The Spanish Society of Epidemiology and Oral Public Health.

In 2019 the Sociedad Española de Epidemiología y Salud Pública Oral (SESPO, The Spanish Society of Epidemi- ology and Oral Public Health) approached BASCD and EADPH about using Community Dental Health as their offi cial journal. Very pleasant discussions between all parties mean that henceforth, SESPO will translate the abstracts of all papers published in Community Dental Health so that they may be placed on our website. The SESPO website will link to the translated abstracts.

Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line.

OBJECTIVE: Staphylococcus aureus is a particularly difficult pathogen to eradicate from the respiratory tract. Previous studies have highlighted the intracellular capacity of S.aureus in several phagocytic and non-phagocytic cells. The aim of this study was to define S.aureus interaction within a murine alveolar macrophage cell line. METHODS: Cell line MH-S was infected with Newman strain. Molecular mechanisms involved in phagocytosis were explored. To assess whether S.aureus survives intracellularly quantitative (gentamicin protection assays and bacterial plating) and qualitative analysis (immunofluorescence microscopy) were performed. Bacterial colocalization with different markers of the endocytic pathway was examined to characterize its intracellular trafficking. RESULTS: We found that S.aureus uptake requires host actin polymerization, microtubule assembly and activation of phosphatidylinositol 3-kinase signaling. Time course experiments showed that Newman strain was able to persist within macrophages at least until 28.5 h post infection. We observed that intracellular bacteria are located inside an acidic subcellular compartment, which co-localizes with the late endosome/lysosome markers Lamp-1, Rab7 and RILP. Colocalization counts with TMR-dextran might reflect a balance between bacterial killing and intracellular survival. CONCLUSIONS: This study indicates that S.aureus persists and replicates inside murine alveolar macrophages, representing a privileged niche that can potentially offer protection from antimicrobial activity and immunological host defense mechanisms.

Diagnostic accuracy study of multiplex PCR for detecting tuberculosis drug resistance.

OBJECTIVE: To study the diagnostic accuracy of a multiplex real-time PCR (Anyplex II MTB/MDR/XDR, Seegene, Corea) that detects Mycobacterium tuberculosis resistant to isoniazid (INH), rifampicin (RIF), fluoroquinolones (FLQ) and injectable drugs (kanamycin [KAN], amikacin [AMK] and capreomycin [CAP]) in isolates and specimens. METHODS: One hundred fourteen cultured isolates and 73 sputum specimens were retrospectively selected. Results obtained with multiplex PCR were compared with those obtained with BACTEC. Discordant results between multiplex PCR and BACTEC were tested by alternative molecular methods. RESULTS: Sensitivity and specificity of multiplex PCR for detecting drug resistance in isolates were 76.5% and 100%, respectively, for INH; 97.2% and 96.0%, respectively, for RIF; 70.4% and 87.9%, respectively, for FLQ; 81.5% and 84.8%, respectively, for KAN; 100% and 60%, respectively, for AMK, and 100% and 72.3%, respectively, for CAP. Sensitivity and specificity of Anyplex for detecting drug resistance in specimens were 93.3% and 100%, respectively, for INH; 100% and 100%, respectively, for RIF; 50.0% and 100%, respectively, for FLQ; and 100% and 94.4%, respectively, for both KAN and CAP. Among the discordant results, 87.7% (71/81) of results obtained with the multiplex PCR were concordant with at least one of the alternative molecular methods. CONCLUSIONS: This multiplex PCR may be a useful tool for the rapid identification of drug resistant tuberculosis in isolates and specimens, thus allowing an initial therapeutic approach. Nevertheless, for a correct management of patients, results should be confirmed by a phenotypic method.

AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples.

