Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives.

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

A role for human leucocyte antigens in the susceptibility to SARS-Cov-2 infection observed in transplant patients.

We analysed data from 80 patients who tested positive for SARS-CoV-2 RNA who had previously been HLA typed to support transplantation. Data were combined from two adjacent centres in Manchester and Leeds to achieve a sufficient number for early analysis. HLA frequencies observed were compared against two control populations: first, against published frequencies in a UK deceased donor population (n = 10,000) representing the target population of the virus, and second, using a cohort of individuals from the combined transplant waiting lists of both centres (n = 308), representing a comparator group of unaffected individuals of the same demographic. We report a significant HLA association with HLA- DQB1*06 (53% vs. 36%; p < .012; OR 1.96; 95% CI 1.94-3.22) and infection. A bias towards an increased representation of HLA-A*26, HLA-DRB1*15, HLA-DRB1*10 and DRB1*11 was also noted but these were either only significant using the UK donor controls, or did not remain significant after correction for multiple tests. Likewise, HLA-A*02, HLA-B*44 and HLA-C*05 may exert a protective effect, but these associations did not remain significant after correction for multiple tests. This is relevant information for the clinical management of patients in the setting of the current SARS-CoV-2 pandemic and potentially in risk-assessing staff interactions with infected patients.

An emerging opportunistic infection: fatal astrovirus (VA1/HMO-C) encephalitis in a pediatric stem cell transplant recipient.

Neuroinvasive astrovirus (VA1-HMO-C) is an emerging life-threatening infection in immunocompromised hosts. We describe an 8-month-old child who died of VA1/HMO-C encephalitis following bone marrow transplantation. The diagnosis was only made post-mortem using RNA deep sequencing of the brain. Repeat analysis of the post-mortem brain tissue using polymerase chain reaction specific primers for VA1/HMO-C was positive. Astrovirus VA1/HMO-C should be included in the evaluation of patients with similar encephalitis.

Genome stability of adenovirus types 3 and 7 during a simultaneous outbreak in Greater Manchester, UK.

A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five genome regions. Of these, 62 isolates were HAdV-B3 and HAdV-B7 collected during a simultaneous 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 and other species B adenovirus types collected in different years following the outbreak. The phylogenetic analysis results of all the isolates in the structural regions hexon L2, penton, and fiber knob were found to be consistent and no mismatches were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable even when different types are circulating in a limited geographical area simultaneously.

Incidence and clinical impact of respiratory viruses in adults with cystic fibrosis.

BACKGROUND: Viral respiratory infection (VRI) is a common cause of pulmonary exacerbations in children with cystic fibrosis (CF). The importance of VRI in adult CF populations is unclear. OBJECTIVE: To determine the incidence and clinical impact of VRI among adults with CF. METHODS: One hundred adults with CF were followed up prospectively for 12 months. Sputum, nose swabs and throat swabs were collected every 2 months and at onset of pulmonary exacerbation. PCR assays for adenovirus, influenza A&B, human metapneumovirus, parainfluenza 1-3, respiratory syncytial virus and human rhinovirus were performed on each sample. Symptom scores, spirometry and inflammatory markers were measured at each visit. RESULTS: One or more respiratory viruses were detected in 191/626 (30.5%) visits. Human rhinovirus accounted for 72.5% of viruses. Overall incidence of VRI was 1.66 (95% CI 1.39 to 1.92) cases/patient-year. VRI was associated with increased risk of pulmonary exacerbation (OR=2.19; 95% CI 1.56 to 3.08; p<0.001) and prescription of antibiotics (OR=2.26; 95% CI 1.63 to 3.13; p<0.001). Virus-positive visits were associated with higher respiratory symptom scores and greater C-reactive protein levels. Virus-positive exacerbations had a lower acute fall in FEV1 than virus-negative exacerbations (12.7% vs 15.6%; p=0.040). The incidence of exacerbations, but not VRI, was associated with greater lung function decline over 12 months (-1.79% per pulmonary exacerbation/year; 95% CI -3.4 to -0.23; p=0.025). CONCLUSION: VRI is common in adults with CF and is associated with substantial morbidity. Respiratory viruses are a potential therapeutic target in CF lung disease.

