Etiology and the challenge of diagnostic testing of community-acquired pneumonia in children and adolescents.

BACKGROUND: Pneumonia is the leading cause of mortality in pediatric population. The etiology of pneumonia in this population is variable and changes according to age and disease severity and where the study is conducted. Our aim was to determine the etiology of community-acquired pneumonia (CAP) in children aged 1 month to 17 years admitted to 13 Colombian hospitals. METHODS: Prospective cohort study. Hospitalized children with radiologically confirmed CAP and ≤ 15 days of symptoms were included and followed together with a control group. Induced sputum (IS) was submitted for stains and cultures for pyogenic bacteria and Mycobacterium tuberculosis, and multiplex PCR (mPCR) for bacteria and viruses; urinary antigens for pneumococcus and Legionella pneumophila; nasopharyngeal swabs for viruses, and paired serology for atypical bacteria and viruses. Additional cultures were taken at the discretion of primary care pediatricians. RESULTS: Among 525 children with CAP, 71.6% had non-severe pneumonia; 24.8% severe and 3.6% very severe pneumonia, and no fatal cases. At least one microorganism was identified in 84% of children and 61% were of mixed etiology; 72% had at least one respiratory virus, 28% pyogenic bacteria and 21% atypical bacteria. Respiratory syncytial virus, Parainfluenza, Rhinovirus, Influenza, Mycoplasma pneumoniae, Adenovirus and Streptococcus pneumoniae were the most common etiologies of CAP. Respiratory syncytial virus was more frequent in children under 2 years and in severe pneumonia. Tuberculosis was diagnosed in 2.3% of children. IS was the most useful specimen to identify the etiology (33.6%), and blood cultures were positive in 3.6%. The concordance between all available diagnostic tests was low. A high percentage of healthy children were colonized by S. pneumoniae and Haemophilus influenzae, or were infected by Parainfluenza, Rhinovirus, Influenza and Adenovirus. CONCLUSIONS: Respiratory viruses are the most frequent etiology of CAP in children and adolescents, in particular in those under 5 years. This study shows the challenges in making an etiologic diagnosis of CAP in pediatric population because of the poor concordance between tests and the high percentage of multiple microorganisms in healthy children. IS is useful for CAP diagnosis in pediatric population.

High transient colonization by Pneumocystis jirovecii between mothers and newborn.

The aim of the study was to explore the frequency and dynamics of acquisition and colonization of Pneumocystis jirovecii among neonates, as well as the epidemiological and genotypic characteristics in mother-child binomial. In a prospective enrolled cohort of women in their third trimester of pregnancy, nasopharyngeal swabs (NPS) and clinical and epidemiological data were collected at four different times: 17 days, 2nd, 4th, and 6th month of life of the newborn. P. jirovecii was detected by nested-PCR for the mtLSU-rRNA gene in each NPS; the genotypes were determined amplifying four genes. Forty-three pairs and 301 NPS were included. During the third trimester, 16.3% of pregnant women were colonized. The rate of colonization in mothers at delivery was 16, 6, 16, and 5% and in their children 28, 43, 42, and 25%, respectively. Within pregnant women, 53% remained negative throughout follow-up, and among these, 91% of their children were positive in at least one of their samples. In both, mothers and children, the most frequent genotype of P. jirovecii was 1. CONCLUSION: The frequency of colonization by P. jirovecii was higher in newborns than in their respective progenitors. Colonization of both mothers and children is transitory; however, the mother of the newborn is not necessarily the source of primary infection. What is Known: • We did not find studies comparing P. jirovecii colonization between mothers and children simultaneously, yet the frequency of colonization by serologic and molecular methods in pregnant women has been reported. What is New: • According to our findings, 3/4 of the children had transient colonization during the first 6 months of life, in only half in the mothers, without proof of mother-to-child transmission or vice versa.

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria.

BACKGROUND: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specific PCR commercial kits, paired serology, and urinary antigen. RESULTS: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1%, mPCR-IS = 57.1%, Seeplex®-IS = 52.4%, and Speed-oligo®-NPA/NPS = 11.1%, and the specificities were mPCR-NPS = 97.1%, mPCR-IS = 77.8%, Seeplex®-IS = 92.6%, and Speed-oligo®-NPA/NPS = 96.1%. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs. CONCLUSIONS: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60%; thus, better diagnostic techniques for these three bacteria are required.

Unique cytokine and chemokine patterns in bronchoalveolar lavage are associated with specific causative pathogen among HIV infected patients with pneumonia, in Medellin, Colombia.

