Pericarditis caused by Mycobacterium africanum: case report.

BACKGROUND: Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and is endemic in West Africa, where it causes up to half of all cases of pulmonary tuberculosis. Here, we report the first isolation of Mycobacterium africanum from the pericardial effusion culture of a patient with tuberculous pericarditis. CASE PRESENTATION: A 31-year-old man, native from Senegal, came to the emergency room with massive pericardial effusion and cardiac tamponade requiring pericardiocentesis. M. africanum subtype II was identified in the pericardial fluid. The patient completed 10 months of standard treatment, with a favorable outcome. CONCLUSIONS: We report the first case of tuberculous pericarditis caused by Mycobacterium africanum, which provide evidence that this microorganism can cause pericardial disease and must be considered in patients from endemic areas presenting with pericardial effusion.

Clinical evaluation of a new molecular method for the detection of multidrug-resistant microorganisms.

INTRODUCTION: The main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. METHODS: We developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. RESULTS: Both the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in nasal swabs and 97% in rectal swabs. CONCLUSIONS: The AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms (MDR) from nasal and rectal swab samples.

SARS-CoV-2 vaccine response and rate of breakthrough infection in patients with hematological disorders.

BACKGROUND: The clinical efficacy of SARS-CoV-2 vaccines according to antibody response in immunosuppressed patients such as hematological patients has not yet been established. PATIENTS AND METHODS: A prospective multicenter registry-based cohort study conducted from December 2020 to December 2021 by the Spanish transplant and cell therapy group was used to analyze the relationship of antibody response at 3-6 weeks after full vaccination (2 doses) with breakthrough SARS-CoV-2 infection in 1394 patients with hematological disorders. RESULTS: At a median follow-up of 165 days after complete immunization, 37 out of 1394 (2.6%) developed breakthrough SARS-CoV-2 infection at median of 77 days (range 7-195) after full vaccination. The incidence rate was 6.39 per 100 persons-year. Most patients were asymptomatic (19/37, 51.4%), whereas only 19% developed pneumonia. The mortality rate was 8%. Lack of detectable antibodies at 3-6 weeks after full vaccination was the only variable associated with breakthrough infection in multivariate logistic regression analysis (Odds Ratio 2.35, 95% confidence interval 1.2-4.6, p = 0.012). Median antibody titers were lower in cases than in non-cases [1.83 binding antibody units (BAU)/mL (range 0-4854.93) vs 730.81 BAU/mL (range 0-56,800), respectively (p = 0.007)]. We identified 250 BAU/mL as a cutoff above which incidence and severity of the infection were significantly lower. CONCLUSIONS: Our study highlights the benefit of developing an antibody response in these highly immunosuppressed patients. Level of antibody titers at 3 to 6 weeks after 2-dose vaccination links with protection against both breakthrough infection and severe disease for non-Omicron SARS-CoV-2 variants.

SARS-CoV-2-reactive antibody detection after SARS-CoV-2 vaccination in hematopoietic stem cell transplant recipients: Prospective survey from the Spanish Hematopoietic Stem Cell Transplantation and Cell Therapy Group.

