The Possibility of Differentiating Between Pneumococcal Pneumonia and Colonized <i>S. pneumoniae</i> in Children by Cycle Threshold of Real-time PCR

Decision letter: Asymptomatic Bordetella pertussis infections in a longitudinal cohort of young African infants and their mothers

BackgroundHCT recipients are frequently infected with respiratory viruses (RVs) in the URT; however, diagnostic evaluation of the LRT by bronchoalveolar lavage (BAL) is less common. We sought to determine whether the detection of RVs in the URT is predictive of LRT detection and to identify factors that predict discordance between upper and lower RV detection.MethodsHCT recipients with respiratory symptoms and LRT RV testing via multiplex PCR in BAL from July 2009 to October 2016 were included in the study. RV PCR results, including cycle threshold (CT) values, were compared with URT samples obtained within ±3 days. Logistic regression models were used to analyze risk factors for RV discordance between paired samples.ResultsAmong 1,000 HCT recipients with BAL RV testing, 250 had URT testing within 3 days. In total, 75(30%) sample pairs were concordant for the same RV in both the URT and BAL (P/P); 132 (53%) were negative from both sites. Among 43 discordant pairs, 25 (10%) were only positive by URT but negative by BAL (P/N) and 18 (7%) were positive by BAL but negative by URT (N/P). In pairs with positive RV results in the URT or BAL, discordance was common for HMPV (44%), HRV (33%), and PIV-3 (28%); RSV was almost always concordant (92%) (Figure 1). In a multivariable model, the risk of discordance (P/N or N/P) was increased in the presence of a solitary nodule on radiography (OR 6.8; 95% CI 1.2–38.3) and with lymphocyte count >500/mm3 (OR 3.1; 95% CI 1.08–9.0). Among P/P pairs, the median difference between CT values between URT and BAL samples was 0 (range −12 to +13), with 33 and 29% of subjects having lower and higher CT values (>4, ~1 log10) in the BAL, respectively (Figure 2).ConclusionIn symptomatic HCT recipients with RV PCR testing performed concurrently in the upper and lower tract, discordant results are relatively common, especially for HRV, HMPV, and PIV-3. The presence of a solitary nodule on imaging and the absence of lymphopenia are associated with discordant results, with BAL results more likely being negative in these situations. More than half of the P/P pairs had a >4 difference in CT values between URT and LRT samples. Taken together, these data suggest that RV testing in BAL can provide useful diagnostic information that may guide management in HCT recipients.DisclosureS. A. Pergam, MERCK: Consultant and Investigator, Consulting fee.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay of the upper respiratory tract is used increasingly to diagnose lower respiratory tract infections. The cycle threshold (CT ) values of qRT-PCR are continuous, semi-quantitative measurements of viral load, although interpretation of diagnostic qRT-PCR results are often categorized as positive, indeterminate, or negative, obscuring potentially useful clinical interpretation of CT values. From 2008 to 2010, naso/oropharyngeal swabs were collected from outpatients with influenza-like illness, inpatients with severe respiratory illness, and asymptomatic controls in rural Kenya. CT values of positive specimens (i.e., CT values < 40.0) were compared by clinical severity category for five viruses using Mann-Whitney U-test and logistic regression. Among children <5 years old we tested with respiratory syncytial virus (RSV), inpatients had lower median CT values (27.2) than controls (35.8, P = 0.008) and outpatients (34.7, P < 0.001). Among children and older patients infected with influenza virus, outpatients had the lowest median CT values (29.8 and 24.1, respectively) compared with controls (P = 0.193 for children, P < 0.001 for older participants) and inpatients (P = 0.009 for children, P < 0.001 for older participants). All differences remained significant in logistic regression when controlling for age, days since onset, and coinfection. CT values were similar for adenovirus, human metapneumovirus, and parainfluenza virus in all severity groups. In conclusion, the CT values from the qRT-PCR of upper respiratory tract specimens were associated with clinical severity for some respiratory viruses.

