Acquisition and Development of the Extremely Preterm Infant Microbiota Across Multiple Anatomical Sites.

OBJECTIVES: Microbial communities influencing health and disease are being increasingly studied in preterm neonates. There exists little data, however, detailing longitudinal microbial acquisition, especially in the most extremely preterm (<26 weeks' gestation). This study aims to characterize the development of the microbiota in this previously under-represented cohort. METHODS: Seven extremely preterm infant-mother dyads (mean gestation 23.6 weeks) were recruited from a single neonatal intensive care unit. Oral and endotracheal secretions, stool, and breast milk (n = 157 total), were collected over the first 60 days of life. Targeted 16S rRNA gene sequencing identified bacterial communities present. RESULTS: Microbiota of all body sites were most similar immediately following birth and diverged longitudinally. Throughout the sampling period Escherichia, Enterococcus, Staphylococcus, and an Enterobacteriaceae were dominant and well dispersed across all sites. Temporal divergence of the stool from other microbiota was driven by decreasing diversity and significantly greater proportional abundance of Bifidobacteriaceae compared to other sites. CONCLUSIONS: Four taxa dominated all anatomical sampling sites. Rare taxa promoted dissimilarity. Cross-seeding between upstream communities and the stool was demonstrated, possibly relating to buccal colostrum/breast milk exposure and indwelling tubes. Given the importance of dysbiosis in health and disease of extremely preterm infants, better understanding of microbial acquisition within this context may be of clinical benefit.

Comparison of in situ sequence type analysis of Legionella pneumophila in respiratory tract secretions and environmental samples of a hospital in East Jerusalem.

Legionella pneumophila genotyping is important for epidemiological investigation of nosocomial and community-acquired outbreaks of legionellosis. The prevalence of legionellosis in pneumonia patients in the West Bank was monitored for the first time, and the sequence types (STs) from respiratory samples were compared with STs of environmental samples from different wards of the hospital. Sputum (n = 121) and bronchoalveolar lavage (BAL) (n = 74) specimens were cultured for L. pneumophila; genomic DNA was tested by 16S rRNA polymerase chain reaction (PCR) amplification. Nested PCR sequence-based typing (NPSBT) was implemented on DNA of the respiratory and environmental PCR-positive samples. Only one respiratory specimen was positive for L. pneumophila by culture. BAL gave a higher percentage of L. pneumophila-positive samples, 35% (26/74) than sputum, 15% (18/121) by PCR. NPSBT revealed the following STs: ST 1 (29%, 7/24), ST 461 (21%, 5/24), ST 1037 (4%, 1/24) from respiratory samples, STs from environmental samples: ST 1 (28.5%, 4/14), ST 187 (21.4%, 3/14) and ST 2070, ST 461, ST 1482 (7.1%, 1/14) each. This study emphasises the advantage of PCR over culture for the detection of L. pneumophila in countries where antibiotics are indiscriminately used prior to hospital admission. ST 1 was the predominant ST in both respiratory and environmental samples.

Nasal PCR assay for the detection of Mycobacterium leprae pra gene to study subclinical infection in a community.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Identification of Mycobacterium leprae is difficult in part due to the inability of the leprosy bacillus to grow in vitro. A number of diagnostic methods for leprosy diagnosis have been proposed. Both serological tests and molecular probes have shown certain potential for detection and identification of Mycobacterium leprae in patients. In this study, we have investigated whether Mycobacterium leprae DNA from the nasal secretion of healthy household contacts and the non contacts could be detected through PCR amplification as a method to study the sub clinical infection in a community. A total of 200 samples, 100 each from contacts and non contacts representing all age groups and sex were included in this study. The M. leprae specific primer (proline-rich region) of pra gene was selected and PCR was performed using extracted DNA from the sample. A total of 13 samples were found to be positive for nasal PCR for pra gene among the male and female contacts out of which 7% were males and 6% were females. Even though several diagnostic tools are available to detect the cases of leprosy, they lack the specificity and sensitivity. PCR technology has demonstrated the improved diagnostic accuracy for epidemiological studies and requires minimal time. Although nasal PCR studies have been reported from many countries it is not usually recommended due to the high percentage of negative results in the contact.

Antifungal activity of the pygidial gland secretion of Laemostenus punctatus (Coleoptera: Carabidae) against cave-dwelling micromycetes.

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.

Performance of Xpert MTB/RIF and Alternative Specimen Collection Methods for the Diagnosis of Tuberculosis in HIV-Infected Children.