Characterisation of acute respiratory infections at a United Kingdom paediatric teaching hospital: observational study assessing the impact of influenza A (2009 pdmH1N1) on predominant viral pathogens.

BACKGROUND: According to the World Health Organisation, influenza A (2009 pdmH1N1) has moved into the post-pandemic phase, but there were still high numbers of infections occurring in the United Kingdom in 2010-11. It is therefore important to examine the burden of acute respiratory infections at a large children's hospital to determine pathogen prevalence, occurrence of co-infection, prevalence of co-morbidities and diagnostic yield of sampling methods. METHODS: This was a retrospective study of respiratory virus aetiology in acute admissions to a paediatric teaching hospital in the North West of England between 1st April 2010 and 31st March 2011. Respiratory samples were analysed either with a rapid RSV test if the patient had symptoms suggestive of bronchiolitis, followed by multiplex PCR testing for ten respiratory viruses, or with multiplex PCR testing alone if the patient had suspected other ARI. Patient demographics and data regarding severity of illness, presence of co-morbidities and respiratory virus sampling method were retrieved from case notes. RESULTS: 645 patients were admitted during the study period. 82/645 (12.7%) patients were positive for 2009 pdmH1N1, of whom 24 (29.2%) required PICU admission, with 7.3% mortality rate. Viral co-infection occurred in 48/645 (7.4%) patients and was not associated with more severe disease. Co-morbidities were present more frequently in older children, but there was no significant difference in prevalence of co-morbidity between 2009 pdmH1N1 patients and those with other ARI. NPA samples had the highest diagnostic yield with 192/210 (91.4%) samples yielding an organism. CONCLUSIONS: Influenza A (2009 pdmH1N1) is an ongoing cause of occasionally severe disease affecting both healthy children and those with co-morbidities. Surveillance of viral pathogens provides valuable information on patterns of disease.

Evaluation of a custom designed hybridisation assay for whole genome sequencing of human adenoviruses direct from clinical samples.

BACKGROUND: Human Adenoviruses are a common cause of disease and can cause significant morbidity and mortality in immunocompromised patients. Nosocomial transmission events can occur with whole genome sequencing playing a crucial role. This study evaluates the performance of a custom designed SureSelectXT target enrichment assay based on 14 adenovirus genomes for sequencing direct from clinical samples. METHODS: Modifications were made to the SureSelectXT low input protocol to enhance performance for viral targets. Consensus sequences were generated using an in-house designed three stage bioinformatics pipeline. We assessed, percentage of on target reads, average depth of coverage and percentage genome coverage to determine assay performance across a range of sample matrices. RESULTS: Whole genome sequences were successfully generated for 91.6 % of samples assessed. Adenovirus DNA concentration was a good indicator of enrichment success. Highly specific enrichment was observed with only 6 % of samples showing < 50 % on target reads. Respiratory and faecal samples performed well where bloods showed higher levels of non-specific enrichment likely confounded by low adenovirus DNA concentrations. Protocol performance did not appear impacted by Adenovirus type or species. CONCLUSION: Overall performance of this modified SureSelectXT protocol appears in line with previously published works although there are some confounding factors requiring further investigation. The use of a small RNA bait set has the potential to reduce associated costs which can be prohibitive.

Chronic rhinovirus infection in an adult with cystic fibrosis.

Rhinovirus is a common cause of exacerbations of cystic fibrosis (CF) and is usually considered a self-limiting infection. We report a case of chronic infection with rhinovirus A type 33 in a 43-year-old male with CF which has persisted for over 2 years.

Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.

In this study, real-time PCR was used to quantify adenovirus DNA in cell suspensions prepared from 106 right and left tonsils and 10 adenoids obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty-four (72.4%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load ranged from 2.8 × 10(2) to 2.6 × 10(6) copies/10(7) cells and varied up to sixfold between the right and left tonsils. In some cases, only one tonsil was positive and the viral load was lower in older children. Seventy-eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C (types 1, 2, and 5) were detected in 84.1% of the patients followed by types 3 and 7 of species B (6.8%), HAdV-E4 (6.8%), and HAdV-F41 (2.3%). In one patient adenovirus C2 was found in the left tonsil and adenovirus C5 in the right tonsil. No DNA methylation was detected in either the E1A promoter or the major late promoter region of adenovirus DNA from six tonsils and adenoids samples and two clinical isolates.

