Purpose. Platelet concentrates are at risk of transfusion-related sepsis. The microbial detection methods currently available have reached their limits, as they do not completely prevent transfusion-related bacterial contamination.
The aim of this study was to develop a new strategy to detect the risk of platelet transfusion-related bacterial contamination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Methodology. In vitro, platelet concentrates were seeded with known concentrations of bacterial strains. Protein mass profiles were acquired by using a Microflex MALDI-TOF instrument. Dedicated 'Platelet' software was used as a spectrum subtraction tool to reveal specific peaks caused by the presence of pathogens in samples.
Results. The MALDI-TOF spectra of platelets were characterized and the reproducibility over time, regardless of the blood donor, was demonstrated with a positive predictive value of 100 %. In addition, the negative predictive value of the total number of specific peaks to predict contamination was 100 %.
Conclusion. Detecting bacteria in platelet concentrates using the MALDI-TOF approach and analysing spectra with the Platelet software present the advantage of combining the precocity of results and sufficient sensitivity (10 c.f.u. ml−1). Further research will be conducted to compare this novel method with the current conventional method in order to validate our results, the objective being to reduce the risk of platelet transfusion-related bacterial contamination.
Purpose. To determine the timing of the emergence of macrolide-resistant mutations after macrolide treatment in individuals with Mycoplasma pneumoniae infections.
Methodology. Between October 2011 and December 2013, serial pharyngeal swab specimens were collected before and after macrolide treatment from 21 otherwise healthy children infected with M. pneumoniae without macrolide-resistant mutations. The copy numbers of a M. pneumoniae gene and the proportion of clones showing macrolide-resistance mutations were determined for each specimen.
Results. After macrolide treatment (10–15 mg kg−1 day−1 clarithromycin for 5–10 days or 10 mg kg−1 day−1 azithromycin for 3 days), fever resolved in 19 (90 %) of 21 children within 1 to 2 days, and the M. pneumoniae gene copy number decreased in all but one specimen in the second set of specimens relative to the number in the corresponding initial specimens. None of the second specimens, which were collected 2–4 days after initiation of macrolide treatment, showed mutations in the 23S rRNA gene. However, the proportion of mutant clones with A2063G and A2064G mutations in the specimens collected 7–24 days after initiation of treatment increased to 100 %. We identified a family in which three members had M. pneumoniae infections. The analysis of transmission in this household indicated that the M. pneumoniae harbouring a macrolide-resistant mutation that developed in the index patient after macrolide treatment was not transmitted to the family members.
Conclusion. A macrolide-resistant population might develop in individual patients up to 24 days after initiation of macrolide treatment. However, the decrease in M. pneumoniae load after macrolide administration effectively reduces interpersonal transmission.
Objective. We compared the recovery of potential respiratory bacterial pathogens and normal flora from nasopharyngeal specimens collected from children during health and at the onset of acute otitis media (AOM) by selective direct-plating and overnight broth-enrichment.
Methods. Overall, 3442 nasal wash (NW) samples collected from young children were analysed from a 10-year prospective study. NWs were cultured by (1) direct-plating to TSAII/5 % sheep blood agar and chocolate agar plates and (2) overnight broth-enrichment in BacT/ALERT SA-broth followed by plating. Standard microbiology techniques were applied to identify three dominant respiratory bacterial pathogens: Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hflu) and Moraxella catarrhalis (Mcat) as well as two common nasal flora, Staphylococcus aureus (SA) and alpha-haemolytic Streptococci (AHS).
Results/Key findings. Direct-plating of NW resulted in isolation of Spn from 37.8 %, Hflu from 13.6 % and Mcat from 33.2 % of samples. In comparison, overnight broth-enrichment isolated fewer Spn (30.1 %), Hflu (6.2 %) and Mcat (16.2 %) (P<0.001–0.0001). Broth-enrichment resulted in significant increased isolation of SA (6.0 %) and AHS (30.1 %) (P<0.0001). Competition between bacterial species in broth when both species were detected by direct-plating was assessed, and it was found that SA and AHS out-competed other species during broth-enrichment when samples were collected from healthy children but not during AOM. In middle ear fluids (MEF) at the onset of AOM, broth-enrichment resulted in higher recovery of Spn (+10.4 %, P<0.001), Hflu (+4.4 %, P=0.39) and Mcat (+13.5 %, <0.001).