This is a multicenter prospective observational study that included a large cohort (n = 397) of allogeneic (allo-HSCT; (n = 311) and autologous (ASCT) hematopoietic stem cell transplant (n = 86) recipients who were monitored for antibody detection within 3-6 weeks after complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination from February 1, 2021, to July 20, 2021. Most patients (n = 387, 97.4%) received mRNA-based vaccines. Most of the recipients (93%) were vaccinated more than 1 year after transplant. Detectable SARS-CoV-2-reactive antibodies were observed in 242 (78%) of allo-HSCT and in 73 (85%) of ASCT recipients. Multivariate analysis in allo-HSCT recipients identified lymphopenia < 1 × 109 /ml (odds ratio [OR] 0.33, 95% confidence interval [95% CI] 0.16-0.69, p = .003), active graft versus host disease (GvHD; OR 0.51, 95% CI 0.27-0.98, p = .04) and vaccination within the first year of transplant (OR 0.3, 95% CI 0.15-0.9, p = .04) associated with lower antibody detection whereas. In ASCT, non-Hodgkin's lymphoma (NHL; OR 0.09, 95% CI 0.02-0.44, p = .003) and active corticosteroid therapy (OR 0.2, 95% CI 0.02-0.87, p = .03) were associated with lower detection rate. We report an encouraging rate of SARS-CoV-2-reactive antibodies detection in these severe immunocompromised patients. Lymphopenia, GvHD, the timing of vaccine, and NHL and corticosteroids therapy should be considered in allo-HSCT and ASCT, respectively, to identify candidates for SARS-CoV-2 antibodies monitoring.

Clinical Performance of a Standardized Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Interferon-γ Release Assay for Simple Detection of T-Cell Responses After Infection or Vaccination.

BACKGROUND: We evaluated a standardized interferon-γ (IFN-γ) release assay (IGRA) for detection of T-cell immune response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. METHODS: This prospective study included patients with coronavirus disease 2019 (COVID-19) with different severity of illness and follow-up (FU), vaccinated subjects, and healthy unvaccinated persons. SARS-CoV-2 T-cell response was measured using a specific quantitative IGRA in whole blood (Euroimmun, Germany) and TrimericS-IgG and neutralizing antibodies with validated serological platforms. Positivity of reverse transcription-polymerase chain reaction or vaccination was considered as the reference standard. RESULTS: A total of 239 individuals were included (152 convalescent, 54 vaccinated, and 33 uninfected unvaccinated). Overall sensitivity, specificity, and positive- and negative-predictive values (95% confidence interval) of the IGRA were 81.1% (74.9-86%), 90.9% (74.5-97.6%), 98.2% (94.5-99.5%), and 43.5% (31.8-55.9%), respectively. All vaccinated SARS-CoV-2-naive subjects had positive IGRA at 3 months. In convalescent subjects the magnitude of IFN-γ responses and IGRA accuracy varied according to disease severity and duration of FU, with the best performance in patients with severe COVID-19 at 3 months and the worst in those with mild disease at 12 months. The greatest contribution of IGRA to serological tests was observed in patients with mild disease and long-term FU (incremental difference, 30.4%). CONCLUSIONS: The IGRA was a reliable method of quantifying T-cell response after SARS-COV-2 infection or vaccination. In convalescent patients, the sensitivity is largely dependent on disease severity and time since primary infection. The assay is more likely to add clinical value to serology in patients with mild infections.

Late reinfection with a different severe acute respiratory syndrome coronavirus-2 clade in a patient with refractory arterial hypertension: a case report.

BACKGROUND: Differentiating between persistent infection with intermittent viral shedding and reinfection with severe acute respiratory syndrome coronavirus 2 remains challenging. Although a small number of cases with genomic evidence of second infection have been reported, limited information exists on frequency and determinants of reinfection, time between infections, and duration of immunity after the primary infection. CASE PRESENTATION: We report a reinfection with severe acute respiratory syndrome coronavirus 2 in a 52-year-old caucasian male whose primary infection was diagnosed in May 2020, during the first wave of the pandemic in Spain, and the second occurred 8 months later, in January 2021. We present a complete dataset including results from real-time polymerase chain reaction, serology, and genome sequencing confirming reinfection with a different clade. Noteworthy was that the patient was immunocompetent but had multiple cardiometabolic comorbidities, including refractory arterial hypertension, that might increase the individual risk in coronavirus disease 2019. CONCLUSIONS: This case of reinfection with severe acute respiratory syndrome coronavirus 2 occurring several months after the primary infection reports the longest time interval between reinfection and initial infection described to date. It raises concerns on the duration of protective immunity, suggesting that it may begin to wane in patients who acquired the initial infection during the first wave of the pandemic. The potential contributing role of arterial hypertension and cardiometabolic comorbidities as risk factors for reinfection deserves investigation.

