentally infected cows
1993, Veterinary Microbiology
Shedding patterns of and serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis (L. hardjobovis) have been studied in experimentally infected cows treated with streptomycin in comparison to experimentally infected cows receiving no such treatment. Fourteen cows were experimentally infected with L. hardjobovis, and blood and urine samples were collected weekly for 24 weeks. The microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. The polymerase chain reaction (PCR) was used to determine bacterial shedding in urine. Six weeks after infection six cows were treated with the antibiotic streptomycin (25 mg/kg body weight/day); three cows were treated only once, and the remaining three were treated for five consecutive days. After treatment all six cows had lower serologic responses compared to the untreated cows. The treated cows became also PCR-negative two days after the first treatment, whereas the eight untreated cows remained PCR-positive for at least 70 days. Cows that stopped shedding did not resume shedding within the observation period. Since streptomycin treatment reduces the period of shedding, transmission of leptospira via contaminated urine might be prevented by a single treatment of an infected herd.
Comparison of diagnostic procedures for porcine leptospirosis
1992, Veterinary Microbiology
Kidneys and matched serum samples were obtained from 368 pigs slaughtered at three Victorian abattoirs, and originating from 42 farms. Macroscopic lesions (white spots) were observed on 102 of the kidneys. Serum samples were tested by the microscopic agglutination test (MAT) and by an IgM enzyme immunoassay (EIA). Kidneys were cultured for leptospires, examined histologically after Warthin-Starry silver staining and after immunogold silver staining (IGSS), and tested for leptospiral DNA by DNA hybridization. Forty-four infected pigs were identified by culture or immunogold silver staining of kidneys or by high MAT titres (⩾ 1024). Infection was demonstrated in 7.5% of visibly normal kidneys, in 23.5% of kidneys with white spots, and in 48% of kidneys with large white spots, of 1 cm diameter or greater. The apparent (maximum) sensitivities of diagnostic procedures for detecting infection were as follows: MAT (at a titre of either 64 or 1024) 95%; IgM EIA 82%; culture 61%; presence of white spots 55%; IGSS 52%; presence of large white spots 30%; Warthin-Starry silver staining 20%. IGSS, Warthin-Starry staining and DNA hybridization all appeared to be highly specific. Of 22 kidney sections identified as positive by IGSS, 13 showed intact leptospires, and these kidneys were all culture-positive. Nine others showed leptospiral antigen in the kidney tubules but no intact leptospires. Only five of these kidneys were culture-positive.
Recommended articles (6)
Research article
A novel approach for differentiating pathogenic and non-pathogenic Leptospira based on molecular fingerprinting
Journal of Proteomics, Volume 119, 2015, pp. 1-9
Leptospirosis is a worldwide, deadly zoonotic disease. Pathogenic Leptospira causes leptospirosis. The rapid and accurate identification of pathogenic and non-pathogenic Leptospira strains is essential for appropriate therapeutic management and timely intervention for infection control. The molecular fingerprint is a simple and rapid alternative tool for microorganisms identification, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, molecular fingerprint was performed to identify pathogenic strains of Leptospira. Phylogenetic analysis based on 16S rRNA gene sequences was used as the reference method. In addition, a label-free technique was used to reveal the different proteins of pathogenic or non-pathogenic Leptospira. A reference database was constructed using 30 Leptospira strains, including 16 pathogenic strains and 14 non-pathogenic strains. Two super reference spectra that were associated with pathogenicity were established. Overall, 33 Leptospira strains were used for validation, and 32 of 33 Leptospira strains could be identified on the species level and all the 33 could be classified as pathogenic or non-pathogenic. The super reference spectra and the major spectra projection (MSP) dendrogram correctly categorized the Leptospira strains into pathogenic and non-pathogenic groups, which was consistent with the 16S rRNA reference methods. Between the pathogenic and non-pathogenic strains, 108 proteins were differentially expressed. molecular fingerprint is an alternative to conventional molecular identification and can rapidly distinguish between pathogenic and non-pathogenic Leptospira strains. Therefore, molecular fingerprint may play an important role in the clinical diagnosis, treatment, surveillance, and tracking of epidemic outbreaks of leptospirosis.