Conclusion. Broth-enrichment significantly reduces the accurate detection of bacterial respiratory pathogens and increases identification of SA and AHS in NW. Broth-enrichment improves detection of bacterial respiratory pathogens in MEF samples.
Purpose. This study aims to investigate the synergistic antimicrobial activity of four phytoalexins in combination with fluoroquinolones against Ureaplasma spp., a genus of cell wall-free bacteria that are intrinsically resistant to many available antibiotics, making treatment inherently difficult.
Methodology. A total of 22 958 urogenital tract specimens were assessed for Ureaplasma spp. identification and antimicrobial susceptibility. From these, 31 epidemiologically unrelated strains were randomly selected for antimicrobial susceptibility testing to determine the minimum inhibitory concentration (MIC) of four fluoroquinolones and the corresponding quinolone resistance-determining regions (QRDRs). Synergistic effects between fluoroquinolones and four phytoalexins (reserpine, piperine, carvacrol and biochanin A) were evaluated by fractional inhibitory concentration indices (FICIs).
Results. Analysis of the QRDRs suggested a vital role for the mutation of Ser-83→Leu in ParC in fluoroquinolone-resistant strains, and the occurrence of mutations in QRDRs showed significant associations with the breakpoint of levofloxacin. Moreover, diverse synergistic effects of the four phytoalexins with ofloxacin or ciprofloxacin were observed and biochanin A was able to enhance the antimicrobial activity of fluoroquinolones significantly.
Conclusion. This is the first report of the antimicrobial activity of biochanin A in combination with fluoroquinolones against a pathogenic mycoplasma, and opens up the possibility of using components of biochanin A as a promising therapeutic option for treating antibiotic-resistant Ureaplasma spp. infections.
Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant A cinetobacter, Pseudomonas, Stenotrophomonas and E nterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture medium (SuperPolymyxin).
Methodology. The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media.
Results. Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 101 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1.
Conclusion. CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance.
Purpose. This study examined the risk factors for, and molecular mechanisms underlying, the increase in carbapenem minimum inhibitory concentrations (MICs) in clinical isolates of Pseudomonas aeruginosa.
Methodology. Consecutive clinical isolates of P. aeruginosa were collected. The MicroScan WalkAway system detected more than fourfold increases in the MICs of carbapenems in P. aeruginosa isolates serially recovered from some patients during their clinical course. The clinical risk factors associated with this increase were examined by multiple logistic regression analysis. Western blot analysis and nucleotide sequencing of the oprD gene of 19 clonally related and paired P. aeruginosa isolates from the same patients were undertaken to examine the mechanisms underlying the increase in MICs.
Results. The results showed that prior use of carbapenems (OR, 2.799; 95 % CI, 1.088–7.200; P=0.033) and the use of ventilators or tracheostomies (OR, 2.648; 95 % CI, 1.051–6.671; P=0.039) were risk factors for increased carbapenem MICs. Analysis of the underlying mechanisms revealed that loss of functional OprD protein due to mutation of the oprD gene tended to occur in P. aeruginosa isolates with imipenem MICs of more than 8 µg ml−1; a reduction in OprD expression was observed in P. aeruginosa isolates with imipenem MICs of 4 or 8 µg ml−1. This difference in the resistance mechanism was not correlated with the MICs of meropenem.
Conclusion. This difference in the resistance mechanism of P. aeruginosa indicates a critical breakpoint at an imipenem MIC of 8 µg ml−1, in accordance with EUCAST criteria. Reducing carbapenem use will prevent P. aeruginosa clinical isolates from developing resistance to carbapenems.
Purpose. Sporothrix brasiliensis, the most virulent species in the Sporothrix schenckii complex, is responsible for the ongoing epidemics of human and animal sporotrichosis in Brazil. Feline outbreaks are usually driven by S. brasiliensis and followed by extensive transmission to humans. Itraconazole is the first-line treatment for both feline and human sporotrichosis; however, reduced sensitivity is an emerging issue. Thus, we investigated the effect of the widely used antifungal clotrimazole – alone or in combination with itraconazole – against the pathogenic (yeast) form of feline and human S. brasiliensis isolates, in vitro.
