Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease (original) (raw)
Related papers
2018
Currently, the direct detection of Leptospira infection can be done in clinical laboratories by a conventional nested polymerase chain reaction method (nested PCR), which is labourious and time-consuming. To overcome these drawbacks, we tested a set of paired samples of serum and urine from 202 patients presenting at a hospital located in an area endemic for leptospirosis using high resolution melting (HRM). The results were compared with those obtained by nested PCR for direct detection of the pathogen in both specimens and with the gold standard test used for indirect detection of anti-Leptospira antibodies in serum (the microscopic agglutination test, MAT). The HRM assay results were positive for 46/202 (22.7%) samples, whereas 47/202 (23.3%) samples were positive by nested PCR. As expected in recently infected febrile patients, MAT results were positive in only 3/46 (6.5%) HRM-positive samples. We did a unique comparative analysis using a robust biobank of paired samples of seru...
A rapid and quantitative method for the detection of Leptospira species in human leptospirosis
FEMS Microbiology Letters, 2005
Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0 · 10 1 -3.9 · 10 4 leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.
Optimizing real-time PCR method to detect Leptospira spp. in human blood and urine specimens
Medical Journal of Indonesia, 2012
Latar belakang: Leptospirosis adalah penyakit infeksi akut yang dapat menyerang manusia yang disebabkan bakteri Leptospira spp. dan digolongkan sebagai zoonosis. Gejala klinis leptospirosis yang tidak spesifik dan sulitnya uji laboratorium untuk konfirmasi diagnosis mengakibatkan penyakit ini seringkali tidak terdiagnosis. Oleh karenanya perlu dilakukan optimasi uji diagnostik molekuler menggunakan real-time PCR sebagai deteksi cepat Leptospira patogen yang sensitif dan spesifik. Penelitian ini di disain untuk mengoptimatisasi uji diagnostik molekuler menggunakan real time-PCR sebagai metode yang sensitif dan spesifik untuk mendeteksi Leptospira. Metode: DNA bakteri di dalam spesimen diektraksi menggunakan DNA extraction kit dengan prosedur sesuai dengan petunjuk manualnya. Primer dan probe yang digunakan berdasarkan penelitian terpublikasi terdahulu. Penelitian ini menggunakan mesin PCR-IQTM5, iCycler Multicolor real-time PCR detection system. Spesifisitas primer diuji menggunakan DNA bakteri patogen lain yang mungkin dapat ditemukan pada kedua spesimen dan dapat memberikan gejala klinis yang mirip. Hasil: Hasil uji real-time PCR menunjukkan bahwa kadar DNA Leptospira spp. terendah yang dapat terdeteksi adalah 0,375 fg/μl. Hasil uji juga menunjukkan bahwa primer yang digunakan untuk deteksi bakteri Leptospira spp. tidak beraksi silang dengan genom bakteri uji lainnya. Konsentrasi minimal DNA bakteri standar yang masih dapat dideteksi dengan pemeriksaan ini adalah 150 fg/μl, sedangkan dalam urin adalah 1470 fg/μl. Kesimpulan: Uji real-time PCR adalah metode yang cepat dan akurat untuk deteksi Leptospira spp. patogen pada spesimen manusia. Penelitian lebih lanjut diperlukan untuk mengetahui sensitivitas dan spesifisitas dari uji real-time PCR dibandingkan dengan metode diagnostik lain.
Rapid diagnosis of leptospirosis by multiplex PCR
The Malaysian journal of medical sciences : MJMS, 2012
Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance. In this study, we developed a multiplex PCR (mPCR) assay for detecting Leptospira's DNA. The mPCR assay detects both the 16S rRNA gene and the major outer membrane lipoprotein gene, which is known as LipL32. Representative serovars were tested from 10 species of Leptospira and 23 other species of bacteria. A positive result was obtained from all leptospiral serovars. The amplification sensitivity for the multiplex assay was 21.8 pg and 1 ...
A Multiplex PCR for the Rapid Diagnosis of Leptospirosis
2012
Background: Diagnosis of leptospirosis has traditionally relied on the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular methods including conventional and real-time Polymerase Chain Reaction (PCR) have been developed for the specific detection of pathogenic Leptospira. PCR is a sensitive, specific and rapid technique that has been successfully used to detect several microorganisms, including those of clinical significance. Objective: In this research, we developed of a multiplex PCR (mPCR) for detecting leptospira DNA. The mPCR detects both the 16S rRNA gene as well as major outer membrane lipoprotein LipL32 gene. Representative serovars from 10 species of Leptospira and 23 species of other bacteria species were tested. Results: Positive results were obtained from all leptospiral serovars. The amplification sensitivity for multiplex assay was 21.8 pg and 1 x 103 leptospires/ml. This mPCR has a potential to facil...
PLoS ONE, 2009
Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first -for treatment important -4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
Journal of Microbiological Methods, 2012
In the acute phase of leptospirosis, the diagnosis can be established with high sensitivity by testing blood and urine samples with polymerase chain reaction (PCR). However, only few real-time PCR assays have been validated for diagnostic use. The diagnostic accuracy of a novel TaqMan® PCR (LipL32 real-time PCR) targeting the lipl32 gene (or hap-1) and a previously described TaqMan® PCR (16S real-time PCR) targeting the rrs gene coding for 16S rRNA was evaluated when applied to both urine and blood specimens from humans suspected of leptospirosis. Applied to at least two blood cultures LipL32 real-time PCR had a sensitivity of 86%, and a specificity of 100%; and 16S real-time PCR had a sensitivity of 100%, and a specificity of 97%. Applied to urine samples, patients that were positive by the reference methods were also positive by both real-time PCR assays (n=4). For LipL32 real-time PCR the specificity was 100%, while for 16S real-time PCR it was only 91.5% due to unexpected cross-reactions with other bacteria. The analytical sensitivity was close to the theoretical limit-of-detection for both assays detecting all described human pathogenic species. We report a specific real-time PCR assay for detection of Leptospira, i.e., LipL32 real-time PCR that has been validated for diagnostic application in both urine and blood specimens from humans. We further show that a previously described 16S real-time PCR no longer can be recommended for diagnostic use due to a low specificity.
The American journal of tropical medicine and hygiene, 2016
Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N = 109) and negative (N = 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specifi...
Transactions of the Royal Society of Tropical Medicine and Hygiene, 2011
Given the protean manifestations of leptospirosis, adequate laboratory support for diagnosis is necessary. Traditionally, the gold standard is the microscopic agglutination test (MAT) using a panel of Leptospira isolates representing a broad range of serogroups and serovars. It has been proposed that screening with serovars circulating in a region would enhance test performance. We assessed the diagnostic usefulness of MAT using both regionally obtained clinical Leptospira isolates and the specific isolates recovered from the tested patients. Serum obtained from 41 acute febrile patients (obtained on average 7.2 days [SD ± 5.2] after onset of fever) was tested using a standard panel of 24 serovars along with regional isolates recovered from human and animal blood cultures from different regions in Egypt and a patient's own isolate, if available, to establish additional MAT panels. Serum samples tested by a standard 24 panel with a cut-off of >1:800 revealed five patients with positive serology. Only one patient had a positive result using a regional panel or patient's own culture developed MAT. However, the serovar with the highest titers did not match the cultured serovar. Region-specific MATs did not appear to be reliable in detection of infection or in identifying the infecting serovar.