Diagnostic benefits of adding EspC, EspF and Rv2348-B to the QuantiFERON Gold In-tube antigen combination.

Interferon (IFN)-γ release assays (IGRAs) are used to diagnose latent tuberculosis (TB) infection (LTBI). To improve the accuracy of these tests, different approaches, such as alternative cytokine detection and using different antigens, are considered. Following this purpose, this study aims to evaluate the addition of EspC, EspF and Rv2348-B to those present in the QuantiFERON-TB Gold In-Tube (QFN-G-IT). We included 115 subjects: 74 active TB patients, 17 LTBI individuals and 24 healthy controls. Whole blood samples were collected in QFN-G-IT and in-house tubes containing different combinations of EspC, EspF and Rv2348-B, together with ESAT-6, CFP-10, and TB7.7. After overnight incubation at 37 ºC, plasma was harvested and IFN-γ quantified. IFN-γ levels in the QFN-G-IT and in-house tubes correlated very good (Spearman Rho(r) > 0.86). In-house antigen combinations distinguished healthy individuals from those with active TB and LTBI (specificities and sensitivities higher than 87.5% and 96.3%, respectively [AUC > 0.938]). Adding EspC, EspF and Rv2348-B, increased the sensitivity of the test, being the addition of EspC and Rv2348-B the combination that yielded a higher sensitivity with no specificity loss. Addition of these antigens could improve diagnosis in patients with impaired or immature immune response who are at high risk of developing TB.

Use of IP-10 detection in dried plasma spots for latent tuberculosis infection diagnosis in contacts via mail.

The aim of this study was to test the use of IP-10 detection in dried plasma from contact studies individuals (contacts of smear positive patients), by comparing it with IP-10 and IFN-γ detection in direct plasma, to establish IP-10 detection in DPS as a useful assay for LTBI diagnosis. Whole blood samples were collected from 80 subjects: 12 with active tuberculosis (TB), and 68 from contact studies. The amount of IFN-γ produced by sensitized T cells was determined in direct plasma by QuantiFERON Gold In-Tube test. IP-10 levels were determined in direct and dried plasma by an in-house ELISA. For dried plasma IP-10 determination, two 25 µl plasma drops were dried in Whatman903 filter paper and sent by mail to the laboratory. Regarding TB patients, 100.0%, 91.7% and 75.0% were positive for IFN-γ detection and IP-10 detection in direct and dried plasma, respectively. In contacts, 69.1%, 60.3% and 48.5% had positive results after IFN-γ and IP-10 in direct and dried plasma, respectively. The agreement among in vitro tests was substantial and IP-10 levels in direct and dried plasma were strongly correlated (r = 0.897). In conclusion, IP-10 detection in dried plasma is a simple and safe method that would help improve LTBI management.

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid.

OBJECTIVE: Loop-mediated isothermal amplification (LAMP) is a simple and sensitive technique for rapid microbiological diagnosis. The aim of this study was to evaluate the analytical and diagnostic performance of the LAMP eazyplex MRSA test for the direct detection and differentiation of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) in synovial/pleural fluid. METHODS: Analytical validation included the determination of the limit of detection (LoD) and analytical specificity of the eazyplex MRSA test. A diagnostic evaluation of the eazyplex test against bacterial culture was performed on routine pleural/synovial samples collected prospectively from patients aged less than 18 years with complicated pneumonia with empyema or arthritis admitted to the Children's Hospital Sant Joan de Déu in Barcelona, Spain, between April 2015 and May 2016. RESULTS: The new system appropriately detected a quality control panel of clinical samples with DNA of MSSA, MRSA, and other pathogens. The LoD was established at 6.4×103 CFU/ml for S. aureus and 1.0×104 CFU/ml for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70min. CONCLUSIONS: The eazyplex MRSA assay was found to be a useful test for the rapid detection of S. aureus in invasive samples such as pleural/synovial fluid.

Homeopathic mistletoe adverse reaction mimics nodal involvement in 18F-FDG PET/CT performed for evaluation of response to chemotherapy in lymphoma.

Some patients use complementary medicine. We present a patient with Hodgkin's lymphoma, scanned with 18F-FDG PET/CT for evaluation of response after chemotherapy, who was self-administering mistletoe as a homeopathic medicine product. The careful review of the images of the entire scan and patient collaboration in anamnesis were crucial to avoid a false positive result. A review of the published scientific data on the effects of mistletoe is also presented.

Respiratory monitoring with electrical impedance tomography for lung protective ventilation and alveolar recruitment maneuver in a patient with a single lung transplant and early graft dysfunction. / Monitorización respiratoria con tomografía de impedancia eléctrica durante la ventilación de protección pulmonar y maniobra de reclutamiento alveolar en un paciente con trasplante unipulmonar y disfunción precoz del injerto.