Epstein-barr virus coinfection in cerebrospinal fluid is associated with increased mortality in Malawian adults with bacterial meningitis.

Mortality from adult bacterial meningitis exceeds 50% in sub-Saharan Africa. We postulated that-particularly in individuals infected with human immunodeficiency virus (HIV)-herpes simplex virus, varicella zoster virus, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in the cerebrospinal fluid (CSF) contribute to poor outcome. CSF from 149 Malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction. EBV was detected in 79 of 149 bacterial meningitis patients. Mortality (54%) was associated with higher CSF EBV load when adjusted for HIV (P = .01). CMV was detected in 11 of 115 HIV-infected patients, 8 of whom died. The mechanisms by which EBV and CMV contribute to poor outcome require further investigation.

Genomic epidemiology of syphilis in England: a population-based study.

BACKGROUND: Syphilis is a sexually transmitted bacterial infection caused by Treponema pallidum subspecies pallidum. Since 2012, syphilis rates have risen dramatically in many high-income countries, including England. Although this increase in syphilis prevalence is known to be associated with high-risk sexual activity in gay, bisexual, and other men who have sex with men (GBMSM), cases are rising in heterosexual men and women. The transmission dynamics within and between sexual networks of GBMSM and heterosexual people are not well understood. We aimed to investigate if whole genome sequencing could be used to supplement or enhance epidemiological insights around syphilis transmission. METHODS: We linked national patient demographic, geospatial, and behavioural metadata to whole T pallidum genome sequences previously generated from patient samples collected from across England between Jan 1, 2012, and Oct 31, 2018, and performed detailed phylogenomic analyses. FINDINGS: Of 497 English samples submitted for sequencing, we recovered 240 genomes (198 from the UK Health Security Agency reference laboratory and 42 from other laboratories). Three duplicate samples (same patient and collection date) were included in the main phylogenies, but removed from further analyses of English populations, leaving 237 genomes. 220 (92·8%) of 237 samples were from men, nine (3·8%) were from women, and eight (3·4%) were of unknown gender. Samples were mostly from London (n=118 [49·8%]), followed by southeast England (n=29 [12·2%]), northeast England (n=24 [10·1%]), and southwest England (n=15 [6·3%]). 180 (76·0%) of 237 genomes came from GBMSM, compared with 25 (10·5%) from those identifying as men who have sex with women, 15 (6·3%) from men with unrecorded sexual orientation, nine (3·8%) from those identifying as women who have sex with men, and eight (3·4%) from people of unknown gender and sexual orientation. Phylogenomic analysis and clustering revealed two dominant T pallidum sublineages in England. Sublineage 1 was found throughout England and across all patient groups, whereas sublineage 14 occurred predominantly in GBMSM older than 34 years and was absent from samples sequenced from the north of England. These different spatiotemporal trends, linked to demography or behaviour in the dominant sublineages, suggest they represent different sexual networks. By focusing on different regions of England we were able to distinguish a local heterosexual transmission cluster from a background of transmission in GBMSM. INTERPRETATION: These findings show that, despite extremely close genetic relationships between T pallidum genomes globally, genomics can still be used to identify putative transmission clusters for epidemiological follow-up. This could be of value for deconvoluting putative outbreaks and for informing public health interventions. FUNDING: Wellcome funding to the Sanger Institute, UK Research and Innovation, National Institute for Health and Care Research, European and Developing Countries Clinical Trials Partnership, and UK Health Security Agency.

Paediatric acute hepatitis of unknown aetiology: a national investigation and adenoviraemia case-control study in the UK.

