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PubMed2026

Application of metagenomic next-generation sequencing in children with pneumonia of unknown etiology.

OBJECTIVE: To investigate the pathogen spectrum and clinical application value of metagenomic next-generation sequencing (mNGS) in lower respiratory tract specimens from children with pneumonia of unknown etiology. METHODS: A retrospective analysis was conducted on children hospitalized in the intensive care unit (ICU) and respiratory department ward of Children's Hospital of Chongqing Medical University from January 2025 to December 2025. All enrolled cases presented negative results for conventional respiratory pathogen tests and received mNGS testing of lower respiratory tract specimens for etiological identification. The mNGS findings and clinical data of the included children were analyzed. RESULTS: A total of 92 children were enrolled, including 54 males and 38 females, with ages ranging from 2 months to 13 years and 8 months. Causative pathogens were detected in 77 cases (83.7%). The clinically adjudicated etiological diagnosis rates of bacteria, viruses, fungi and atypical pathogens were 75.0% (69/92), 37.0% (34/92), 13.0% (12/92) and 5.4% (5/92), respectively. Thirty-eight cases were complicated with polymicrobial infection, among which bacterial-viral infection was predominant, accounting for 23.1% (24/92). Children with immunocompromised conditions exhibited higher incidences of clinically adjudicated bacterial, fungal and polymicrobial infection than immunocompetent patients. The most common clinically confirmed causative pathogens in immunocompromised children were Streptococcus pneumoniae, human cytomegalovirus, Haemophilus influenzae, Stenotrophomonas maltophilia and Enterococcus faecalis. Treatment regimens were adjusted in 58 cases (63.0%) based on mNGS findings, switching to pathogen-targeted anti-infective therapy. CONCLUSION: For pediatric pneumonia with negative conventional etiological tests, mNGS of lower respiratory tract specimens significantly enhances pathogen detection rates, effectively identifies polymicrobial infection and opportunistic pathogens. Immune status serves as a critical stratification factor influencing pathogen spectrum and infection patterns, with immunocompromised children being more susceptible to opportunistic infections. Adjustment of anti-infective regimens based on mNGS results can effectively facilitate personalized anti-infective therapy.

Humans↗
PubMed1979

Distribution of hemolytic streptococci in respiratory specimens.

One hundred thirty-seven isolates of beta-hemolytic streptococci were recovered from 623 pharyngeal cultures. Twenty-nine percent of these were group A, 10% were group B, 31% were group C, 11% were group F, 12% were group G, and 7% could not be grouped. The significance of non-group A isolates in pharyngitis could not be evaluated in the absence of viral and serological studies. Hemolytic streptococci were recovered from 9% of 799 lower respiratory cultures. All except one were non-group A, and other potential respiratory pathogens were also present in these specimens. It is our impression that the presence of hemolytic streptococci in lower respiratory tract specimens usually represents pharnygeal contamination.

Adult↗
PubMed2025

Performance evaluation of a commercial multiplex pathogen panel for detection of bacteria in sputum specimens from non-ICU patients with suspected lower respiratory tract infection.

Rapid diagnostic testing can improve pathogen detection and lead to targeted antibiotics. The BioFire FilmArray Pneumonia Panel (BFPP) is a multiplex PCR that has displayed strong concordance with traditional microbiologic techniques. However, most existing literature focuses on deep respiratory specimens, and there is sparse literature on performance in sputum specimens. This retrospective, single-center study included adult patients between 1 September 2022 and 31 August 2024 who had collection of a BFPP with standard of care (SOC) culture from a sputum specimen on a non-intensive care unit (ICU) floor or in the emergency department if admitted to a non-ICU floor. Out of 189 BFPPs performed on 189 sputum specimens, a total of 141 bacterial targets were detected. Between the BFPP and SOC culture, the overall positive percent agreement and negative percent agreement (NPA) were 96.3% and 54.9%, respectively. The positive predictive value (PPV) was 26.3% while the negative predictive value was 98.9%. Patients with greater than 24 h of antibiotic exposure prior to BFPP collection had a lower PPV compared to patients with less than 24 h or no exposure (13.6% vs 29.6% vs 30.4%). The lowest concordance was observed for Haemophilus influenzae (15.4%), Moraxella catarrhalis (18.2%), Streptococcus pneumoniae (19%), and Staphylococcus aureus (22.7%), several of which are fastidious in culture. BFPP showed a high NPA, with all bacterial targets having an NPA greater than 90%, except H. influenzae (82%). Based on these data, a negative BFPP in sputum specimens could help to rule out a bacterial pneumonia, but the benefit of a positive test remains unclear.IMPORTANCEThis study evaluates the BioFire FilmArray Pneumonia Panel (BFPP) by comparing its performance to standard of care cultures exclusively in sputum specimens from non-intensive care unit patients with suspected lower respiratory tract infection. Findings show an overall high positive percent agreement and negative predictive value but a low negative percent agreement and positive predictive value, suggesting that a negative test in sputum specimens could be beneficial when attempting to rule out a bacterial infection, but the benefit of a positive test remains unclear, particularly if common airway colonizing bacteria are detected and at low semi-quantitative thresholds. Clinical symptoms should guide test interpretation in patients with positive BFPP results but negative culture growth.