BACKGROUND: The diagnosis of tuberculosis in human immunodeficiency virus (HIV)-infected children is challenging. We assessed the performance of alternative specimen collection methods for tuberculosis diagnosis in HIV-infected children using Xpert MTB/RIF (Xpert). METHODS: HIV-infected children aged ≤13 years with suspected intrathoracic tuberculosis were enrolled in 8 hospitals in Burkina Faso, Cambodia, Cameroon, and Vietnam. Gastric aspirates were taken for children aged <10 years and expectorated sputum samples were taken for children aged ≥10 years (standard samples); nasopharyngeal aspirate and stool were taken for all children, and a string test was performed if the child was aged ≥4 years (alternative samples). All samples were tested with Xpert. The diagnostic accuracy of Xpert for culture-confirmed tuberculosis was analyzed in intention-to-diagnose and per-protocol approaches. RESULTS: Of 281 children enrolled, 272 (96.8%) had ≥1 specimen tested with Xpert (intention-to-diagnose population), and 179 (63.5%) had all samples tested with Xpert (per-protocol population). Tuberculosis was culture-confirmed in 29/272 (10.7%) children. Intention-to-diagnose sensitivities of Xpert performed on all, standard, and alternative samples were 79.3% (95% confidence interval [CI], 60.3-92.0), 72.4% (95% CI, 52.8-87.3), and 75.9% (95% CI, 56.5-89.7), respectively. Specificities were ≥97.5%. Xpert combined on nasopharyngeal aspirate and stool had intention-to-diagnose and per-protocol sensitivities of 75.9% (95% CI, 56.5-89.7) and 75.0% (95% CI, 47.6-92.7), respectively. CONCLUSIONS: The combination of nasopharyngeal aspirate and stool sample is a promising alternative to methods usually recommended by national programs. Xpert performed on respiratory and stools samples enables rapid confirmation of tuberculosis diagnosis in HIV-infected children. CLINICAL TRIALS REGISTRATION: The ANRS (Agence Nationale de Recherche sur le Sida) 12229 PAANTHER (Pediatric Asian African Network for Tuberculosis and HIV Research) 01 study is registered at ClinicalTrials.gov (NCT01331811).

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens.

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resource-poor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n = 252) and unextracted respiratory specimens (n = 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community.

Sputum and bronchial secretion samples are equally useful as bronchoalveolar lavage samples for the diagnosis of invasive pulmonary aspergillosis in selected patients.

In the absence of histopathology studies of lung biopsies, the bronchoalveolar lavage (BAL) sample is preferred for the diagnosis of invasive pulmonary aspergillosis. Isolation of Aspergillus fumigatus from sputum and bronchial secretion samples are commonly interpreted as colonization or laboratory contamination, particularly in nonneutropenic patients. We studied if sputum/bronchial secretions and BAL samples are equally useful for the diagnosis of invasive pulmonary aspergillosis. We retrospectively selected 14 patients with proven (n = 1) or probable (n = 13) invasive pulmonary aspergillosis from whose samples A. fumigatus had been simultaneously isolated in BAL and sputum/bronchial secretions between 2006 and 2012. The isolates were identified by sequencing the ß-tubulin gene and genotyped using the STRAf assay. Matches between BAL and sputum/bronchial secretions were observed in patients with identical genotypes in BAL and sputum/bronchial secretions. All patients had clinically suspected pneumonia, before the diagnosis of invasive pulmonary aspergillosis. The sample from which A. fumigatus was initially isolated was collected as a result of the presence of fever (50%), abnormal radiological findings (100%), and/or pneumonia that did not respond to antibiotics (36%). The underlying conditions varied, although the most common predisposing factors were hematological malignancies (21.5%) and COPD (43%). In 13 of the 14 patients (93%), we found matching genotypes in the BAL and the sputum/bronchial secretion samples. Genotyping showed that samples of sputum or bronchial secretions were equally useful as samples of BAL for the diagnosis of invasive pulmonary aspergillosis.

Evaluation of two commercial real-time PCR assays for detection of Mycoplasma genitalium in urogenital specimens.

The performance of two commercial real-time PCR kits for the detection of Mycoplasma genitalium was evaluated in comparison to an in-house real-time PCR assay. Concordances of 96% and 93% were found for the TIB MOLBIOL and the Diagenode assays, respectively, compared to the results of the in-house assay.

Detection and characterization of Mycoplasma pneumoniae during an outbreak of respiratory illness at a university.