BACKGROUND: An increase in acute severe hepatitis of unknown aetiology in previously healthy children in the UK in March, 2022, triggered global case-finding. We aimed to describe UK epidemiological investigations of cases and their possible causes. METHODS: We actively surveilled unexplained paediatric acute hepatitis (transaminase >500 international units per litre) in children younger than 16 years presenting since Jan 1, 2022, through notifications from paediatricians, microbiologists, and paediatric liver units; we collected demographic, clinical, and exposure information. Then, we did a case-control study to investigate the association between adenoviraemia and other viruses and case-status using multivariable Firth penalised logistic regression. Cases aged 1-10 years and tested for adenovirus were included and compared with controls (ie, children admitted to hospital with an acute non-hepatitis illness who had residual blood samples collected between Jan 1 and May 28, 2022, and without known laboratory-confirmed diagnosis or previous adenovirus testing). Controls were frequency-matched on sex, age band, sample months, and nation or supra-region with randomised selection. We explored temporal associations between frequency of circulating viruses identified through routine laboratory pathogen surveillance and occurrence of cases by linear regression. SARS-CoV-2 seropositivity of cases was examined against residual serum from age-matched clinical comparison groups. FINDINGS: Between Jan 1 and July 4, 2022, 274 cases were identified (median age 3 years [IQR 2-5]). 131 (48%) participants were male, 142 (52%) were female, and one (<1%) participant had sex data unknown. Jaundice (195 [83%] of 235) and gastrointestinal symptoms (202 [91%] of 222) were common. 15 (5%) children required liver transplantation and none died. Adenovirus was detected in 172 (68%) of 252 participants tested, regardless of sample type; 137 (63%) of 218 samples were positive for adenovirus in the blood. For cases that were successfully genotyped, 58 (81%) of 72 had Ad41F, and 57 were identified as positive via blood samples (six of these were among participants who had undergone a transplant). In the case-control analysis, adenoviraemia was associated with hepatitis case-status (adjusted OR 37·4 [95% CI 15·5-90·3]). Increases in the detection of adenovirus from faecal samples, but not other infectious agents, in routine laboratory pathogen surveillance correlated with hepatitis cases 4 weeks later, which independently suggested an association (ß 0·06 [95% CI 0·02-0·11]). No association was identified for SARS-CoV-2 antibody seropositivity. INTERPRETATION: We observed an association between adenovirus 41F viraemia and paediatric acute hepatitis. These results can inform diagnostic testing recommendations, clinical management, and exploratory in vitro or clinical studies of paediatric acute hepatitis of unknown aetiology. The role of potential co-factors, including other viruses and host susceptibility, requires further investigation. FUNDING: None.

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated from AIDS patients.

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated between 1986 and 1997 from AIDS patients has been performed. Sequencing the L2 part of the hexon gene of 51 adenoviruses isolated between 1986 and 1997 from AIDS patients revealed only one type each from species A and C and two types from species B with all the remaining isolates from species D. Further sequencing and phylogenetic analysis of the fiber knob region of these species D adenoviruses revealed that 28/46 were intermediate strains with conflicting hexon and fiber sequences. When the penton regions of these intermediate strains were sequenced, it became clear that some had originated from a third adenovirus type presumably by intergene recombination events. Evidence from sequencing the L1 hexon and fiber shaft regions showed no evidence of intragene recombination but penton sequences showed that recombination between the hypervariable region (HVR) and RGD regions was common. Six isolates appear to be from three new adenovirus types. Five AIDS patients showed sequential infection with different adenovirus variants and six such variants were isolated from a single patient in 2 years.

Evaluation of a novel direct RT-LAMP assay for the detection of SARS-CoV-2 from saliva samples in asymptomatic individuals.

Large scale screening of health care workers and the general population for asymptomatic COVID-19 infection requires modalities that are amenable to testing at scale while retaining acceptable levels of sensitivity and specificity. This study evaluated a novel COVID-19 Direct-RT LAMP assay using saliva samples in asymptomatic individuals by comparison to RT-PCR. Additional studies were performed using VTM collected from routine diagnostic testing. Analytical sensitivity was determined for Direct RT-LAMP assay using the WHO International Standard. Finally, quantified results from RT-PCR testing of 9177 nose and throat swabs obtained from routine diagnostic testing were used to estimate the sensitivity of Direct RT-LAMP using the limit of detection curve obtained from the analytical sensitivity data. Results from saliva testing demonstrated a sensitivity of 40.91% and a specificity of 100% for Direct RT-LAMP. The sensitivity and specificity for nose and throat swabs were 44.85% and 100% respectively. The 95% limit of detection (LOD) for Direct RT-LAMP was log 7.13 IU/ml (95% 6.9-7.5). The estimated sensitivity for Direct-RT LAMP based on the results of 9117 nose and throat swabs was 34% and 45% for saliva and VTM respectively. The overall diagnostic sensitivity of Direct RT-LAMP was low compared to RT-PCR. Testing of nose and throat swabs and estimating the sensitivity based on a large cohort of clinical samples demonstrated similar results. This study highlights the importance of utilising the prospective collection of samples from the intended target population in the assessment of diagnostic sensitivity.