Humans↗
PubMed2025

Rapid pan-microbial metagenomics for pathogen detection and personalised therapy in the intensive care unit: a single-centre prospective observational study.

BACKGROUND: Most clinical metagenomic studies do not provide rapid results, detect pathogens from all microbial kingdoms, or measure clinical impacts. We aimed to evaluate the feasibility, performance, and clinical impacts of a rapid pan-microbial respiratory metagenomic service for patients admitted to intensive care units (ICUs). METHODS: This was a single-centre observational study of a rapid metagenomics service that tests respiratory samples from ICU patients at Guy's and St Thomas' hospitals, London, UK, between Dec 5, 2023, and April 12, 2024. Testing used a previously published pan-microbial metagenomics workflow, which simultaneously detects bacteria, fungi, and DNA and RNA viruses; provides same-day preliminary results after 2 h; and provides final results after 24 h. Patients were included if they were aged 18 years or older, admitted to the ICU, had confirmed respiratory failure requiring supplemental oxygen or advanced airway support, and had at least one of the following: (1) clinical suspicion of lower respiratory tract infection based on clinical, biochemical, or radiological findings, (2) sepsis of unknown origin, and (3) concern from an intensive care physician regarding inflammatory pathology. Patients with a suspected or confirmed containment level three organism were excluded. The outcome was performance characteristics of the metagenomic test compared with routine diagnostic testing, detection of additional pathogens by metagenomics, change in antimicrobial prescribing within 24 h of testing, and initiation of immunomodulation. FINDINGS: We processed 114 samples (1-5 per day) from 74 patients (39 [53%] female and 35 [47%] male). 107 (94%) of 114 samples passed quality control, of which 101 (94%) provided same-day preliminary results. Bacteria were detected in 45 (43%) of 104 tested specimens, fungal organisms in 17 (16%) of 104 tested specimens, and viruses in 28 (34%) of 83 tested specimens. Sensitivity in lower respiratory tract samples after 24 h was 97% (95% CI 87-100) for bacteria, 89% (65-99) for fungi, and 89% (71-98) for viruses, with only one false positive for bacteria. Metagenomics identified 42 pathogens not detected by other tests in 32 (30%) of 107 samples. Antimicrobial therapy was changed after metagenomic results from 30 (28%) of 107 samples: 22 (21%) were de-escalated and eight (7%) were escalated. Metagenomics contributed to the initiation of immunomodulation in 15 (20%) of 74 patients for a range of inflammatory conditions. Pathogens with clinical significance to local infection control or national public health were found in ten (14%) of 74 patients, including three invasive Group A streptococci, two parvovirus B19, and one each of HIV-1, measles virus, Mycobacterium tuberculosis, Neisseria meningitidis, and Mycoplasma pneumoniae. INTERPRETATION: Respiratory metagenomics for ICU patients showed good performance and turnaround time, and diverse clinical and public health benefits. This ability to inform both personalised patient therapy and infectious disease surveillance needs evaluation in multicentre studies. FUNDING: None.

Humans↗
PubMed1978

Bacteriology of expectorated sputum with quantitative culture and wash technique compared to transtracheal aspirates.