An outbreak at a university in Georgia was identified after 83 cases of probable pneumonia were reported among students. Respiratory specimens were obtained from 21 students for the outbreak investigation. The TaqMan array card (TAC), a quantitative PCR (qPCR)-based multipathogen detection technology, was used to initially identify Mycoplasma pneumoniae as the causative agent in this outbreak. TAC demonstrated 100% diagnostic specificity and sensitivity compared to those of the multiplex qPCR assay for this agent. All M. pneumoniae specimens (n=12) and isolates (n=10) were found through genetic analysis to be susceptible to macrolide antibiotics. The strain diversity of M. pneumoniae associated with this outbreak setting was identified using a variety of molecular typing procedures, resulting in two P1 genotypes (types 1 [60%] and 2 [40%]) and seven different multilocus variable-number tandem-repeat analysis (MLVA) profiles. Continued molecular typing of this organism, particularly during outbreaks, may enhance the current understanding of the epidemiology of M. pneumoniae and may ultimately lead to a more effective public health response.

Metagenomic sequencing of expressed prostate secretions.

To investigate which microorganisms may be present in expressed prostate secretions (EPS) metagenomic sequencing (MGS) was applied to prostate secretion samples from five men with prostatitis and five matched control men as well as to combined expressed prostate secretion and urine from six patients with prostate cancer and six matched control men. The prostate secretion samples contained a variety of bacterial sequences, mostly belonging to the Proteobacteria phylum. The combined prostate secretion and urine samples were dominated by abundant presence of the JC polyomavirus, representing >20% of all detected metagenomic sequence reads. There were also other viruses detected, for example, human papillomavirus type 81. All combined prostate secretion and urine samples were also positive for Proteobacteria. In summary, MGS of expressed prostate secretion is informative for detecting a variety of bacteria and viruses, suggesting that a more large-scale use of MGS of prostate secretions may be useful in medical and epidemiological studies of prostate infections.

Special structures of hoopoe eggshells enhance the adhesion of symbiont-carrying uropygial secretion that increase hatching success.

Animals live in a bacterial world, and detecting and exploring adaptations favouring mutualistic relationships with antibiotic-producing bacteria as a strategy to fight pathogens are of prime importance for evolutionary ecologists. Uropygial secretion of European hoopoes (Upupa epops, Linnaeus) contains antimicrobials from mutualistic bacteria that may be used to prevent embryo infection. Here, we investigated the microscopic structure of hoopoe eggshells looking for special features favouring the adhesion of antimicrobial uropygial secretions. We impeded female access to the uropygial gland and compared microscopic characteristics of eggshells, bacterial loads of eggs and of uropygial secretion, and hatching success of experimental and control females. Then, we explored the link between microbiological characteristics of uropygial secretion and these of eggs of hoopoes, as well as possible fitness benefits. The microscopic study revealed special structures in hoopoes' eggshells (crypts). The experimental prevention of females' gland access demonstrated that crypts are filled with uropygial secretion and that symbiotic enterococci bacteria on the eggshells come, at least partially, from those in the female's uropygial gland. Moreover, the experiment resulted in a higher permeability of eggshells by several groups of bacteria and in elimination of the positive relationships detected for control nests between hatching success and density of symbiotic bacteria, either in the uropygial secretion of females or on the eggshell. The findings of specialized crypts on the eggshells of hoopoes, and of video-recorded females smearing secretion containing symbiotic bacteria at a high density onto the eggshells strongly support a link between secretion and bacteria on eggs. Moreover, the detected associations between bacteria and hatching success suggest that crypts enhancing the adhesion of symbiont-carrying uropygial secretion likely protect embryos against infections.

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR.

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision.

[Application of FilmArray assay for detection of respiratory tract infections in immunocompromised persons]. / Wykorzystanie techniki FilmArray w diagnostyce zakazen dróg oddechowych u osób z niedoborami odpornosci.

INTRODUCTION: A variety of viruses and bacteria are responsible for acute upper and lower respiratory tract infections worldwide. Severe and even fatal disease can occur especially in group ofimmunocompromised individuals. Accurate pathogen identification allows clinicians to determine the need for ancillary diagnostic testing, antibacterial and/or antiviral therapy and can motivate decisions regarding hospitalization and infection control measures. METHODS: We compared the diagnostic performance of FilmArray Respiratory Panel highly multiplexed nucleic acid amplification test with previous used direct immunofluorescence assay. Both assays were performed on a panel of 6 nasopharyngeal-secretion specimens and 6 BALF samples, collected from 12 patients, subjected to allogeneic haematological stem cells transplantation, with lower respiratory tract symptoms. RESULTS AND CONCLUSIONS: Among viruses detectable by both assays were especially influenzaA virus, parainfluenza viruses type 3 and respiratory syncytial virus. In conclusion, the FilmArray assay is rapid and extremely user-friendly system, with results available in just over one hour with almost no labor involved. In few laboratories its low throughput and qualitative results may be a disadvantage in some clinical settings.