Kidney Transplant-Associated Viral Infection Rates and Outcomes in a Single-Centre Cohort.

BACKGROUND: Opportunistic infections remain a significant cause of morbidity and mortality after kidney transplantation. This retrospective cohort study aimed to assess the incidence and predictors of post-transplant DNA virus infections (CMV, EBV, BKV and JCV infections) in kidney transplant recipients (KTR) at a single tertiary centre and evaluate their impact on graft outcomes. METHODS: KTR transplanted between 2000 and 2021 were evaluated. Multivariate logistic regression analysis and Cox proportional hazard analyses were used to identify factors associated with DNA virus infections and their impact on allograft outcomes respectively. A sub-analysis of individual viral infections was also conducted to describe the pattern, timing, interventions, and outcomes of individual infections. RESULTS: Data from 962 recipients were evaluated (Mean age 47.3 ± 15 years, 62% male, 81% white). 30% of recipients (288/962) had infection(s) by one or more of the DNA viruses. Individually, CMV, EBV, BKV and JCV viruses were diagnosed in 13.8%. 11.3%, 8.9% and 4.4% of recipients respectively. Factors associated with increased risk of post-transplant DNA virus infection included recipient female gender, higher number of HLA mismatch, lower baseline estimated glomerular filtration rate (eGFR), CMV seropositive donor, maintenance with cyclosporin (rather than tacrolimus) and higher number of maintenance immunosuppressive medications. The slope of eGFR decline was steeper in recipients with a history of DNA virus infection irrespective of the virus type. Further, GFR declined faster with an increasing number of different viral infections. Death-censored graft loss adjusted for age, gender, total HLA mismatch, baseline eGFR and acute rejection was significantly higher in recipients with a history of DNA virus infection than those without infection (adjusted hazard ratio (aHR, 1.74, 95% CI, 1.08-2.80)). In contrast, dialysis-free survival did not differ between the two groups of recipients (aHR, 1.13, 95% CI, 0.88-1.47). CONCLUSION: Post-transplant DNA viral infection is associated with a higher risk of allograft loss. Careful management of immunosuppression and close surveillance of at-risk recipients may improve graft outcomes.

An Overview of SARS-CoV-2 Molecular Diagnostics in Europe.

The COVID-19 pandemic has led to the rapid development of a plethora of molecular diagnostic assays with real-time polymerase chain reaction (RT-PCR) at the forefront. In this review, we will discuss the history and utility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) molecular diagnostics and the associated current and future regulatory process in Europe. We will assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests currently used in Europe with a focus on as rapid molecular platforms, stand-alone RT-PCR kits, the role of low-throughput and high-throughput end-to-end testing platforms, and the rapidly evolving field of SARS-CoV-2 variant of concern identification.

Evaluation of the artus® Prep&Amp UM RT-PCR for detection of SARS-CoV-2 from nasopharyngeal swabs without prior nucleic acid eluate extraction.