Coronavirus disease 2019 can spread rapidly. Surgery in the oral cavity poses a high risk of transmission of severe acute respiratory syndrome coronavirus 2. The American Dental Association and the Centers for Disease Control and Prevention recommend the use of mouthwash containing 1.5% hydrogen peroxide (H 2O 2) or 0.2% povidone iodine (PI) to reduce the viral load in the upper respiratory tract and decrease the risk of transmission. The aim of the present study was to analyze the effect of mouth rinsing and gargling with mouthwash containing 1% PI, 0.5% PI, 3% H 2O 2, or 1.5% H 2O 2 and water on the cycle threshold (CT) value obtained by real-time reverse transcription polymerase chain reaction (RT-PCR). This study is a randomized single blind controlled clinical trial which has been registered in the International Standard Randomized Controlled Trial Number (ISRCTN) registry on the 3 rd February 2022 (Registration number: ISRCTN18356379). In total, 69 subjects recruited from Persahabatan General Hospital who met the inclusion criteria were randomly assigned to one of four treatment groups or the control group. The subjects were instructed to gargle with 15 mL of mouthwash for 30 s in the oral cavity followed by 30 s in the back of the throat, three times per day for 5 days. CT values were collected on postprocedural days 1, 3, and 5. The results of the Friedman test significantly differed among the groups (n=15). The CT values increased from baseline (day 0) to postprocedural days 1, 3, and 5. Mouth rinsing and gargling with mouthwash containing 1% PI, 0.5% PI, 3% H 2O 2, or 1.5% H 2O 2 and water increased the CT value.

Background: Coronavirus disease 2019 can spread rapidly. Surgery in the oral cavity poses a high risk of transmission of severe acute respiratory syndrome coronavirus 2. The American Dental Association and the Centers for Disease Control and Prevention recommend the use of mouthwash containing 1.5% hydrogen peroxide (H2O2) or 0.2% povidone iodine (PI) to reduce the viral load in the upper respiratory tract and decrease the risk of transmission. The aim of the present study was to analyze the effect of mouth rinsing and gargling with mouthwash containing 1% PI, 0.5% PI, 3% H2O2, or 1.5% H2O2 and water on the cycle threshold (CT) value obtained by real-time reverse transcription polymerase chain reaction (RT-PCR). Methods: This study is a randomized single blind controlled clinical trial which has been registered in the International Standard Randomized Controlled Trial Number (ISRCTN) registry on the 3rd February 2022 (Registration number: ISRCTN18356379). In total, 69 subjects recruited from Persahabatan General Hospital who met the inclusion criteria were randomly assigned to one of four treatment groups or the control group. The subjects were instructed to gargle with 15 mL of mouthwash for 30 s in the oral cavity followed by 30 s in the back of the throat, three times per day for 5 days. CT values were collected on postprocedural days 1, 3, and 5. Results: The results of the Friedman test significantly differed among the groups (n=15). The CT values increased from baseline (day 0) to postprocedural days 1, 3, and 5. Conclusions: Mouth rinsing and gargling with mouthwash containing 1% PI, 0.5% PI, 3% H2O2, or 1.5% H2O2 and water increased the CT value.

Decision letter: SARS-CoV-2 shedding dynamics across the respiratory tract, sex, and disease severity for adult and pediatric COVID-19

