Real-time PCR optimization to identify Mycobacterium tuberculosis complex strains in clinical samples.

Resumen del artículo publicado en FEBS Journal,http://dx.doi.org/10.1111/febs.12919During recent years several molecular techniques have become available for Mycobacterium tuberculosis complex (MTC) detection,both for clinical samples and for isolates. One of the techniques more widely used is real time PCR in combination with nucleic acid amplification protocols. There are numerous studies based on PCR for the diagnosis of tuberculosis although the different protocols and primers used in the laboratory, together with the variability in the diagnostic performance of the methods tested, require that a comparative study be performed. Furthermore,the fact that the detection from clinical samples requires using highly sensitive targets suggests that this type of study should include multicopy targets to compare their efficiency with respect to the single copy. Our aim was to identify the members of the MTC using real-time PCR assays based on SYBR Green,among a large panel of isolated bacterial strains and clinical samples.We chose three targets (IS6110, senx3-regx3 and cfp32) and the optimal values for each PCR assay were empirically defined by testing in triplicate different concentrations of MgCl2 and primer sets and different annealing temperatures. These conditions were determined based on the specific amplification reactions that showed a lower Ct value, higher fluorescence and absence of non-specific PCR products. The analytical sensitivity was evaluated by ten-fold serial dilutions of DNA from MTC and the specificity was tested by 62 different microorganisms, including bacteria related with the MTC. The diagnostic yield was evaluated in 66 specimens from patients with suspected tuberculosis;30 had tuberculosis and 36 (control group) had different diseases.Under the conditions that resulted in optimization, standard curves showed that senx3-regx3 assay was the most efficient, followed by IS6110 and cfp32. However, the detection of bacterial DNA was faster with the repetitive element IS6110, with Ct values of up to 3 and 9 cycles of difference with respect to senx3-regx3 and cfp32. The analytical specificity, done only with the senx3-regx3 and IS6110 targets, was in the order of 100 and 93.5%, since IS6110 amplified various non-tuberculous micobacteria.For all the clinical samples studied, the sensitivity of both assays was identical (93.3%) but the specificity of senx3-regx3(100%) was higher than that of IS6110 (94.7%). In conclusion,real time PCR assay-SYBR Green based on the targets senx3-regx3 is highly reproducible and more sensitive and specific than the assays based on IS6110 or cfp32. The protocol developed in this study provides an appropriate and rapid tool to identify the strains of MTC in different clinical isolates and specimens.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis

Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis.

Journal Article; Research Support, Non-U.S. Gov't;BACKGROUND Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods. METHODS This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene. RESULTS The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively. CONCLUSION Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.This work received financial support from the Consejería de Innovación Ciencia y Empresa (grant CTS-276 and P-08-CTS-3969) both of theJunta de Andalucía (Spain).Ye

Comparative Clinical Study of Different Multiplex Real Time PCR Strategies for the Simultaneous Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis

<div><p>Background</p><p>Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis.</p><p>Methodology/Principal Findings</p><p>We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting <i>bcsp31</i> and the <i>IS711</i> sequence detecting all pathogenic species and biovars of <i>Brucella</i> genus, the <i>IS6110</i> sequence detecting <i>Mycobacterium</i> genus, and the intergenic region senX3-regX3 specifically detecting <i>Mycobacterium tuberculosis complex</i>. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the <i>Brucella</i> genus and <i>M. tuberculosis complex</i>. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%).</p><p>Conclusions/Significance</p><p>In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of <i>M. tuberculosis</i> and <i>Brucella</i> spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.</p></div

Standard monoplex assay curves.

<p>Panel A. Comparison between standard curves of the three real-time PCR for <i>Brucella</i> spp. Panel B. Comparison between standard curves of the three real-time PCR for <i>M. tuberculosis</i> complex.</p

Values of reproducibility and repeatability for the combinations senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711.

*<p>Only amplification of <i>M. bovis</i>.</p

Evaluation of the M RT-PCR assay and analysis of amplicons by agarose gel electrophoresis.

<p>A total of 9 M RT-PCR reactions based on SG were evaluated using the different primer combinations. The omp2a, bcsp31 and IS711 genes were used for the identification of <i>Brucella</i> spp and the IS6110, cfp32 and senX3-regX3 genes were targeted for the detection of members of the MTC. The orange lines represent the positive controls of <i>M. bovis</i>, blue lines indicate the positive control of <i>Brucella</i> spp, a yellow line represents the negative control and green lines a mixture of both pathogens at different concentrations. Below each image of melting peaks is shown the corresponding electrophoresis. Lanes: Mw, molecular size DNA ladder XIII; 1, negative controls; 2 to 3, positive controls for <i>M</i>. <i>bovis</i>; 4 to 5, positive controls for <i>B. abortus</i> B-19; 6 to 9, multiplex DNA of MTC and <i>Brucella</i>.</p

Primer sequences used for amplification by real-time PCR.

<p>Primer sequences used for amplification by real-time PCR.</p

Results of M RT-PCR assays with clinical samples for gene combinations senX3-regX3+bcsp31 (A), senX3-regX3+IS711 (B) and IS6110+IS711 (C).

<p>Blue lines, positive control of <i>B. abortus</i>; orange lines, positive controls of <i>M. tuberculosis</i>; yellow lines, negative controls. Panel A. Red lines, synovial fluid from a patient with brucellosis arthritis; green lines, vertebral tissue from a patient with tuberculous vertebral osteomyelitis; and grey lines, CFS from a patient with neurosyphilis. Panel B. Purple lines, samples from a patient with Brucellar hepatosplenic abscesses; and grey and green lines, pericardial tissue and CFS from a patient with tuberculosis meningitis. Panel C. Pink and green lines, synovial fluid and CFS from two patients with brucellosis; turquoise lines, vertebral tissue from a patient with vertebral tuberculosis; and grey lines, vertebral tissue from a patient with vertebral osteomyelitis caused by <i>Mycobacterium xenopi</i>.</p