Techniques to improve the reliability of expectorated sputum culture were evaluated in 46 patients using a wash technique, quantitative culture, and a combination of washing and quantification. The wash procedure consisted of a jet of tap water over the specimen contained in a tea strainer. The true lower respiratory tract flora was determined by percutaneous transtracheal aspirates, which served as the basis for evaluating the methods of processing expectorated specimens. Either washing alone or quantification alone decreased the number of specimens with organisms that were not present in companion transtracheal aspirates; however, results that were sufficiently improved to be clearly advantageous for clinical interpretation were achieved only when washing and quantification were combined. Washed sputum specimens yielded oropharyngeal "contaminants" in concentrations exceeding 10(6) bacteria per ml in 12 of 46 specimens (26 per cent). Using this technique, only one specimen contained a misleading potential pathogen, and only one specimen failed to yield a potential pathogen that was recovered with the transtracheal aspirate. The wash procedure decreased the mean concentrations of contaminants approximately 100-fold for all specimens and 1,000-fold for purulent specimens.

Anaerobiosis↗
PubMed1977

Fiberoptic bronchoscopy and culture bacteria from the lower respiratory.

Forty-five specimens were obtained by sequential translaryngeal aspiration and fiberoptic bronchoscopy from 31 clinically unifected patients with lung cancer in order to evaluate the reliability of routine fiberoptic bronchoscopy for culture of the lower respiratory tract. Bacteria were recovered brom 98 percent (44) of the specimens obtained via fiberoptic bronchoscopy and from 58 percent (26) of the specimens obtained by the preceding translaryngeal aspiration. The microorganisms grown from cultures of specimens obtained by fiberoptic bronchoscopy consisted of mixtures of both nonpathogenic and potenitally pathogenic bacteria. Potentially pathogenic bacteria were present in 87 percent (39) of the specimens from fiberoptic bronchoscopy and 31 percent (14) of specimens from translayngeal aspiration. The results of cultures from the two procedures agreed completely in only a single instance. Culture of washings or secretions obtained by routine fiberoptic bronchoscopy is not recommended because it provides inaccurate and clinically confusing information about the presence or types of bacteria in the lower respiratory tract prior to instrumentation.

Aged↗
PubMed1976

Should fiberoptic bronchoscopy aspirates be cultured?

The reliability of fiberoptic bronchoscopy as a method to study the bacteriology of the lower respiratory tract was tested. The procedure used was suction aspiration through the inner channel after topical anesthesia with lidocaine. To detect contamination by oropharyngeal bacteria, the aspirates were cultured in patients with no evidence of active infection, comparison was made with results of transtracheal aspiration cultures, and the aspirate was tested for the presence of an oral dye marker. Results with all 3 methods of analysis indicated contamination with oropharyngeal bacteria that were presumably introduced during instrumentation through the upper airways. An additional factor studied was the effect of topical anesthetics. Analysis of aspirates showed that as much as 96 per cent of the specimen was anesthetic solution. Lidocaine also proved toxic to lower respiratory tract pathogens, although there were significant differences between bacterial species. It was concluded that fiberoptic bronchoscopy as performed in this study does not reliably reflect the bacteriology of the lower respiratory tract.

Anti-Bacterial Agents↗
PubMed1975

Clinically useful method for the isolation of respiratory syncytial virus.

A simple method for the isolation of respiratory syncytial virus (RSV) is reported; it is relatively rapid and results in a high frequency of recovery of virus. A nasal secretion specimen with high titers of virus is inoculated at the bedside onto susceptible cell lines to avoid loss of viral infectivity due to liability of the virus. During an outbreak of RSV, viral specimens were obtained by this method from all young children admitted to the hospital with lower respiratory tract disease. RSV or influenza A virus was recovered from 89% of these 45 children. RSV was isolated from 87% of those with pneumonia. RSV was recovered 60% less often from specimens obtained simultaneously by conventional nasopharyngeal swabs. Identification of RSV cytopathic effect was more rapid with use of the bedside nasal wash method and was accomplished in an average of four days. Hence, this information was available to the clinicician when it was still useful in the management of the patient's illness.

Animals↗
PubMed1977

A reappraisal of the ultrastructure of the human respiratory nasal mucosa.