Pneumococcal Bacteremia: Lessons Learned, Yet More to Learn

BackgroundThis study sought to explore the value of hematological indexes [i.e., the neutrophil count/lymphocyte count ratio (NLR), the platelet count/lymphocyte count ratio (PLR), and red cell distribution width (RDW) index] in the diagnosis of bacterial infectious pneumonia in children.MethodsFifty cases of mycoplasma infectious pneumonia, 50 cases of bacterial infectious pneumonia and 50 healthy children were enrolled in this study. The differences between the NLR, PLR, and RDW index values in each group were compared using the Mann-Whitney test. The correlation coefficients of the NLR, PLR, and RDW index with the interleukin-6 (IL-6) and procalcitonin (PCT) were analyzed using the Spearman’s rank test. The specificity and sensitivity of the NLR, PLR, and RDW index in the diagnosis of bacterial pneumonia in children were evaluated by receiver operating characteristic (ROC) curves.ResultsThe NLR value of the children with bacterial infectious pneumonia was significantly higher than that of the children with mycoplasma infectious pneumonia (P<0.05) and healthy children (P<0.05), while the PLR value of the children with bacterial infectious pneumonia was significantly lower than that of the children with mycoplasma infectious pneumonia (P<0.05) and healthy children (P<0.05). There was no significant difference in the RDW index values of the healthy control children and the children with mycoplasma infectious pneumonia and bacterial infectious pneumonia (P>0.05). There was a positive correlation between NLR and serum IL-6 (R=0.203; P=0.041), and a negative correlation between PLR and serum PCT (R=–0.291; P=0.037). In addition, there was no significant correlation between the RDW index and serum IL-6, and the RDW index and serum PCT in children with bacterial infectious pneumonia. When the 3 indicators were each used to differentiate between healthy children and children with bacterial pneumonia, the area under the PLR curve was the largest for the ROC curve [0.898, 95% confidence interval (CI): 0.815–0.953]. In the differential diagnosis of mycoplasma pneumonia and bacterial pneumonia, the area under the PLR curve was also the largest (0.803, 95% CI: 0.577–0.780).ConclusionsThe PLR has clinical value in the diagnosis of bacterial infectious pneumonia in children.

Pneumocystis jirovecii is a human-specific species and causes fatal infections like P.jirovecii pneumonia (PCP) in immunocompromised persons. Although direct microscopy is the gold standard in the diagnosis of the microorganism, molecular methods such as polymerase chain reaction (PCR) are needed in non-human immune deficiency virus (HIV) immunosuppresive patients with low P.jirovecii burden. In this study, we aimed to evaluate the value of real-time PCR (Rt-PCR) in the laboratory diagnosis of P.jirovecii. Bronchoalveolar lavage (BAL) specimens of 658 patients sent to Dokuz Eylul University Hospital Central Medical Parasitology Laboratory on suspicion of PCP were included in the study. BAL fluids were evaluated for identification of P.jirovecii mitochondrial gene coding ribosomal large subunit (mtLSUrRNA) using Rt-PCR. In addition, Giemsa and Gomori's methenamine silver (GMG) staining assays were applied to all samples and nested PCR (n-PCR) assay was applied to positive samples detected by real time PCR. Ninety-two (14.3%) of these samples were positive by Rt-PCR. Of these 92 patients, 85 (92.4%) were positive with n-PCR. Only seven of the specimens had P.jirovecii cysts and trophozoites with microscopic examination. The mean cycle threshold (CT ) value of Rt-PCR positive patients was 29.7 (18.17 ≤ CT ≤ 37.96). P.jirovecii load in these patients was calculated as 2.6 x 101-6.15 x 107 copies/ml. The difference between the mean CT values of n-PCR positive and negative results was statistically significant (p< 0.01). The CT values of Rt-PCR of the samples with positive microscopy were; 18.2, 20.9, 22.2, 24.3, 24.7, 26.5, 29.7. The difference between the CT means of the samples with positive and negative microscopy was statistically significant (p< 0.05). When positive patients were grouped according to their diagnosis; the lowest mean CT value (CTmean= 24.8) was found in HIV-positive patients. On the other hand, CT values were found to be significantly lower in the organ transplantation patients (CTmean= 26.15) and in the collagen-vascular-inflammatory patient group (CTmean= 27.8). This study demonstrated that Rt-PCR was the effective method in the diagnosis of P.jirovecii in the laboratory. Conventional n-PCR method was found to be more unsuccessful than Rt-PCR in the presence of very low density organism; direct microscopy is generally found to be positive in samples with a higher burden of P.jirovecii.