OBJECTIVES: To determine the sensitivity and specificity of AID TB Resistance line probe assay (AID Diagnostika, Germany) to detect Mycobacterium tuberculosis and its resistance to first- and second-line drugs in clinical samples using BACTEC 460TB as the reference standard. METHODS: The test consists on three strips to detect resistance to isoniazid/rifampicin, fluoroquinolones/ethambutol, and kanamycin/amikacin/capreomycin/streptomycin, respectively. This test was performed on 65 retrospectively selected clinical samples corresponding to 32 patients. RESULTS: A valid result was obtained for 92.3% (60/65), 90.8% (59/65) and 78.5% (51/65) of the samples tested, considering the three strips, respectively. Global concordance rates between AID and BACTEC for detecting resistance to isoniazid, rifampicin, fluoroquinolones, ethambutol, kanamycin/capreomycin and streptomycin were 98.3% (59/60), 100% (60/60), 91.5% (54/59), 72.9% (43/59), 100% (51/51) and 98.0% (50/51), respectively. Regarding the discordant results obtained between AID and BACTEC, the alternative molecular methods performed (GenoType MTBDRplus, GenoType MTBDRsl [Hain Lifescience, Germany] and/or pyrosequencing) confirmed the genotypic result in 90.9% (20/22) of the cases. CONCLUSIONS: AID line probe assay is a useful tool for the rapid detection of drug resistance in clinical samples enabling an initial therapeutic approach. Nevertheless, for a correct management of drug resistant tuberculosis patients, molecular results should be confirmed by a phenotypic method.

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.

Usefulness of consecutive biomarkers measurement in the management of community-acquired pneumonia.

The aim of this study was to investigate whether procalcitonin (PCT), neopterin, C-reactive protein (CRP), and mid regional pro-atrial natriuretic peptide (MR-proANP) levels at admission and during the clinical course can be useful for the management of patients with pneumonia. The study population consisted of 75 patients with clinical and radiological diagnosis of pneumonia. Serum samples were collected at admission and during hospitalization. Complications were defined as intensive care unit (ICU) admission or death. The levels of PCT were significantly higher in pneumonia of definite bacterial origin in comparison to probable bacterial or unknown origin. The PCT levels were higher in pneumococcal pneumonia. The PCT and MR-proANP levels increased significantly according to the Pneumonia Severity Index (PSI). All biomarkers levels are higher in patients developing complications and who were dying. The serial levels of MR-proANP remain significantly elevated in patients developing complications and in patients classified in PSI and CURB-65 risk groups. In patients not developing complications, there is a significant decrease in the PCT levels. PCT can be useful for identifying pneumonia etiology. PCT and MR-proANP levels correlate with pneumonia severity rules. PCT and MR-proANP serial measurements can be useful for predicting short-term prognosis. Systemic biomarkers can provide additional information regarding clinical evolution, because these are dynamic and can be measured daily.

GenoType MTBDRsl for molecular detection of second-line-drug and ethambutol resistance in Mycobacterium tuberculosis strains and clinical samples.

The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

Pyrosequencing for rapid molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical specimens.

The aim of this study was to evaluate a pyrosequencing method for the detection of Mycobacterium tuberculosis isolates resistant to rifampin and isoniazid using both clinical strains and clinical samples, comparing the results with those of the Bactec 460TB and GenoType MTBDRplus assays. In comparison to Bactec 460TB as the gold standard, the sensitivity of pyrosequencing for detecting isoniazid and rifampin resistance was 76.9% and 97.2%, respectively, for clinical strains, and the specificity was 97.2 and 97.9%, respectively. For clinical specimens, the sensitivity and specificity for both drugs were 85.7% and 100%, respectively. The overall concordance between pyrosequencing and the GenoType MTBDRplus assay for clinical strains was 99.1%, and for clinical samples, it was 98.2%. Pyrosequencing is a valuable tool for rifampin and isoniazid resistance detection.

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.

Usefulness of two new methods for diagnosing metapneumovirus infections in children.

Human metapneumovirus (hMPV) is associated with acute respiratory tract infections, mainly in paediatric patients. The aim of this study was to evaluate the usefulness of two new commercial techniques available for the detection of hMPV in clinical samples from children: an enzyme immunoassay, hMPV EIA (Biotrin International Ltd), and a molecular assay, real-time RT-PCR (Pro hMPV Real Time Assay Kit; Prodesse). A total of 184 nasopharyngeal aspirate specimens from 173 children aged less than 5 years who were hospitalized with acute wheezing were analysed. Respiratory syncytial virus was detected in 27% of the samples, followed by influenza A virus (6%), parainfluenza virus (PIV)3 (2.2%), adenovirus (2%), PIV1 (1.1%), PIV2 (1.1%), and influenza B virus (0.5%). The presence of hMPV was tested in all samples, using the real-time RT-PCR and EIA. Real-time RT-PCR detected 13 hMPV-positive samples (8%), and EIA detected 17 (9.3%). When the EIA results were compared with those of real-time RT-PCR for the detection of hMPV, a good correlation was found (94%). A relatively low co-infection rate (15%) was observed in our patients. RT-PCR and EIA provide robust methods for the diagnosis of hMPV infection in children.