We wanted to investigate the pro-inflammatory cytokine/chemokine profile associated with the etiological agents identified in HIV patients. Immunosuppressed patients admitted to two hospitals in Medellin, Colombia, with clinical and radiographic diagnosis of pneumonia were enrolled in the study. After consent, bronchoalveolar lavage (BAL) was collected for bacterial, mycobacterial and fungal diagnosis. All patients were followed for a year. A stored BAL sample was used for cytokine/chemokine detection and measurement using commercial, magnetic human cytokine bead-based 19-plex assays. Statistical analysis was performed by assigning cytokine/chemokine concentrations levels into <25 percentile (lower), 25-75 percentile (normal) and >75 percentile (higher). Principal component analysis (PCA) and Kruskal-Wallis analysis were conducted to identify the clustering of cytokines with the various infectious etiologies (fungi, Mycobacterium tuberculosis - MTB, and bacteria). Average age of patients was 35, of whom 77% were male, and the median CD4 count of 33cells/µl. Of the 57 HIV infected patients, in-hospital mortality was 12.3% and 33% died within a year of follow up. The PCA revealed increased IL-10, IL-12, IL-13, IL-17, Eotaxin, GCSF, MIP-1α, and MIP-1ß concentrations to be associated with MTB infection. In patients with proven fungal infection, low concentrations of IL-1RA, IL-8, TNF-α and VEGF were identified. Bacterial infections displayed a distinct cytokine pattern and were not misclassified using the MTB or fungi cytokine patterns (p-value<0.0001). Our results indicate a unique pattern of pro-inflammatory cytokine/chemokine, allowing differentiation between bacterial and non-bacterial pathogens. Moreover, we found distinct, if imperfectly discriminatory, cytokine/chemokine patterns associated with MTB and fungal infections.

Does the Recovery of Respiratory Viruses Impact Pulmonary Function at Baseline and 1-, 6-, and 12-Month Follow-Up in People Living with HIV and Pneumonia?

The frequency of respiratory viruses in people living with HIV (PLHIV) and their impact on lung function remain unclear. We aimed to determine the frequency of respiratory viruses in bronchoalveolar lavage and induced sputum samples in PLHIV and correlate their presence with lung function. A prospective cohort of adults hospitalized in Medellín between September 2016 and December 2018 included three groups: group 1 = people diagnosed with HIV and a diagnosis of community-acquired pneumonia (CAP), group 2 = HIV, and group 3 = CAP. People were followed up with at months 1, 6, and 12. Clinical, microbiological, and spirometric data were collected. Respiratory viruses were detected by multiplex RT-PCR. Sixty-five patients were included. At least 1 respiratory virus was identified in 51.9%, 45.1%, and 57.1% of groups 1, 2 and 3, respectively. Among these, 89% of respiratory viruses were detected with another pathogen, mainly Mycobacterium tuberculosis (40.7%) and Pneumocystis jirovecii (22.2%). The most frequent respiratory virus was rhinovirus (24/65, 37%). On admission, 30.4% of group 1, 16.6% of group 2, and 50% of group 3 had airflow limitation, with alteration in forced expiratory volume at first second in both groups with pneumonia compared to HIV. Respiratory viruses are frequent in people diagnosed with HIV, generally coexisting with other pathogens. Pulmonary function on admission was affected in patients with pneumonia, improving significantly in the 1st, 6th, and 12th months after CAP onset.

Induced sputum as an adequate clinical specimen for the etiological diagnosis of community-acquired pneumonia (CAP) in children and adolescents.

OBJECTIVES: This study aimed to evaluate the utility of induced sputum (IS) for the diagnosis of community-acquired pneumonia (CAP) in pediatric population. METHODS: This cross-sectional study included pediatric population aged between 1 month and 17 years who were hospitalized with a diagnosis of CAP in 13 hospitals in Colombia, in whom an IS sample was obtained. Gram staining, aerobic bacterial and mycobacterial culture tests, and polymerase chain reaction (PCR) for 6 atypical bacteria and 15 respiratory viruses were performed. We evaluated the quality of IS samples. RESULTS: IS samples were collected in 516 of 525 children included in this study. The median age was 32 months, 38.6% were younger than 2 years, and 40.9% were between 2 and 5 years. Two patients had transient hypoxemia during the procedure. The quality of the IS obtained was good in 48.4% and intermediate in 24.5%. Identification of a respiratory pathogen was achieved with an IS sample (with Gram staining, culture test, and PCR) in 372 of 516 children with CAP. CONCLUSION: Our study shows that IS is an adequate sample for the diagnosis of CAP in pediatric population that required hospitalization. The procedure was safe, well tolerated, and with better diagnostic yields compared with the rest of the samples obtained.

Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs.