Pneumonia is the leading cause of death among children less than 5 years old worldwide. A wide range of viral, bacterial and fungal agents can cause pneumonia: although viruses are the most common etiologic agent, the severity of clinical symptoms associated with bacterial pneumonia and increasing antibiotic resistance makes bacterial pneumonia a major public health concern. Bacterial pneumonia can follow upper respiratory viral infection and complicate lower respiratory viral infection. Secondary bacterial pneumonia is a major cause of influenza-related deaths. In this review, we evaluate the following hypotheses: (i) respiratory viruses influence the etiology of pneumonia by altering bacterial community structure in the upper respiratory tract (URT) and (ii) respiratory viruses promote or inhibit colonization of the lower respiratory tract (LRT) by certain bacterial species residing in the URT. We conducted a systematic review of the literature to examine temporal associations between respiratory viruses and bacteria and a targeted review to identify potential mechanisms of interactions. We conclude that viruses both alter the bacterial community in the URT and promote bacterial colonization of the LRT. However, it is uncertain whether changes in the URT bacterial community play a substantial role in pneumonia etiology. The exception is Streptococcus pneumoniae where a strong link between viral co-infection, increased carriage and pneumococcal pneumonia has been established.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to a wide spectrum of clinical severity. The gold standard diagnosis of infection is reverse transcription polymerase chain reaction of nasopharyngeal swabs, which also provides a semiquantitative assessment of viral loads by measuring cycle threshold (CT) values. To assess whether CT values at admission can predict mortality and oxygen needs among individuals hospitalized for coronavirus disease 2019 (COVID-19). The retrospective study included adults hospitalized for COVID-19 between 1 August 2020 and 30 April 2021 at Barzilai University Medical Center. Patients were categorized according to initial CT values as high (≥ 25) or low (< 25) values. The primary outcome was the association between CT values during admission and overall mortality. The study group included 636 patients, with a mean age of 67.2 years, 54.4% males. Overall mortality of patients with CT values < 25 was significantly higher (odds ratio for mortality 1.78 vs. patients with CT ≥ 25, P = 0.002). Significantly more patients in the low CT group required oxygen support than in the high CT group, 50% vs. 31.9% (P < 0.001). An inverse association between CT values and mortality rates remained significant in multivariate regression analysis, such that a 1-unit decrease in CT was associated with a 6% increased mortality. Lower CT values at admission were associated with increased mortality among patients hospitalized for COVID-19. CT values can be used to predict outcomes among such patients.

We initiated a prospective study with a group of practitioners to assess the etiology, clinical presentation, and outcome of community-acquired pneumonia in patients diagnosed in the outpatient setting. All patients with signs and symptoms suggestive of pneumonia and an infiltrate on chest X-ray underwent an extensive standard workup and were followed over 4 weeks. Over a 4-year period, 184 patients were eligible, of whom 170 (age range, 15-96 yr; median, 43 yr) were included and analyzed. In 78 (46%), no etiologic agent could be demonstrated. In the remaining 92 patients, 107 etiologic agents were implicated: 43 were due to "pyogenic" bacteria (39 Streptococcus pneumoniae, 3 Haemophilus spp., 1 Streptococcus spp.), 39 were due to "atypical" bacteria (24 Mycoplasma pneumoniae, 9 Chlamydia pneumoniae, 4 Coxiella burnetii, 2 Legionella spp.), and 25 were due to viruses (20 influenza viruses and 5 other respiratory viruses). There were only a few statistically significant clinical differences between the different etiologic categories (higher age and comorbidities in viral or in episodes of undetermined etiology, higher neutrophil counts in "pyogenic" episodes, more frequent bilateral and interstitial infiltrates in viral episodes). There were 2 deaths, both in patients with advanced age (83 and 86 years old), and several comorbidities. Only 14 patients (8.2%) required hospitalization. In 6 patients (3.4%), the pneumonia episode uncovered a local neoplasia. This study shows that most cases of community-acquired pneumonia have a favorable outcome and can be successfully managed in an outpatient setting. Moreover, in the absence of rapid and reliable clinical or laboratory tests to establish a definite etiologic diagnosis at presentation, the spectrum of the etiologic agents suggest that initial antibiotic therapy should cover both S. pneumoniae and atypical bacteria, as well as possible influenza viruses during the epidemic season.