Here we describe a retrospective clinical evaluation of the QIAGEN artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay that detects SARS-CoV-2 RNA without the need for a nucleic acid eluate extraction procedure. Using Roche SARS-CoV-2 RT-PCR on the cobas® 8800 platform as a reference standard, a total of 225 confirmed SARS-CoV-2 positive and 320 negative nasopharyngeal swabs in viral transport media, were used to evaluate the artus® assay. Using the RT-PCR cycle threshold as a semi-quantitative marker of viral load, an assessment of over 370,000 SARS-CoV-2 RT-PCR positive results was used in the design of the reference positive specimen cohort. The viral load of all reference positive specimens used in the evaluation was a unique and accurate representation of the range and levels of SARS-CoV-2 positivity observed over a 13-month period of the COVID-19 pandemic. The artus® RT-PCR detects the presence of SARS-CoV-2 RNA, an internal control, and the human RNase P gene to ensure specimen quality. The diagnostic sensitivity of artus® was 92.89% with a specificity of 100%. To assess the analytical sensitivity, a limit of detection was performed using the 1st WHO NIBSC SARS-CoV-2 international standard, recording a 95% LOD of 1.1 × 103 IU/ml. The total invalid rate of specimens was 7.34% due to a lack of detectable RNase P (Ct >35). The artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay is a new rapid RT-PCR assay, which may be considered to produce acceptable levels of diagnostic sensitivity and specificity whilst potentially halving the laboratory processing time.

Bacterial genomic detection within cerebrospinal fluid of patients with meningococcal disease is influenced by microbial and host characteristics.

Among 384 patients with confirmed meningococcal disease, the likelihood of detecting Neisseria meningitidis DNA in cerebrospinal fluid (CSF) increased with age, serogroup B infection, and prehospitalization antibiotic treatment. Plasma and CSF genomic bacterial loads of non-B N. meningitidis serogroups correlated significantly. Serogroup B-infected patients with genotype TNF2 (-308A) had significantly higher CSF bacterial loads.

Sequence typing of adenovirus from samples from hematological stem cell transplant recipients.

Adenovirus infections are usually mild or even asymptomatic, but infections with the virus are being recognized increasingly as a major cause of mortality and morbidity in the immunocompromised, particularly hematological stem cell transplant patients where infections can be life threatening and mortality may reach 60%. Typing by sequencing the HVR7 region of the hexon was established and validated using 60 isolates of different serotypes from the six of the seven species which had been typed previously by serum neutralization. Analysis of nucleotide sequences was used to type 227 samples from 41 hematological stem cell transplant recipients. Types from six species were detected but species C types were detected in 51.4% and species A in 34.3% of patients. Seven patients were infected with different adenovirus types sequentially and a further six patients had evidence of simultaneous multiple infections. Many of the sequences had several differences from the prototype strains which will allow tracing of outbreaks and provide evidence for cross-infection in a hospital setting. In this study, the phylogenetic analysis of adenovirus sequences from hematological stem cell transplant patients' samples showed evidence of two possible cross-infection incidents involving three and five patients, respectively.

Molecular exploration for Mycoplasma amphoriforme, Mycoplasma fermentans and Ureaplasma spp. in patient samples previously investigated for Mycoplasma pneumoniae infection.

OBJECTIVES: To determine the presence and genotypic macrolide susceptibility of Mycoplasma amphoriforme, and the presence of Ureaplasma spp. and Mycoplasma fermentans among clinical samples from England previously investigated for Mycoplasma pneumoniae. METHODS: Quantitative and conventional PCR methods were used to retrospectively screen a collection of 160 clinical samples previously submitted to Public Health England (PHE) for the detection of M. pneumoniae between October 2016 and December 2017. Samples which were positive for M. amphoriforme DNA were further investigated for mutations associated with genotypic macrolide resistance by sequencing domain V of the 23s rRNA. RESULTS: M. amphoriforme was detected in 10/160 samples (6.3%), Ureaplasma parvum was detected in 4/160 samples (2.5%), and M. fermentans was not detected in any samples (0/160). Of the nine individuals (two samples were from the same patient) in which M. amphoriforme was detected, eight were male (age range 10-60 years) and one was female (age range 30-40 years). One individual with cystic fibrosis was positive for both M. amphoriforme and U. parvum. All M. amphoriforme DNA was genotypically susceptible to macrolides. CONCLUSIONS: Mycoplasma amphoriforme was found in clinical samples, including lower respiratory tract samples of patients with pneumonia. In the absence of other respiratory pathogens, these data suggest a potential role for this organism in human disease, with no evidence of acquired macrolide resistance. Ureaplasma parvum was detected in cerebrospinal fluid and respiratory tract samples. These data suggest that there is a need to consider these atypical respiratory pathogens in future diagnostic investigations.