Timely and accurate diagnostics are essential to fight the COVID-19 pandemic, but no test satisfies both conditions. Dogs can scent-identify the unique odors of volatile organic compounds generated during infection by interrogating specimens or, ideally, the body of a patient. After training 6 dogs to detect SARS-CoV-2 by scent in human respiratory secretions (in vitro diagnosis), we retrained 5 of them to search and find the infection by scenting the patient directly (in vivo screening). Then, efficacy trials were designed to compare the diagnostic performance of the dogs against that of the rRT-PCR in 848 human subjects: 269 hospitalized patients (COVID-19 prevalence 30.1%), 259 hospital staff (prevalence 2.7%), and 320 government employees (prevalence 1.25%). The limit of detection in vitro was lower than 10-12 copies ssRNA/mL. During in vivo efficacy experiments, our 5 dogs detected 92 COVID-19 positive patients among the 848 study subjects. The alert (lying down) was immediate, with 95.2% accuracy and high sensitivity (95.9%; 95% C.I. 93.6-97.4), specificity (95.1%; 94.4-95.8), positive predictive value (69.7%; 65.9-73.2), and negative predictive value (99.5%; 99.2-99.7) in relation to rRT-PCR. Seventy-five days after finishing in vivo efficacy experiments, a real-life study (in vivo effectiveness) was executed among the riders of the Metro System of Medellin, deploying the human-canine teams without previous training or announcement. Three dogs were used to examine the scent of 550 volunteers who agreed to participate, both in test with canines and in rRT-PCR testing. Negative predictive value remained at 99.0% (95% C.I. 98.3-99.4), but positive predictive value dropped to 28.2% (95% C.I. 21.1-36.7). Canine scent-detection in vivo is a highly accurate screening test for COVID-19, and it detects more than 99% of infected individuals independent of key variables, such as disease prevalence, time post-exposure, or presence of symptoms. Additional training is required to teach the dogs to ignore odoriferous contamination under real-life conditions.

Mycoplasma pneumoniae in Children With and Without Community-acquired Pneumonia. What do PCR and Serology Say?

BACKGROUND: IgM titers of Mycoplasma pneumoniae can remain high for months or years, and specific DNA can be detected in asymptomatic people. METHODS: We compared the performance of serology and PCR in children with and without community-acquired pneumonia (CAP) for the diagnosis of M. pneumoniae. RESULTS: In children with CAP, a positive test by M. pneumoniae (PCR and/or paired serology or both) were found in 13.9%. Of these, 10.3% were positive by multiplex PCR (Seeplex-Seegen), and 6.7% exhibited quadrupled titers (22 for IgG, 6 for IgM and 5 for both). Both tests were positive in 2.8% of cases. In the group without CAP, 3.3% were positive by PCR. Thirty-two percent of children with CAP and 38.3% of healthy children had IgM titers >11 in the acute phase. CONCLUSIONS: The detection of IgM is not useful for diagnosing acute M. pneumoniae infection, and a positive PCR result can be due to colonization and not infection. New and better diagnostic techniques are required.

Inflammatory Cell Differentiation and Chemotaxis and Extracellular Tissue Repair Markers Are Correlated with Pulmonary Dysfunction in HIV Infected Individuals Presenting with Community-Acquired Pneumonia.

Prior studies have shown that HIV patients develop permanent pulmonary dysfunction following an episode of community-acquired pneumonia (CAP). However, the mechanism causing pulmonary dysfunction remains an enigma. HIV patients experience chronic inflammation. We hypothesized that CAP exacerbates inflammation in HIV patients resulting in an accelerated decline in lung function. A prospective cohort pilot study enrolled HIV patients hospitalized in Medellin, Colombia, with a diagnosis of CAP. Sixteen patients were eligible for the study; they were split into 2 groups: HIV and HIV+CAP. Plasma, sputum, and pulmonary function test (PFT) measurements were retrieved within 48 h of hospital admission and at 1 month follow-up. The concentrations of 13 molecules and PFT values were compared between the 2 cohorts. The HIV+CAP group had lower lung function compared to the HIV group; forced vital capacity (FVC)% predicted and forced expiratory volume in 1 s (FEV1)% predicted decreased, while FEV1/FVC remained constant. APRIL, BAFF, CCL3, and TIMP-1 correlated negatively with FVC% predicted and FEV1% predicted; the relationships however were moderate in strength. Furthermore, the concentrations of BAFF, CCL3, and TIMP-1 were statistically significant between the 2 groups (P ≤ 0.05). Our results indicate that HIV patients with CAP have a different inflammatory pattern and lower lung function compared to HIV patients without CAP. BAFF, CCL3, and TIMP-1 were abnormally elevated in HIV patients with CAP. Future studies with larger cohorts are required to verify these results. In addition, further investigation is required to determine if BAFF, CCL3, and TIMP-1 play a role in the process causing pulmonary dysfunction.