Specimen collection for the diagnosis of pediatric pneumonia.

Diagnosing the etiologic agent of pneumonia has an essential role in ensuring the most appropriate and effective therapy for individual patients and is critical to guiding the development of treatment and prevention strategies. However, establishing the etiology of pneumonia remains challenging because of the relative inaccessibility of the infected tissue and the difficulty in obtaining samples without contamination by upper respiratory tract secretions. Here, we review the published and unpublished literature on various specimens available for the diagnosis of pediatric pneumonia. We discuss the advantages and limitations of each specimen, and discuss the rationale for the specimens to be collected for the Pneumonia Etiology Research for Child Health study.

Recovery of Gram-negative bacilli in stored endotracheal aspirates.

This study assessed the recovery rates of Gram-negative bacilli from stored endotracheal aspirates frozen with and without glycerol. Samples frozen with glycerol showed a significant difference in isolate recovery, 89.7% versus 69.2% (P = 0.02). This study demonstrates that it is possible to achieve high recovery rates of potentially pathogenic organisms from endotracheal aspirates when stored with glycerol, thus broadening the scope of active surveillance cultures for both clinical and research purposes.

Alternative sampling methods for detecting bacterial pathogens in children with upper respiratory tract infections.

Nasopharyngeal sampling is used for detecting bacteria commonly involved in upper respiratory tract infections, but it requires training and may not always be well tolerated. We sampled children (n = 66) of ages 0 to 4 years, with rhinorrhea, by using a nasopharyngeal swab, a nasal swab, and nose blowing/wiping into a paper tissue. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus were cultured at similar rates across methods with high concordance (80 to 97%), indicating that they are reliably detected by alternative means.

Rapid detection of Staphylococcus aureus in lower respiratory tract secretions from patients with suspected ventilator-associated pneumonia: evaluation of the Cepheid Xpert MRSA/SA SSTI assay.

A preclinical evaluation was conducted to evaluate the performance of the Cepheid Xpert assay on 135 lower respiratory tract secretions for detection of methicillin-resistant Staphylococcus aureus and S. aureus. Compared with the quantitative culture, the sensitivity, specificity, and positive and negative predictive values were 99.0%, 72.2%, 90.7%, and 96.3%, respectively.

Evaluation of the Cobas TaqMan MTB test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

The Cobas TaqMan MTB test, based on real-time PCR technology, was evaluated for direct detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. A total of 1,093 samples from 446 patients, including 118 acid-fast smear-positive and 975 acid-fast smear-negative specimens, were investigated. Diagnostic cultures performed with 7H11 agar, Löwenstein-Jensen medium, and the Bactec MGIT 960 system were considered the reference methods. When discrepant results between the Cobas TaqMan MTB test and culture occurred, additional results from the BD MGIT TBc identification test and the GenoType Mycobacterium CM test performed on growth-positive and acid-fast-stain-positive MGIT tubes and review of the patient's medical history were used for discrepancy analysis. The overall sensitivity, specificity, positive predictive value, and negative predictive value for the Cobas TaqMan MTB test were 91.5%, 98.7%, 91.5%, and 98.7%, respectively. In general, the performance of the new Cobas TaqMan MTB test was comparable to that of the replaced Cobas Amplicor MTB system. The most prominent feature of the new system was its extraordinarily high sensitivity (79.5%) for detecting MTBC in smear-negative specimens; out of 44 smear-negative but culture-positive specimens, 35 were positive by the new system. The Cobas TaqMan MTB assay, including DNA extraction, can be completed within 3 h.

Evaluation of the Bactec MGIT 960 system in combination with the MGIT TBc identification test for detection of Mycobacterium tuberculosis complex in respiratory specimens.

The sensitivity and specificity of the MGIT TBc identification (TBc ID) test for Mycobacterium tuberculosis complex (MTC) detection in positive Bactec MGIT cultures were 95.2% and 99.2%, respectively. When MTC-positive results obtained from two additional molecular methods were included, the sensitivity of the MGIT TBc ID test was 85.4%, while that of culture was 95.7%.