Surveillance for Enteroviruses Associated with Hand, Foot, and Mouth Disease, and Other Mucocutaneous Symptoms in Spain, 2006-2020.

Hand, foot, and mouth disease (HFMD) is a mild illness caused by enteroviruses (EV), although in some Asian countries, large outbreaks have been reported in the last 25 years, with a considerable incidence of neurological complications. This study describes epidemiological and clinical characteristics of EV infections involved in HFMD and other mucocutaneous symptoms from 2006 to 2020 in Spain. EV-positive samples from 368 patients were included. EV species A were identified in 85.1% of those typed EV. Coxsackievirus (CV) A6 was the prevalent serotype (60.9%), followed by EV-A71 (9.9%) and CVA16 (7.7%). Infections affected children (1-6 years old) mainly, and show seasonality with peaks in spring-summer and autumn. Clinical data indicated few cases of atypical HFMD as well as those with neurological complications (associated with the 2016 EV-A71 outbreak). Phylogenetic analysis of CVA6 VP1 sequences showed different sub-clusters circulating from 2010 to present. In conclusion, HFMD or exanthemas case reporting has increased in Spain in recent years, probably associated with an increase in circulation of CVA6, although they did not seem to show greater severity. However, EV surveillance in mucocutaneous manifestations should be improved to identify the emergence of new types or variants causing outbreaks and more severe pathologies.

Clinical evaluation of a new molecular method for the detection of multidrug-resistant microorganisms. / Evaluación clínica de un nuevo método molecular para la detección de microorganismos multirresistentes.

INTRODUCTION: The main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip® starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. METHODS: We developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. The completion time of the full analysis is 3 hours. RESULTS: Both the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in rectal swabs and 97% in nasal swabs. CONCLUSIONS: The AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms from nasal and rectal swab samples.

Performance of Saliva Specimens for the Molecular Detection of SARS-CoV-2 in the Community Setting: Does Sample Collection Method Matter?

Data on the performance of saliva specimens for diagnosing coronavirus disease 2019 (COVID-19) in ambulatory patients are scarce and inconsistent. We assessed saliva-based specimens for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcriptase PCR (RT-PCR) in the community setting and compared three different collection methods. This prospective study was conducted in three primary care centers. RT-PCR was performed on paired nasopharyngeal swabs (NPS) and saliva samples collected from outpatients with a broad clinical spectrum of illness. To assess differences in collection methods, saliva specimens were obtained in a different way in each of the participating centers: supervised collection (SVC), oropharyngeal washing (OPW), and self-collection (SC). Pairs of NPS and saliva samples from 577 patients (median age, 39 years; 44% men; 42% asymptomatic) were collected and tested, and 120 (20.8%) gave positive results. The overall agreement with NPS results and kappa coefficients (κ) for saliva samples obtained by SVC, OPW, and SC were 95% (κ = 0.85), 93.4% (κ = 0.76), and 93.3% (κ = 0.76), respectively. The sensitivities (95% confidence intervals [95% CI]) of the saliva specimens ranged from 86% (72.6% to 93.7%) for SVC to 66.7% (50.4% to 80%) for SC samples. Sensitivity was higher for samples with lower cycle threshold (CT ) values. The best RT-PCR performance was observed for SVC, with sensitivities (95% CI) of 100% (85.9% to 100%) in symptomatic individuals and 88.9% (50.7% to 99.4%) in asymptomatic individuals at CT values of ≤30. We conclude that saliva is an acceptable specimen for the detection of SARS-CoV-2 in the community setting. Specimens collected under supervision perform comparably to NPS and can effectively identify individuals at higher risk of transmission under real-life conditions.

Asthma prevalence in patients with SARS-CoV-2 infection detected by RT-PCR not requiring hospitalization.