Methodology. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were determined for treatment with clotrimazole and itraconazole, as monotherapy or in combination. In addition, the effect of the drugs on neutral lipid levels and the yeast ultrastructure were evaluated by flow cytometry and transmission electron microscopy (TEM), respectively.
Results. The MIC and MFC values show that clotrimazole was more effective than itraconazole against feline S. brasiliensis isolates, while human isolates were more sensitive to itraconazole. Similarly to itraconazole, treatment with clotrimazole induced statistically significant neutral lipid accumulation in S. brasiliensis yeasts, and treated yeasts displayed irregularities in the cell membrane and a thicker cell wall when observed by TEM. Clotrimazole increased the antifungal activity of itraconazole in combination assays, with a synergistic effect for two feline isolates.
Conclusion. The strong activity of clotrimazole against feline S. brasiliensis isolates suggests that this drug is potentially a new alternative for the treatment of feline sporotrichosis, alone or in combination with itraconazole.
Purpose. We sought to explore the current status of antifungal stewardship (AFS) initiatives across National Health Service (NHS) Trusts within England, the challenges and barriers, as well as ways to improve current AFS programmes.
Methodology. An electronic survey was sent to all 155 acute NHS Trusts in England. A total of 47 Trusts, corresponding to 30 % of English acute Trusts, responded to the the survey; 46 Trusts (98 %) had an antimicrobial stewardship (AMS) programme but only 5 (11 %) had a dedicated AFS programme. Overall, 20 (43 %) Trusts said they included AFS as part of their AMS programmes. From those conducting AFS programmes, 7 (28 %) have an AFS/management team, 16 (64 %) monitor and report on antifungal usage, 5 (20 %) have dedicated AFS ward rounds and 12 (48 %) are directly involved in the management of invasive fungal infections.
Results/Key findings. Altogether, 13 acute Trusts (52 %) started their AFS programme to manage costs, whilst 12 (48 %) commenced the programme due to clinical need; 27 (73 %) declared that they would increase their AFS initiatives if they could. Of those without an AFS programme, 14 (67 %) responded that this was due to lack of resources/staff time. Overall, 12 Trusts (57 %) responded that the availability of rapid diagnostics and clinical support would enable them to conduct AFS activities.
Conclusion. Although a minority of Trusts conduct dedicated AFS programmes, nearly half include AFS as part of routine AMS activities. Cost issues are the main driver for AFS, followed by clinical need. The availability of rapid diagnostics and clinical support could help increase AFS initiatives.
Purpose. The aim of this study was to develop a multiplex PCR (mPCR) for simultaneous detection (single reaction) of six clinically relevant streptococcal species: Streptococcus pneumoniae, Streptococcus suis, Streptococcus gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, Streptococcus intermedius and Streptococcus anginosus/constellatus.
Methods. mPCR with primers specific for S. pneumoniae (lytA), S. suis (recN), S. gallolyticus subsp. gallolyticus (tanB), S. gallolyticus subsp. pasteurianus (SGPB0680 cell wall surface protein), S. intermedius (ily) and S. anginosus/constellatus (moaC) was employed with 37 reference bacterial strains and 442 clinical streptococcal isolates collected from seven tertiary hospitals in north-east Thailand. Results from this mPCR were compared to those obtained with the API 20 Strep and conventional biochemical tests.
Results. The six clinically relevant streptococcal species gave the expected amplification products of 229, 362, 531, 723, 819 and 978 bp for S. pneumoniae, S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, S. suis, S. intermedius and S. anginosus/S. constellatus, respectively. Non-specific reactions were not observed with the other bacterial species tested. For the 442 clinical streptococci, this mPCR assay confirmed the identity of the species in accordance with results obtained with the API 20 Strep and conventional biochemical tests.
Conclusion. This mPCR can be applied to the rapid identification of pure cultures of these six streptococci. The test was shown to be rapid, simple and reliable for the identification of these streptococci at the species level. This assay should be useful for laboratory identification and surveillance of human infections by these bacterial species.