A case is presented on a patient who underwent left single lung transplantation for emphysema type COPD. There was early graft dysfunction gradeiii during the immediate postoperative period, which required the implantation of an extracorporeal membrane oxygenator (ECMO). Respirator ventilatory parameters were adjusted to avoid lung distension, low tidal volume (Vc) (280ml), high respiratory rates (20rpm), and a positive pressure at end expiration (PEEP) level of 8cmH2O. On monitoring the pulmonary tidal volume distribution by bedside electrical impedance tomography (EIT), it was noted that most of the tidal volume was distributed in the native lung emphysema. An alveolar recruitment manoeuvre was performed, under control of the EIT, that enabled the current volume and distribution and the pressures required to ventilate the transplanted lung to be observed.

Diagnosis of tuberculosis infection by interferon-gamma release assays in patients with psoriasis.

OBJECTIVE: In this study, we have performed a direct comparison between both T-cell based assays (QFN-G-IT and T-SPOT.TB) and TST in patients with psoriasis taking different immunosuppressant drug-regimens. METHODS: We have prospectively studied 103 patients with moderate-to-severe psoriasis who required latent tuberculosis infection (LTBI) screening before starting systemic immunosuppressive treatment or during its sustained use. RESULTS: Overall number of positive results was 16.5%, 17.5% and 8.7% using T-SPOT.TB, QFN-G-IT and TST, respectively. Differences in the percentage of positive results between TST with T-SPOT.TB and QFN-G-IT were significant (p = 0.005 and p = 0.008, respectively). A total of 24.3% of the subjects enrolled were positive for at least one of the three tests performed. Sixteen patients with negative TST (17%) were positive for one of the two IGRAs. We obtained seven indeterminate results by T-SPOT.TB and two by QFN-G-IT. Seven patients with negative TST presented indeterminate results by either of two IFN-γ assays. Positive TST, T-SPOT.TB and QFN-G-IT results were not affected by clinical therapeutic profile. CONCLUSIONS: Our results reveal that in vitro assays are useful methods for LTBI diagnosis in patients with psoriasis, suggesting that they might be less influenced by immunosuppression than TST.

IP-10 is an accurate biomarker for the diagnosis of tuberculosis in children.

OBJECTIVE: Performance of IFN-γ assays in children is compromised. Therefore, we investigated the utility of IP-10 for the detection of active tuberculosis (TB) and latent tuberculosis infection (LTBI) diagnosis in children; comparing its positivity with QuantiFERON-TB Gold In-Tube (QFN-G-IT) and T-SPOT.TB. METHODS: We studied 230 children from three groups: active TB, screening (healthy children without known exposure to active TB patient screened at school or by their paediatrician) and contact-tracing studies. IFN-γ release was determined by QFN-G-IT and T-SPOT.TB. IP-10 was detected in QFN-G-IT supernatants by ELISA. RESULTS: When combining QFN-G-IT and IP-10 assays, positive results improved significantly from 38.3% in QFN-G-IT and 33.9% in IP-10 to 41.3%. Age and type of contact were significant risk factors associated with positive QFN-G-IT and IP-10 results. IP-10 levels after antigen-specific stimulation were significantly higher in comparison to IFN-γ levels. Correlation between the three assays was good (κ = 0.717-0.783). CONCLUSIONS: IP-10 cytokine is expressed in response to TB specific-antigens used in QFN-G-IT. In conclusion, the use of IFN-γ T-cell based assays in combination with an additional IP-10 assay detection could be useful for diagnosing active TB and LTBI in children.

Interferon γ assays in the diagnosis of tuberculosis infection in psoriasis patients who are candidates for biologic therapies.

Although there is no doubt that biologic agents are an effective alternative for the treatment of moderate and severe psoriasis, anti-tumor necrosis factor α therapy has been associated with reactivation of latent tuberculosis infection. Tuberculin skin testing (TST) is used to diagnose tuberculosis infection but it has low specificity in patients who have received the Mycobacterium bovis BCG vaccine and low sensitivity in patients with altered cell-mediated immunity. In vitro assays based on the detection of interferon γ released by T cells stimulated by specific Mycobacterium tuberculosis antigens have emerged as an option for the diagnosis of tuberculosis infection. The results to date show that they are a viable alternative to TST thanks to their higher specificity and sensitivity. Furthermore, these assays are also proving to have high negative predictive value, meaning that we might be able to use them without TST in the short to medium term.