INTRODUCTION: The prevalence of asthma in patients hospitalized with SARS-CoV-2 has been studied and varies widely in the different series. However, the prevalence in SARS-infected patients not requiring hospitalization is not known. The objective of this study was to analyze the presence of asthma in a consecutive series of patients who tested positive in the RT-PCR assay for SARS-CoV-2 and did not require hospital admission. METHODS AND RESULTS: A total of 218 patients (58% of those who tested positive) did not require hospitalization; they had a median age of 45 years (IQR 34-57) and 57% were female. Six patients (2.8%) had a previous diagnosis of asthma. Only one patient developed a mild aggravation of asthma symptoms associated with SARS-CoV-2 infection. CONCLUSIONS: Few patients with asthma were infected by SARS-CoV-2, and this infection was not a significant cause of asthma exacerbation.

Relationship between tobacco, cagA and vacA i1 virulence factors and bacterial load in patients infected by Helicobacter pylori.

BACKGROUND AND AIM: Several biological and epidemiological studies support a relationship between smoking and Helicobacter pylori (H. pylori) to increase the risk of pathology. However, there have been few studies on the potential synergistic association between specific cagA and vacA virulence factors and smoking in patients infected by Helicobacter pylori. We studied the relationship between smoking and cagA, vacA i1 virulence factors and bacterial load in H. pylori infected patients. METHODS: Biopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients' clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (ORadjusted) were estimated by unconditional logistic regression. RESULTS: cagA was significantly associated with active-smoking at endoscope: ORadjusted 4.52. Evidence of association was found for vacA i1 (ORadjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell). CONCLUSIONS: The association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors.

[Characterization of gene expression associated with both the AcrAB/TolC system and the membrane permeability, in Salmonella spp isolates with and without gyrA mutation]. / Caracterización de la expresión de genes asociados con el sistema AcrAB/TolC y la permeabilidad de membrana en aislados de Salmonella spp. con y sin mutación en gyrA.

Introduction. The marA, soxS, ramA, acrB and ompF genes have been studied in order to characterize mechanisms of AcrAB-TolC active efflux pumps and membrane permeability alterations that reduce fluoroquinolones susceptibility in Salmonella spp. Methods. Mutations in marA, soxS, ramA, acrB and ompF genes were detected, as well as their expression levels in presence and absence of ciprofloxacin, calculating the level of change between them by qPCR. Data were analysed by using SPSS 19.0. Results. No mutations in these genes were found, but both AcrAB-TolC regulatory genes and structural acrB gene expression were affected by ciprofloxacin in both mutant strains and wild type bacterial strains (WT). The activation of the marA gene in presence of drug was higher in WT strains (level of change 0.823) than in mutants strains (level of change 0.158; p=0.049). In gyrA mutants, a reduction in ompF gene expression in presence of ciprofloxacin was found (level of change -0.949 p=0.017). Conclusion. The reduction of fluoroquinolones susceptibility in Salmonella spp is a complex process, in which several different bacterial mechanisms are involved. This study has found a high difference in the degree of participation among studied mechanisms, between bacterial strains with and without gyrA mutation. Whereas WT strains activated efflux pumps especially through marA gene, mutants supressed ompF gene expression related to porins.

Influenza vaccine effectiveness in preventing influenza A(H3N2)-related hospitalizations in adults targeted for vaccination by type of vaccine: a hospital-based test-negative study, 2011-2012 A(H3N2) predominant influenza season, Valencia, Spain.