Leptospirosis is a worldwide zoonosis that is caused by spirochetes of the genus Leptospira. Leptospirosis is a serious zoonotic disease that has become an important public health problem. Traditional serological methods are the gold standard for the detection of pathogenic strains of Leptospira. However, serological procedures are cumbersome, require more complex experimental techniques, and are based on a large number of international and domestic reference strains. Additionally, these experiments involve the immunization of animals with antigens from different serotypes to produce immune serum, and improper techniques may result in a rapid decrease in antibody titer, which would affect the final results. It is difficult to perform cumbersome detection procedures in a basic laboratory. Therefore, the use of conventional serological methods is limited, which significantly impacts daily leptospirosis epidemic surveillance, prevention, and control. Molecular biology methods, such as 16S rRNA and PCR-based methods, can be used to identify the pathogenic Leptospira. However, DNA extraction and gene sequencing methods are laborious and time consuming. Therefore, more rapid and reliable high-throughput identification methods are urgently needed for the clinical diagnosis of leptospirosis to improve epidemic control. Here, molecular fingerprinting technique was use to identify the pathogenicity. We constructed the reference spectra database and the super reference spectra of pathogenic and non-pathogenic Leptospira, which can rapidly identified Leptospira at the species level and the pathogenicity of these isolates can be simultaneously confirmed. Furthermore, the protein components of Leptospira pathogenicity were revealed. These findings thus provide a new way for Leptospira pathogenicity identification.
Research article
Outcomes Following Single-Stage Posterior Vertebral Column Resection for Severe Thoracic Kyphosis
World Neurosurgery, Volume 119, 2018, pp. e551-e559
Thoracic kyphosis can result in neurologic deficits, pain, and cardiopulmonary dysfunction. Vertebral column resection (VCR) is a powerful technique that can be employed for large curves and fixed deformities. This study reports the outcomes of posterior VCR for adult spinal deformity with severe thoracic kyphosis.
A retrospective review of all patients with adult spinal deformity who underwent posterior VCR for severe thoracic kyphosis (defined as segmental kyphosis greater than 80°) was performed. Patients with kyphosis secondary to trauma, tumor, or infection were excluded. Perioperative, radiographic, and minimum 2-year outcomes were assessed.
Nineteen patients were included. Mean age was 57.1 years and 31.6% were male. Mean preoperative sagittal vertical axis was 57.7 mm and thoracic kyphosis was 92.2°. Among 19 patients, 24 VCR were performed. Mean blood loss was 2188 mL. Perioperative complication rate was 36.8% and mortality rate was 5.3%. Mean postoperative sagittal vertical axis was 42.3 mm and thoracic kyphosis was 58.1°. Incidence of junctional failure at 2-year follow-up was 14.8%: 1 proximal and 2 distal. All patients with junctional disease required reoperation. At mean 35.7-month follow-up, 61.1% of patients reported a significant reduction of back pain and 50.0% were able to reduce their dose of opioid medications.
Single-stage posterior VCR is a powerful technique for the correction of severe thoracic kyphosis. Perioperative morbidity can be high, but a majority of patients fare well at follow-up. Junctional disease occurs both proximal and distal; surgeons should continue to implement strategies to minimize distal junctional disease.
Research article
Adalimumab versus infliximab for the treatment of moderate to severe ulcerative colitis in adult patients naïve to anti-TNF therapy: An indirect treatment comparison meta-analysis
Journal of Crohn's and Colitis, Volume 8, Issue 7, 2014, pp. 571-581
To compare the efficacy of adalimumab and infliximab for the treatment of moderate to severe ulcerative colitis using indirect treatment comparison meta-analysis.
A systematic review and Bayesian indirect treatment comparison meta-analyses were performed for seven patient-important clinical outcomes at 8weeks and 52weeks. Odds ratio (OR) estimates and associated 95% credible intervals (CrIs) were produced.