Interferon γ Assays in the Diagnosis of Tuberculosis Infection in Psoriasis Patients Who Are Candidates for Biologic Therapies. / Técnicas basadas en la detección de IFN-γ en el diagnóstico de la infección tuberculosa en pacientes con psoriasis candidatos a terapias biológicas.

Although there is no doubt that biologic agents are an effective alternative for the treatment of moderate and severe psoriasis, anti-tumor necrosis factor α therapy has been associated with reactivation of latent tuberculosis infection. Tuberculin skin testing (TST) is used to diagnose tuberculosis infection but it has low specificity in patients who have received the Mycobacterium bovis BCG vaccine and low sensitivity in patients with altered cell-mediated immunity. In vitro assays based on the detection of interferon γ released by T cells stimulated by specific Mycobacterium tuberculosis antigens have emerged as an option for the diagnosis of tuberculosis infection. The results to date show that they are a viable alternative to TST thanks to their higher specificity and sensitivity. Furthermore, these assays are also proving to have high negative predictive value, meaning that we might be able to use them without TST in the short to medium term.

Diagnosing TB infection in children: analysis of discordances using in vitro tests and the tuberculin skin test.

The aim of the present study was to compare the performance of the interferon (IFN)-γ tests (QuantiFERON®-TB Gold In-Tube (QFT-G-IT) and T-SPOT®.TB) with the tuberculin skin test (TST) in diagnosing tuberculosis (TB) infection in children, and to analyse discordant results. This was a prospective study including 98 children from contact-tracing studies and 68 children with TST indurations ≥ 5 mm recruited during public health screenings. Positive IFN-γ tests results were associated with risk of exposure (p<0.0001). T-SPOT.TB was positive in 11 (78.6%) out of 14 cases with active TB and QFT-G-IT in nine (64.3%) out of 14 cases. Sensitised T-cells against Mycobacterium avium were detected in six out of 12 children not vaccinated with bacille Calmette-Guérin (BCG), a TST induration 5-9 mm in diameter and both IFN-γ tests negative. In concordant IFN-γ tests results, a positive correlation was found (p = 0.0001) between the number of responding cells and the amount of IFN-γ released. However, in discordant IFN-γ tests results this correlation was negative (p = 0.371): an increase in the number of spot-forming cells correlated with a decrease in the amount of IFN-γ released. The use of IFN-γ tests is helpful for the diagnosis of TB infection, avoiding cross-reactions with BCG immunisation and nontuberculous mycobacterial infections. The analysis of highly discordant results requires further investigation to elucidate possible clinical implications.

Evaluating the non-tuberculous mycobacteria effect in the tuberculosis infection diagnosis.

The aim of the present study was to determine the role of previous non-tuberculous mycobacteria sensitisation in children as a factor of discordant results between tuberculin skin test (TST) and an in vitro T-cell based assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille Calmette-Guérin-vaccinated paediatric patients for suspicious of latent tuberculosis infection (LTBI). These patients yielded a positive TST and a negative T-SPOT.TB. Cells were stimulated with Mycobacterium avium sensitin (having cross-reaction with Mycobacterium intracellulare and Mycobacterium scrofulaceum) and the presence of reactive T-cells was determined by an ex vivo ELISPOT. From the 21 patients, in 10 cases (47.6%), we obtained a positive ELISPOT result after stimulation with M. avium sensitin, in six (28.6%) cases, the result was negative and in the remaining five (23.8%) cases, the result was indeterminate. In conclusion, previous non-tuberculous mycobacteria sensitisation induces false-positive results in the TST for diagnosing LTBI and the use of gamma-interferon tests could avoid unnecessary chemoprophylaxis treatment among a child population.

Prospective evaluation of latent tuberculosis with interferon-gamma release assays in drug and alcohol abusers.

In vitro tests have been developed for the diagnosis of tuberculosis (TB) infection. The objective was to analyse latent TB infection in drug and alcohol abusers through two interferon-gamma techniques. One hundred and thirty-nine patients were admitted between February 2006 and May 2007. Mean age was 39.8 years [31% HIV positive]. The enzyme immunoassay (EIA) and enzyme-linked immunospot (ELISPOT) interferon-gamma assays were positive in 34% of patients with an agreement of 83% (kappa=0.63). Tuberculin skin test (TST) was positive in 29% of patients and the agreement of TST with EIA and ELISPOT interferon-gamma assays was 85% (kappa=0.62) and 83% (kappa=0.57), respectively. Almost 50% of patients with history of TB had a positive in vitro test. In conclusion, we observed a high prevalence of latent TB and good agreement between the new in vitro tests that otherwise may continue to be positive long after developing TB disease.

Evaluation of a Legionella urinary antigen enzyme immunoassay for rapid detection of Legionella pneumophila in water samples.