BACKGROUND: Most evidence of the effectiveness of influenza vaccines comes from studies conducted in primary care, but less is known about their effectiveness in preventing serious complications. Here, we examined the influenza vaccine effectiveness (IVE) against hospitalization with PCR-confirmed influenza in the predominant A(H3N2) 2011-2012 influenza season. METHODS: A hospital-based, test-negative study was conducted in nine hospitals in Valencia, Spain. All emergency admissions with a predefined subset of symptoms were eligible. We enrolled consenting adults age 18 and over, targeted for influenza vaccination because of comorbidity, with symptoms of influenza-like-illness within seven days of admission. We estimated IVE as (1-adjusted vaccination odds ratio)*100 after accounting for major confounders, calendar time and recruitment hospital. RESULTS: The subjects included 544 positive for influenza A(H3N2) and 1,370 negative for influenza admissions. Age was an IVE modifying factor. Regardless of vaccine administration, IVE was 72% (38 to 88%) in subjects aged under 65 and 21% (-5% to 40%) in subjects aged 65 and over. By type of vaccine, the IVE of classical intramuscular split-influenza vaccine, used in subjects 18 to 64, was 68% (12% to 88%). The IVE for intradermal and virosomal influenza vaccines, used in subjects aged 65 and over, was 39% (11% to 58%) and 16% (-39% to 49%), respectively. CONCLUSIONS: The split-influenza vaccine was effective in preventing influenza-associated hospitalizations in adults aged under 65. The intradermal vaccine was moderately effective in those aged 65 and over.

Prevalence of methicillin-resistant Staphylococcus aureus : effect of different criteria for elimination of duplicates.

BACKGROUND: The purpose of this study is to compare the effect of different systems for eliminating duplicates in order to optimize the calculation of the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection. METHODS: We compare the Clinical and Laboratory Standards Institute (CLSI) criterion, time criteria and the criterion recommended by the European Antimicrobial Surveillance System (EARSS). RESULTS: Multiple isolates of MRSA are frequently recovered from successive cultures from the same patient (the average isolation rate of MRSA is 2.72), which demonstrates the importance of eliminating duplicates. When CLSI criterion data are compared to those obtained using other criteria, a significant increase in the number of S. aureus isolates was found applying time criteria (up to 36%) or the EARSS criterion (13%). There is also an increase in the methicillin resistance rate (between 3.31 and 3.96%; p < 0.01). CONCLUSIONS: We believe that the EARSS method, with the proper quality controls and latest software tools available, is the best for determining the true situation of MRSA.

Infección respiratoria causada por Alcaligenes xylosoxidans en un paciente con síndrome de Mounier-Kuhn / Case report: respiratory infection due to Alcaligenes xylosoxidans in a patient with Mounier-Kuhn syndrome

Mounier-Kuhn syndrome is a rare entity characterized by abnormal dilatation of the trachea and main bronchi (tracheobronchomegaly). Alcaligenes xylosoxidans is a non fermenting gram-negative pathogen common in extra-and intra-hospital environment, which may be related to immunosuppression states. We describe the case of a 75 years old male, ex-smoker with moderate functional obstruction, chronic respiratory failure and chronic colonization by Pseudomonas aeuriginosa. He had an infectious exacerbation of his disease, reason that previously required several hospital admissions. The patient was treated with antibiotics and his evolution was favourable with negativization in cultures of the pathogen. This is the first description of the isolation of Alcaligenes xylosoxidans as a cause of respiratory infection in a patient with Mounier-Kuhn syndrome.

Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin.