Five eligible RCTs informed clinical remission, response, mucosal healing, quality of life, colectomy, serious adverse events, and discontinuation due to adverse events at 8weeks and 52weeks. At 8weeks of induction therapy, clinical remission (OR=0.42, 95% CrI 0.17–0.97), clinical response (OR=0.45, 95% CrI 0.23–0.89) and mucosal healing (OR=0.46, 95% CrI 0.25–0.86) statistically favored infliximab. However, after 52weeks of maintenance therapy OR estimates showed no significant difference between infliximab and adalimumab. For serious adverse events and discontinuations due to adverse events, adalimumab and infliximab were similar to placebo. Further, the indirect treatment comparison of adalimumab and infliximab yielded odds ratios close to 1.00 with wide credible intervals.
The findings of this indirect treatment comparison meta-analysis suggest that both infliximab and adalimumab are superior to placebo in the treatment of moderate to moderately severe ulcerative colitis. While infliximab is statistically more effective than adalimumab in the induction of remission, response and mucosal healing at 8weeks, infliximab and adalimumab are comparable in efficacy at 52weeks of maintenance treatment.
Research article
The role of Notch signaling in diabetic endothelial progenitor cells dysfunction
Journal of Diabetes and its Complications, Volume 30, Issue 1, 2016, pp. 12-20
To investigate the role of Notch signaling pathway in vasculogenic dysfunction of diabetic EPCs (DM-EPCs).
The study was performed in mice and diabetes was induced with Streptozotocin. The functional consequences of Notch pathway modulation were studied by assessment of colony forming capacity (EPC colony forming assay), EPC differentiation capacity (% of definitive EPC-CFU (dEPC-CFU)), circulating EPCs (EPC culture assay) and migrated cells (migration assay); in the presence of Notch inhibitor (γ-secretase inhibitors (GSI)) compared to control. Notch pathway and VEGF involvement in DM- EPCs were assessed by gene expression (RT-qPCR).
DM demonstrated to increase Notch pathway expression in bone marrow (BM) EPCs followed by lower EPC-CFU number, EPCs differentiation capacity, number of circulating EPCs, migrated cells and VEGF expression compared to control (p<0.05). Inhibition of Notch pathway by GSI rescued vasculogenic dysfunction in DM-EPCs as represented by increase in EPC-CFU number, differentiation capacity and number of circulating EPCs (p<0.05).
Our findings indicate the involvement of Notch pathway in mediating DM-EPCs dysfunction including less number of EPC-CFU, circulating EPCs and migrated cell number compared to control. Further in vitro inhibition of Notch pathway by GSI rescued DM-EPC dysfunction. Therefore targeting Notch pathway in DM may provide a target to restore DM-EPC dysfunction.
Research article
Leptospira Species (Leptospirosis)
Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Volume 2, 2015, pp. 2714-2720.e2
Research article
Amelioration of cisplatin-induced mouse renal lesions by a cyclooxygenase (COX)-2 selective inhibitor
European Journal of Pharmacology, Volume 715, Issues 1–3, 2013, pp. 181-188
In this study, we investigated the effects of the cyclooxygenase (COX)-2 selective inhibitor, meloxicam, on cisplatin-induced inflammation, oxidative stress and renal lesions in BALB/c mice. A single cisplatin injection (13mg/kg, i.p.) significantly increased plasma creatinine, blood urea nitrogen and urinary glucose accompanied by a concomitant increase in COX-2 mRNA and COX-2 protein levels. These changes in renal lesion parameters were diminished by simultaneous treatment of meloxicam (0.7mg/kg/day in drinking water). The expression of oxidative stress markers, p47phox, p67phox, hemoxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and 4-hydroxy-2-nonenal (4-HNE)-modified protein were increased with cisplatin injection. Simultaneous treatment of meloxicam with cisplatin significantly inhibited the increase in p47phox, HO-1 and 4-HNE-modified protein. The phosphorylation of extracellular regulated kinase (ERK) and c-jun-N-terminal kinase (JNK) were increased with cisplatin injection, but these changes were inhibited by meloxicam. Moreover, concomitant meloxicam treatment also prevented the cisplatin-induced infiltration of macrophages to the tubulointerstitial area. These results suggest that meloxicam can ameliorate cisplatin-induced mouse renal lesions, potentially through the inhibition of inflammatory and oxidative stress responses.
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