Despite advances in medium formulations and pretreatment techniques, recovery of Legionella from water samples can still be quite low, difficult and time consuming. The aim of this study was to evaluate the utility of a Legionella urinary antigen enzyme immunoassay (Bartels ELISA, Trinity Biotech, Ireland) for the detection of Legionella in water samples. Reference ATCC Legionella strains were used to spike water samples to a final concentration of 10(4)-10(5)cfu/ml. The lower detection limit of the test for all Legionella pneumophila serogroups was assessed by serial dilutions of spiked water samples. Legionella antigen was detected in all filtered samples except for those spiked with L. bozemanii and L. longbeachae. The lower detection limit for soluble L. pneumophila serogroup 1 antigen was 780cfu/ml. Bartels ELISA could be a useful method for antigen detection in water samples when a high recovery of L. pneumophila is suspected. The test could be used as a rapid screening method for the detection of Legionella in a large number of samples. However, the low sensitivity of the test requires to keep on performing conventional culture for isolation and for further studies on isolated bacteria.

Comparison of a monoclonal with a polyclonal antibody-based enzyme immunoassay stool test in diagnosing Helicobacter pylori infection before and after eradication therapy.

BACKGROUND: Detection of Helicobacter pylori antigen in stool samples has been a subject of controversy. However, it has been included in several clinical guidelines as a recommended non-invasive testing procedure in dyspeptic patients. AIM: To compare a monoclonal enzyme immunoassay for detection of H. pylori stool antigen (Amplified IDEIA HpStAR, DakoCytomation) with a polyclonal enzyme immunoassay (HpSA test, Premier Platinum HpSA, Meridian Diagnostics) in diagnosing infection and in determining H. pylori status after eradication treatment. METHODS: We evaluated stool samples of 198 patients diagnosed with H. pylori infection and of 41 patients without infection. The results of the monoclonal enzyme immunoassay HpStAR were compared with those of the polyclonal enzyme immunoassay HpSA. RESULTS: The sensitivity and specificity of HpStAR were 91.9% and 70.7%, while those of HpSA were 89.4% and 80.5%, respectively. In the 126 patients evaluated 6 weeks after eradication therapy, the overall agreement between urea breath test and HpStAR was 90.5% (P = 0.710) and between urea breath test and HpSA was 76.9% (P = 0.410). CONCLUSIONS: HpStAR is a rapid and easy-to-perform test with similar sensitivity to HpSA in the diagnosis of H. pylori infection, although it had lower specificity. In contrast, HpStAR is more accurate after eradication therapy than HpSA.

Usefulness of pneumococcal antigen detection in pleural fluid samples by immunochromatographic assay for diagnosis of pneumococcal pneumonia.

This study investigated the utility of an immunochromatographic test (ICT) for the detection of Streptococcus pneumoniae antigens in pleural fluid. Antigen was detected in 15 of 19 (79%) patients with pneumococcal pneumonia. The ICT was always negative in patients with non-pneumococcal pneumonia, but was positive in three cases with a non-infectious aetiology. In patients with pneumonia for which no pathogen was identified, antigen was detected in one of 24 pleural fluids tested. The ICT can be a valuable tool for the management of pneumonia because it can detect pneumococcal antigen in pleural effusion samples.

Rapid detection of pneumococcal antigen in serum samples for diagnosing pneumococcal pneumonia.

OBJECTIVES: The aim of the study is to assess the usefulness of C polysaccharide and polysaccharide capsular antigen detection by immunochromatography (ICT) and enzyme immunoassay (EIA), respectively, in serum samples for diagnosing pneumococcal pneumonia. METHODS: Adult patients included in the study were classified in the following groups: In group 1 we studied 101 serum samples from patients with pneumonia due to Streptococcus pneumoniae. In 53 cases the pneumonia was bacteremic. The second group contained 113 serum samples from patients with no pneumococcal pneumonia. Group 3 was made up of 40 serum samples from healthy subjects with no clinical or radiological signs of pneumonia. RESULTS: Using ICT, antigen was detected in 50% of patients with pneumococcal pneumonia, in 64.3 and 40.9% of patients with bacteremic and non-bacteremic pneumococcal pneumonia, respectively. Using EIA, antigens were detected in 35.8% of patients with pneumococcal pneumonia, in 45 and 22.2% of patients with bacteremic and non-bacteremic pneumococcal pneumonia, respectively. CONCLUSIONS: In conclusion, the sensitivity of the tests is low. However, in special situations, where obtaining large volume of urine is difficult, they could be a complementary method in the rapid diagnosis of pneumococcal pneumonia.

Variability and transmission by Aphis glycines of North American and Asian Soybean mosaic virus isolates.

The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.