Utility of the rapid antigen detection BinaxNOW Influenza A&B test for detection of novel influenza A (H1N1) virus.

Nasopharyngeal aspirates, collected during outbreaks, of the novel influenza A (H1N1) virus in Barcelona, were used to compare the accuracy of a rapid antigen-based test (Binax) with the real-time RT-PCR assay developed by the CDC. The sensitivity, specificity and positive predictive value of the rapid test are higher in patients less than 18 years old and during the acute stage of the epidemic than in adult patients.

Performance of VITEK-2 Compact and overnight MicroScan panels for direct identification and susceptibility testing of Gram-negative bacilli from positive FAN BacT/ALERT blood culture bottles.

We describe the reliability of the VITEK-2 Compact and overnight MicroScan panels for direct identification and susceptibility testing from the BacT/ALERT blood culture system when using FAN (FA and FN) bottles. A simple procedure, in two centrifugation steps, was designed to remove the charcoal particles present in FA and FN bottles. A total of 113 positive blood cultures showing Gram-negative rods were investigated. Enterobacteriaceae were isolated in 104 cases, and Pseudomonas aeruginosa in nine. The MicroScan system correctly identified 106 (93.8%) of the 113 isolates. The seven identificaction errors included P. aeruginosa (three), Enterobacter cloacae (one), Escherichia coli (one), Klebsiella oxytoca (one), and Klebsiella pneumoniae (one). The VITEK-2 system correctly identified 109 (96.5%) of the 113 samples obtained directly from the blood culture bottles. The four unidentified isolates were Enterobacter cloacae (two), Escherichia coli (one), and P. aeruginosa (one). MicroScan yielded 4/779 (0.5%) very major errors and 28/2825 (0.9%) minor errors. VITEK-2 yielded 2/550 (0.36%) very major errors, 1/1718 (0.05%) major error, and 32/2373 (1.3%) minor errors. Both systems provided excellent identification (correlation of >90%) and susceptibility (correlation of >98%) results. The average times required to obtain identification and susceptibility results using the direct test applied to the VITEK-2 Compact system were 4.57 +/- 1.37 h and 6.52 +/- 1.64 h, respectively. The VITEK-2 compact system provided results on the same day that the blood culture was found to be positive.

Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant.

Safety is one of the main concerns for attenuated live vaccine candidates. Here we extend the stability and attenuation studies of the promising tuberculosis vaccine candidate based on Mycobacterium tuberculosis phoP mutant strain, SO2. Stability of the phoP mutation was tested after sub-culturing SO2 strain for 6 months in laboratory media and also after 3 months of infection in SCID mice. Results showed no reversion of the phoP mutation either in vitro or in vivo. In addition, SO2 was fully sensitive to four major first-line antituberculous drugs against tuberculosis. Safety and toxicity studies were performed in guinea pigs. Animals were infected with a quantity of SO2 equivalent to 50 vaccination doses (2.5x10(6) CFUs) and weight was monitored for 6 months. All animals survived and no histological lesions were found, showing full attenuation of SO2. Studies in a post-exposure model of guinea pigs and mice, previously infected with M. tuberculosis, were performed and no toxicity effects were found after inoculation of SO2. All these results together confirm that SO2 has a secure safety profile that encourages its use in clinical trials.

Mycobactericidal activity of chlorine dioxide wipes in a modified prEN 14563 test.

Tristel Sporicidal Wipes are chlorine dioxide-based disinfectant wipes for disinfecting non-lumened semi-critical medical devices. In this study, the mycobactericidal activity of this product was assessed by a modified version of the European Standard prEN 14563 carrier test under clean conditions against Mycobacterium avium. The chlorine dioxide concentration in the activated wipe was 200ppm. The results showed that the chlorine dioxide wipes were mycobactericidal in 30s contact time with mechanical action and in 60s without mechanical action, both under clean conditions.