OBJECTIVES: To detect the presence of lnu genes in staphylococcal strains with the unusual phenotype lincosamide resistance/macrolide susceptibility (L(R)/M(S)), and to determine their locations and genetic environments. METHODS: Six staphylococcal strains of human and animal origin with the phenotype L(R)/M(S) were studied. The presence of 15 resistance genes was tested by PCR. SCCmec typing was performed for all methicillin-resistant strains. agr typing, spa typing and multilocus sequence typing were carried out for Staphylococcus aureus strains. Transformation experiments were carried out by electrotransformation. Plasmid or chromosomal gene location was determined by Southern blot analysis and the genetic environments of the lnu genes were studied in all strains. RESULTS: Three methicillin-resistant staphylococcal strains contained the lnu(A) gene. The presence of the pLNU1 plasmid carrying lnu(A) was confirmed in one methicillin-resistant S. aureus (MRSA) ST398-t108 and one methicillin-resistant Staphylococcus sciuri. A novel lnu(A)-carrying plasmid (pUR5425) was identified in one MRSA ST125-t067 strain. Transformants of the three lnu(A)-positive strains presented increased lincomycin MIC values. The remaining three studied staphylococcal strains harboured the lnu(B) gene: two methicillin-susceptible S. aureus (MSSA) ST9-t337 and one MRSA ST398-t011. The lnu(B) gene was embedded in the chromosome in the two MSSA strains and in a large-sized plasmid in the MRSA strain. The same lnu(B) genetic environment was detected in these three strains. CONCLUSIONS: The resistance phenotype L(R)/M(S) seems to be related to S. aureus animal-associated clonal lineages (ST398 and ST9). A novel lnu(A)-carrying plasmid was identified and this is the first detection of the lnu(B) gene in MRSA ST398.

Quantification of Helicobacter pylori in gastric mucosa by real-time polymerase chain reaction: comparison with traditional diagnostic methods.

The aim of this study was to determine the main diagnostic validity parameters of a quantitative real-time polymerase chain reaction (PCR) system for detecting Helicobacter pylori in gastric biopsies. Prospective study. The real-time PCR has an internal control for eliminating the false negatives. Our system has a good diagnostic capacity compared with the gold standard and was superior in antral mucosa: area under the curve was 0.91 for antrum (95% confidence interval [CI] 0.87 to 0.96) and 0.83 for corpus (95% CI 0.77 to 0.9). The optimum cut-off point was 3.56 microorganisms/cell for antrum (sensitivity 83.5% [95% CI 74.2 to 89.9]; specificity 91.3% [95% CI 82.3 to 96.0]; positive predictive value 92.2%; negative predictive value 81.8%). The positive likelihood ratios were 9.61 and 8.52 for antrum and corpus, respectively. With the cut-off point that maximises the Youden index, 8.7% false positives were obtained. Our methodology is useful for diagnosing infection due to H. pylori and the false positives detected probably correspond to patients who were actually infected but the infection was not detected by traditional techniques. The clinical importance of these cases should be studied in greater detail since they may involve colonisations unrelated to the patient's digestive pathology.

Real-time PCR for diagnosing Helicobacter pylori infection in patients with upper gastrointestinal bleeding: comparison with other classical diagnostic methods.

The aim of this study was to determine the diagnostic usefulness of quantification of the H. pylori genome in detection of infection in patients with upper gastrointestinal bleeding (UGB). A total of 158 consecutive patients with digestive disorders, 80 of whom had clinical presentation of UGB, were studied. The number of microorganisms was quantified using a real-time PCR system which amplifies the urease gene with an internal control for eliminating the false negatives. A biopsy sample from the antrum and corpus of each patient was processed. The rapid urease test, culture, histological study, stool antigen test, and breath test were done. The gold standard was a positive culture or positive results in at least two of the other techniques. When a positive result was defined as any number of microorganisms/human cell, the sensitivity of real-time PCR was greater in bleeding patients, especially in the gastric corpus: 68.4% (95% confidence interval [CI], 52.3 to 84.5%) in non-UGB patients versus 91.5% (95% CI, 79.6 to 97.6%) in UGB patients. When a positive result was defined as a number of microorganisms/human cell above the optimal value that maximizes the Youden index (>3.56 microorganisms/human cell in the antrum and >2.69 in the corpus), the sensitivity and specificity in UGB patients were over 80% in both antrum and corpus. Our findings suggest that some bleeding patients with infection caused by H. pylori may not be correctly diagnosed by classical methods, and such patients could benefit from the improved diagnosis provided by real-time PCR. However, the clinical significance of a small number of microorganisms in patients with negative results in classical tests should be evaluated.