Mycoplasma pneumoniae (Mp) is reported to be the most common bacterial cause of community-acquired pneumonia (CAP) in hospitalized U.S. children (1). However, current diagnostic tests, including PCR of upper respiratory tract (URT) specimens and serology, do not differentiate between Mp infection and carriage (2). Mp carriage in the URT is found in up to 56% of healthy children (2, 3). A ≥4-fold increase in IgG levels is still used in most centers to confirm Mp infection but has low sensitivity (4) and is not helpful in acute clinical management (3). In the absence of an accurate diagnostic test, it is not surprising that studies and meta-analyses on the efficacy of antibiotics are inconclusive for Mp CAP in children (5, 6). Circulating antibody-secreting cell (ASC) responses have been demonstrated to be more rapid and shorter-lived than antibody responses (7). We hypothesized that Mp-IgM-ASCs circulate in peripheral blood only for a few days or weeks after Mp infection, whereas Mp-DNA in the URT and serum antibodies persist for months. We aimed to evaluate the measurement of Mp-IgM-ASCs by enzyme-linked immunospot (ELISpot) assay as a new test for diagnosing Mp CAP. Methods Pediatric patients with CAP (n = 152) and control subjects (n = 156) were enrolled from May 2016 to April 2017 after written informed consent. Inclusion criteria for patients with CAP were clinical diagnosis of pneumonia (fever >38.5°C and tachypnea [8]) in previously healthy children aged 3–18 years. Children <3 years were excluded because of a high probability of viral coexistence in the URT (8). Control individuals included healthy children (undergoing elective surgical procedures) and siblings of patients with CAP (with higher chance of being asymptomatic carriers) without recent (≤1 wk) respiratory tract infections. In all enrolled children, pharyngeal swabs were taken for Mp real-time PCR (9). If additional consent was given, blood samples also were collected in control individuals and patients with CAP (before antibiotic treatment) to test for the presence of Mp-IgM-ASCs by ELISpot assay (detailed in the legend of Figure 1) (10) and Mp-IgM, Mp-IgG, and Mp-IgA by ELISA (2). Finally, we only included children with fresh (isolated ≤4 h) peripheral blood mononuclear cells to avoid poor ELISpot assay performance resulting from decreased ASC viability (in case of isolation >4 h after sampling) or reduced ASC recovery (after a freeze–thaw cycle) (10). Samples and clinical data (using a standardized questionnaire) were collected at follow-up visits at <2 weeks, 2 weeks to 2 months, and 2–6 months.

1. Asif Noor, MD* 2. Theresa Fiorito, MD* 3. Leonard R. Krilov, MD*,† 1. *Department of Pediatrics, Children's Medical Center, NYU Winthrop Hospital, Mineola, NY 2. †Department of Pediatrics, State University of New York, Stony Brook School of Medicine, Stony Brook, NY * Abbreviations: AAP: : American Academy of Pediatrics Adv: : adenovirus CDC: : Centers for Disease Control and Prevention FDA: : Food and Drug Administration hMPV: : human metapneumovirus PCR: : polymerase chain reaction PIV: : parainfluenza virus RSV: : respiratory syncytial virus RV: : rhinovirus Clinicians must learn to identify viral infections in children during the winter months and must practice caution with the use of unnecessary medications in such cases. Recognition of the clinical pattern of viral infection (eg, bronchiolitis) in conjunction with judicious use of viral tests (either office-based immunoassays or newer molecular tests) may assist in epidemiological monitoring, cohorting patients in the hospital, withholding unnecessary therapies, and providing a definitive diagnosis. After completing this article, readers should be able to: 1. Review the epidemiological aspects and clinical signs and symptoms of common cold weather viruses. 2. Recognize situations in which viral testing is indicated. 3. Recognize situations in which treatment is indicated. In early November you are evaluating a 9-month-old boy born at 33 weeks of gestation. The infant presents with 2 days of fevers (101°F–102°F [38.3°C–38.8°C]), copious rhinorrhea, and 1 day of coughing with difficulty breathing. He is otherwise feeding well and has had adequate urination. His 4-year-older sister has an upper respiratory tract infection. On physical examination, the infant has a respiratory rate of 45 breaths/min without chest wall retractions. On auscultation there is good air entry with scattered rhonchi bilaterally. What is the most appropriate next step in management? 1. Obtain respiratory syncytial virus (RSV) and influenza antigen testing. 2. Obtain a chest radiograph to look for focal infiltrate. 3. Provide supportive care with nasal …