A study of the ultrastructure of human respiratory nasal mucosa using specimens of simple mucous polyps showed features essentially identical to those described in the lower respiratory tract. Minor forms of epithelial specialization seen in the nasal lining of the other mammalian species are not demonstrable in man. Inflammatory cells, especially lymphocytes, form an integral constituent of the epithelium, and are in close apposition to the epithelial cells. A marked increase in the epithelial surface area is achieved through ridging and clefting, irregularity of the cell's surface and the presence of microvilli. Branching microvilli are admixed with more conventional structures.

Basement Membrane↗
PubMed1975

Quantitative shedding patterns of respiratory syncytial virus in infants.

Quantitative shedding patterns of respiratory syncytial virus in 40 infants hospitalized with acute disease of the lower respiratory tract were determined for elucidation of the pathophysiology of infection with the virus. Nasal wash specimens were collected on admission and daily thereafter and were tested for the presence and quantities of respiratory syncytial virus. The following pattern of shedding was observed. (1) The virus was shed for prolonged periods. For the first seven days of hospitalization, 92%-100% of the infants tested continued to shed virus. At discharge 87% were still shedding the virus. (2) Respiratory syncytial virus was present in high titer in the nasal secretions obtained at the time of admission. The mean titer in these samples was 5.0 log10 TCID50. (3) The titer of respiratory syncytial virus did not fall during the first few days of hospitalization, despite clinical improvement of the infants. Neither peak nor admission titers of virus could be correlated with age or with the severity of disease. However, the mean admission titer in patients with bronchiolitis appeared to be significantly higher than that in those with pneumonia.

Acute Disease↗
PubMed1979

Are anaerobic pleuropulmonary infections uncommon or commonly undetected in Britain?

Despite marked and increasing awareness of the clinical significance of pathogenic anaerobes, the role of anaerobes in pleuropulmonary infections is still largely unrecognised in Britain. A considerable literature testifies that this is not so in America. The reasons for this paradox are explored. British anaerobic methodology is briefly reviewed, with special reference to the range of recognised respiratory pathogens and the anaerobes that can be cultured in simple anaerobic jars. The limitations of sampling and transport procedures are considered. Differences in approach are discussed. The technological problems of the isolation and culture of "difficult" pathogenic anaerobes have been largely solved at the laboratory level; effective approaches to sampling and transit of specimens require more positive consideration and the results might influence our choice of specific antimicrobial therapy. If these points are taken and an increased awareness of the role of the anaerobes in certain pathological conditions of the lower respiratory tract is generated, we might hope either to produce a meaningful parallel to the experience of our American colleagues--or to structure a more valid defence to explain our lack of experience of these infections in the lower respiratory tract.

Bacterial Infections↗
PubMed1976

Interactions of Pseudomonas aeruginosa with immunoglobulins and complement in sputum.

The interactions of Pseudomonas aeruginosa with humoral factors in the sputum of patients with cystic fibrosis were investigated by using an indirect immunofluorescent technique. Fluorescein-conjugated, monovalent antiserum specific to heavy chains of human immunoglobulin A (IgA), IgG, or IgM and to complement C3 were used. All strains of P. aeruginosa recovered from the sputum specimens of patients with cystic fibrosis were found to be coated with antibodies of IgA, IgG, and IgM classes and with C3. The specificity of the antibody coating was determined. The fluorescence was most intense with IgA and was followed in intensity by IgG, IgM, and C3. No difference was noted between rough and mucoid strains of P. aeruginosa. When the subcultured P. aeruginosa was incubated with the sputum eluates, a similar pattern of fluorescence was demonstrated, indicating that these humoral factors are present in the sputum and that the coating process can take place in the lower respiratory tract of the patients. By single radial immunodiffusion, significant quantities of the humoral factors in the sputum eluates were detected. These findings suggest that P. aeruginosa is opsonized in sputum of patients with cystic fibrosis.

Adolescent↗
PubMed1976

Respiratory syncytial virus infections in infants: quantitation and duration of shedding.