Purpose. Bartonella is an increasingly isolated emerging pathogen that can cause severe illness in humans, including cat scratch disease (CSD). The bacteria are difficult to grow and thus many detection methods have been developed, especially molecular. We previously developed a PCR method targeting ribC to identify Bartonella sp. A manufactured kit (RealCycler BART, Progenie Molecular) was commercialised shortly thereafter for the detection of Bartonella infection, including Bartonella henselae.
Methodology. We performed a comparison between this test and our in-house PCR assay on 73 lymphadenopathy samples sent to the laboratory for suspicion of CSD.
Results/Key findings. Among the 28 positive samples for Bartonella, 21 were identified by the two PCR assays, and seven by the commercial kit only.
Conclusion. The performance of this commercial kit suggests that it could be a suitable alternative to our in-house PCR assay, highlighting the importance of the molecular methods used to diagnose CSD.
Introduction. Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal lung disease. One of the risk factors involved in the acquisition of IPF is viral infections, especially respiratory viruses. In the present study, we investigated the detection of respiratory viruses and the possible relationship between these viruses and IPF.
Methods. This cross-sectional study was supported by the Iran University of Medical Sciences (IUMS), Tehran, Iran. A total of 40 respiratory samples (five nasopharyngeal and 35 bronchoalveolar lavage specimens) were obtained from IPF patients referred to IUMS hospitals between April 2015 and December 2016. Assays were performed using the CLART Pneumovir DNA array assay, which made it possible to detect five genera of respiratory viruses simultaneously.
Results/Key findings. Altogether, 22 of the 40 participants were male. Respiratory syncytial virus (RSV), parainfluenza, rhino, corona and influenza viruses were found in 2.5 % (1/40), 7.5 % (3/40), 10 % (4/40), 2.5 % (1/40) and 0% (0/40) of cases, respectively.
Conclusion. Determining a correlation between the viruses and IPF is not an easy task, and therefore, this will require more research. In addition, the CLART Pneumovir DNA array can be considered as a useful method for simultaneous detection of several viral respiratory infections.
We describe a longitudinal study carried out in an adult outbreak-associated cohort to investigate health effects, including post-infectious irritable bowel syndrome, occurring after resolution of acute Cryptosporidium parvum infection. New symptoms self-reported up to 12 months included: weight loss (31 %), abdominal pain (38 %), diarrhoea (33 %), eye pain (9 %), joint pain (33 %), fatigue (22 %) and symptoms consistent with irritable bowel syndrome (IBS) (28 %). Two people were medically diagnosed with IBS. This study describes for the first time sequelae reported by patients up to 12 months after infection with C. parvum, which appear to be similar to those described with C. hominis.
The implementation of PCR for the detection of carbapenemase genes enables rapid results with significant epidemiological implications. Two commercial real-time PCR assays, the Hylabs Hy-CRE and Sacace MDR MBL+KPC/OXA, were evaluated for the detection of the genes bla KPC, bla VIM, bla NDM, bla IMP and bla OXA-48-carbapenemasein a collection of 96 carbapenem-resistant Enterobacteriaceae strains with different resistant mechanisms. Both assays exhibited excellent diagnostic performance, with 100 % sensitivity and specificity.
Recently, human adenovirus type 3 (HAdV-3) has become the most isolated HAdV worldwide. Restriction endonuclease analysis of globally isolated strains of HAdV-3 has uncovered 51 genome types to date. Information on the genome type is important to the epidemiological study of HAdV-3. In this study, analysis of 75 isolates of HAdV- 3 collected over a 24-year period in Fukui revealed: (1) the emergence of three novel genome types (HAdV-3a52, HAdV-3a53 and HAdV-3a54) and two known genome types (HAdV-3a and HAdV-3a54); (2) the spectrum of diseases caused by individual genome types and their major involvement in the paediatric age population; and (3) the co-circulation and replacement of genome types as a usual phenomenon. The rising number of HAdV-3 genome types indicates that the genetic variation of HAdV-3 is more than other HAdVs. Considering the clinical importance of HAdV-3 infection, its genetic diversity underscores the need for its continuous surveillance and genetic characterization.