The tuberculin skin test increases the responses measured by T cell interferon-gamma release assays.

RUTI is a vaccine consisting of Mycobacterium tuberculosis bacilli grown in stress conditions that is fragmented, detoxified and liposomed. RUTI was designed to shorten the treatment of latent tuberculosis infection (LTBI) with isoniazid from 9 months to just 1 month, by additional treatment with two inoculations of RUTI 4 weeks apart. During the validation process for monitoring the immunogenicity of administration of RUTI in a Phase I clinical trial, the question arose whether to introduce the tuberculin skin test (TST) in the screening of non-LTBI volunteers. This study was designed to evaluate the effect of TST on subsequent different T-cell interferon-gamma release assay (TIGRA) responses, using a spectrum of M. tuberculosis-related antigens (ESAT-6, CFP-10, 16 kDa, 19 kDa, MPT64, Ag 85B, 38 kDa, hsp65, PPD and BCG). The results showed an increase in post-TST response even in non-LTBI subjects for most antigens tested, as measured both by whole blood assay (WBA) and ELISPOT. Increased ELISPOT response decreased toward pre-TST levels within 1 month whereas the WBA response did not. Taking into account that there is no definitive correlation between TST and TIGRA tests to diagnose LTBI and the feasibility that TST might alter the immune monitoring included in clinical trials, these data suggest that TST determination should be carefully planned to avoid any interference with TIGRA.

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.

Detection of hepatitis C virus antibodies in oral fluid specimens for prevalence studies.

Within the framework of hepatitis C virus (HCV) prevalence monitoring, we evaluated oral fluid (OF), which is richer in IgG than whole saliva, as a possible alternative to serum for the detection of HCV antibodies. Paired OF and serum samples were collected from 90 individuals, including 45 HCV-positives and 45 HCV-negatives. The detection of HCV antibodies in both serum and OF was performed using the Ortho HCV 3.0 SAVe enzyme-linked immunosorbent assay (ELISA) (Ortho-Clinical Diagnostics, Inc., Raritan, NJ), but a modified, more sensitive protocol was used to process OF. The sensitivity and specificity of this assay were 86.67% (95% confidence interval (CI): 72.51-94.46%) and 100% (95% CI: 90.20-99.80%) in OF and 100% in serum. The correlation obtained between both types of clinical specimens was excellent (k: 0.87, 95% CI: 0.66-1.07). However, the negative predictive value (NPV) of the assay in OF decreased with the prevalence of HCV infection in the population studied. Our results suggest that the modified Ortho HCV 3.0 SAVe ELISA is suitable for the detection of HCV antibodies in OF for epidemiological studies. Using this assay, we observed an unadjusted anti-HCV prevalence of 78.6% among a population of intravenous drug users; when adjusted to account for assay sensitivity, this prevalence may be closer to 90%.

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 acr expression for monitoring latent Mycobacterium tuberculosis bacilli in 'in vitro' and 'in vivo' experimental models.

Real-time RT-PCR was used to quantify the expression of genes possibly involved in Mycobacterium tuberculosis latency in in vitro and murine models. Exponential and stationary phase (EP and SP) bacilli were exposed to decreasing pH levels (from 6.5 to 4.5) in an unstirred culture, and mRNA levels for 16S rRNA, sigma factors sigA,B,E,F,G,H and M, Rv0834c, icl, nirA, narG, fpbB, acr, rpoA, recA and cysH were quantified. The expression of acr was the one that best correlated with the CFU decrease observed in SP bacilli. In the murine model, the expressions of icl, acr and sigF tended to decrease when bacillary counts increased and vice versa. Values from immunodepressed mice (e.g. alpha/beta T cells, TNF, IFN-gamma and iNOs knock out strains), with accelerated bacillary growth rate, confirmed this fact. Finally, the expression of acr was maintained in mice following long-term treatment with antibiotics. The quantification of acr expression could be useful for monitoring the presence of latent bacilli in some murine models of tuberculosis.