Infants hospitalized with respiratory syncytial virus infection were studied to delineate the quantitative shedding patterns and duration of shedding of RSV. Nasal wash specimens collected daily from 19 infants contained a mean maximal titer of 4.34 log10 50% tissue culture infective doses per milliliter. On admission, the mean titer was 4.14 log10 TCID50, with no consistent decline until after Day 6. The mean duration of shedding for 23 patients until they were virus negative was 6.7 days with a range of 1 to 21 days. Quantities of RSV shed were significantly greater in infants less than one month of age and in infants with evidence of pulmonary consolidation on chest roentgenogram. Shedding extended for a significantly longer time in infants with lower respiratory tract disease than in those with clinical manifestations limited to the upper respiratory tract.

Child, Preschool↗
PubMed1979

A fiberoptic bronchoscopy technique to obtain uncontaminated lower airway secretions for bacterial culture.

Seven types of brush catheter were studied in vitro to determine the optimal catheter design for obtaining specimens for bacterial culture using fiberoptic bronchoscopy. The various catheters were inserted through the inner channel, which was contaminated with fresh saliva. The specimen was then obtained by inserting the brush into a broth culture of a marker organism at the distal end of the bronchoscope. Relative merits were based on quantitative bacterial counts of salivary "contaminants" and the marker organism. The catheter that proved superior had telescoping cannulas with a distal occlusion. Twenty-six samplings using catheters with this design uniformly showed no contaminants and high counts of the marker organism. The catheter with telescoping cannulas and a distal occlusion composed of polyethlene glycol was then tested with bronchoscopy performed on 8 healthy volunteers and 6 patients with clinical evidence of a lower airway infection. The specimens yielded likely pulmonary pathogens in high concentrations from patients with pneumonia or lung abscess. Possible contaminants were recovered in low concentrations from 2 of 14 subjects. These in vitro studies and the preliminary clinical trial support the use of this type of catheter for obtaining bronchoscopic specimens for bacterial culture.

Bacteria↗
PubMed1978

A study of acute respiratory disease in the community of Port Chalmers. I. Illnesses within a group of selected families and the relative incidence of respiratory pathogens in the whole community.

A study of respiratory diseases in the semi-isolated community of Port Chamlers, New Zealand, began in April 1973. The intensive surveillance of a selected group fo 26 families involved the weekly reporting of illness, the collection of specimens for virus, Group A streptococci and Mycoplasma pneumoniae isolation and the collection of sera at 6-month intervals. A total of 956 illnesses were reported during 32 months. The median number of illnesses per year were: infants 4.4, children 2.5, female adults 2.4 and male adults 2.0. Of all these illnesses, 57% were upper respiratory, 31% were lower respiratory and 9% were enteric. The severity of these illnesses was not greater than would be expected in open communities. Surveillance by pathogen isolation only of the whole community through the patients in the general practice was carried out concurrently. A total of 640 nasopharyngeal swab specimens were collected from which 161 viruses, 47 Group A streptococci and 2 M. pneumoniae were isolated. The overall isolation rate was 33%. The similarities between the epidemiological patterns of respiratory disease in the open community and the isolated community are discussed.

Adolescent↗
PubMed1976

Factors influencing the establishment of the normal values of the respiratory activities of human liver mitochondria.

Some factors that influence the values of respiratory activities of liver mitochondria isolated from surgical biopsy specimens have been studied. By sedimentating of mitochondria at a lower centrifugal force (5,500 g) than usually used for rat liver mitochondria, and washing the mitochondrial pellet twice, the contamination with lysosomes and microsomes was lowered. At 37 degrees C, and in the presence of hexokinase and glucose, the oxygen uptake was greater than at 25 degrees C and in their absence. The respiratory control was good and the respiratory activities were rather stable during the first 3-4 h after isolation. The respiratory activities of mitochondria isolated from patients with duodenal or gastric ulcers, biliary diseases, and subjects with no digestive diseases (all having normal liver) were compared. Differences in oxygen uptake and acceptor control index values with some substrates were noted. The conditions for selection of controls in studies on subcellular fractions of human liver include: absence of any hepatic antecedents; no clinical evidence of liver involvement; no abnormality in routine liver function tests; a histologic aspect free of pathological conditions, and a normal aspect of the tissue during the homogenization and the fractionation procedure (absence of steatosis or fibrosis). These data provide a basis for the standardization of methods in establishing the reference values of mitochondrial activities for the modifications in a variety of diseases.

Acid Phosphatase↗