- Volume 71, Issue 12, 2022

Japanese encephalitis (JE) is an infection that occurs predominantly in Asia and the Pacific Islands. It is transmitted by mosquito bites, with the main vector being Culex tritaeniorhynchus, and is maintained in enzootic cycles involving pigs, wild birds and mosquitoes. JE is caused by infection with Japanese encephalitis virus (JEV), a zoonotic pathogen that also causes disease in mammals such as pigs and horses. In humans, most symptoms are mild or flu-like but can progress to encephalitis. Pigs are considered amplification hosts, and sows may have gestational complications. Horses may exhibit neurological signs. Detection of the virus can be confirmed by serological or molecular laboratory tests. Vaccination offers protection against JEV infection in humans, pigs and horses. Whilst there is no effective treatment of JE, human cases may require hospitalization for supportive therapy, which may include administration of fluids, oxygen and medication to treat symptoms.

Trichosporon yeasts are classical agents of superficial mycoses, and they are ranked as the first to second predominant basidiomycetous yeast able to cause invasive infections. The clinical presentation of Trichosporon infections varies with the affected anatomical site, with fungaemia present in the majority of invasive trichosporonosis cases. Only a limited number of antifungal compounds can be used to treat Trichosporon infections. Azoles are the first choice due to their intrinsic resistance to echinocandins. Better laboratory methods and up-to-date databases of commercial platforms are required to improve identification, susceptibility testing and surveillance of this potentially threating infection.

Introduction. Colistin is an antibiotic used to treat Gram-negative bacterial infections, particularly those caused by multidrug-resistant bacteria.

Hypothesis/Gap Statement. The broth microdilution (BMD) reference method is the recommended protocol for detecting colistin susceptibility; however, it is laborious and expensive and cannot be performed in all laboratories.

Aim. To evaluate the colistin susceptibility in Klebsiella pneumoniae , Escherichia coli and Pseudomonas aeruginosa using an alternative method, which has been referred to as the drop test.

Methodology. A 16 µg ml−1 colistin solution was deposited on a Mueller–Hinton agar plate previously swabbed with the strain and incubated overnight, and the presence or absence of an inhibition zone was observed.

Results. The categorical agreement (CA) of the drop test with respect to BMD was 100 % when 190 Enterobacterales (19 E. coli and 171 K . pneumoniae ) were used, and no major errors (MEs) or very major errors (VMEs) were detected. The CA of the drop test with respect to the BMD was 99.2 % for 119 P . aeruginosa isolates, while no ME was detected and only 1 VME (6.7%) was observed.

Conclusion. The drop test is an alternative method for antimicrobial susceptibility testing of colistin against K. pneumoniae and E. coli . It is an adequate method for detecting resistant strains of P. aeruginosa , but susceptible isolates should be confirmed using BMD. The drop test is a simpler alternative to the BMD that does not require additional equipment and allows for the testing of numerous isolates in a short period of time.

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM.

Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings.

Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting.

Methodology. CPK isolates (n=181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes.

Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam.

Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Introduction. Bone and joint tuberculosis (BJTB) is rare in developed countries, particularly in the paediatric population.

Hypothesis/Gap Statement. The clinical features and sequelae of paediatric BJTB in Europe are not well characterized and should be assessed to achieve a better approach.

Aim. To assess the management and outcomes of paediatric BJTB.

Methodology. Longitudinal observational study of all paediatric patients (0–17 years old) diagnosed with BJTB between 2008 to 2020 in a tertiary-care hospital.

Results. We identified 18 patients with BJTB, with a median age of 10 years (IQR 6–14.8), 66.7 % male. Most (72 %) were diagnosed after 2015 and were foreign-born (88.9 %), mainly from Portuguese-speaking African countries, and none had HIV. The most common symptoms were pain (77.8 %), fever (50 %) and bone deformity (44.4 %). Spinal TB (STB) affected 13 (72.2 %) and extra-spinal TB (ESTB) 9 (50 %) patients, and 4 (27.7 %) had both conditions. Diagnostic positive procedures included positive nucleic acid amplification technique (NAAT) (44.4 %), Mycobacterium tuberculosis isolation (44.4 %) and compatible histology (33.3 %). All completed antituberculous drugs for a median of 12 months (IQR 12–13) and nine (50 %) had surgery. Overall, acute complications occurred in 16 (88.9 %) patients – 11/13 (84.6 %) with STB and 5/5 (100 %) with ESTB – and included abscesses, spinal compression, spine deformity and pathological fractures. Sequelae were still present at the 12-month follow-up in seven cases (46.7 %), and were more common in foreign-born patients sent to Portugal to receive medical treatment (66.7 vs 20 %).

Conclusions. Paediatric BJTB is difficult to diagnose and has high morbidity, requiring long-term follow-up. Over the last decade, foreign-born TB seems to be increasing, with still longer treatment courses and more acute complications and sequelae.

Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.

Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.

Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.

Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).

Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus /subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.

Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients’ sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.

Introduction. Aggregatibacter are Gram-negative, facultatively anaerobic rods or coccobacilli that are infrequently encountered as pathogens causing infection.

Hypothesis/Gap Statement. The range of invasive infection that Aggregatibacter cause is poorly described. The pathogenicity of species such as Aggregatibacter segnis is debated.

Aim. To identify invasive infection due to Aggregatibacter species in a large healthcare organization and to characterize clinical syndromes, co-morbidities and risk factors.

Methodology. All microbiological samples positive for Aggregatibacter species were identified by conventional culture or 16S rRNA PCR between October 2017 and March 2021. Electronic records for all patients with positive samples were reviewed and the infection syndrome classified for patients with invasive disease.

Results. Twenty-seven patients with invasive infection were identified, with a statistically significant difference in species-specific patterns of invasive infection (P=0.02) and a statistically significant association with residence in the 30 % most deprived households in the UK by postcode (P<0.01). The three most common co-morbidities were periodontitis or recent dental work (29.6%), cardiovascular disease (25.9%) and diabetes (18.5 %).

Conclusion. We describe a novel association of Aggregatibacter segnis with skin and soft tissue infection. The propensity of the Aggregatibacter species to cause invasive infection at different body sites and be associated with deprivation is reported. Aggregatibacter actinomycetemcomitans bacteraemia was associated with infective endocarditis, and Aggregatibacter aphrophilus was implicated in severe appendicitis and noted to cause brain abscess. Areas warranting future research include exploring the risk-factors required for invasive infection and those that may determine the species-specific differences in patterns of invasive disease.

Introduction. Fast and accurate diagnosis is one of the key strategies in the successful control of intramammary infections caused by Staphylococcus aureus . Immunoassays are one of the diagnostic tools that have been proposed for the detection of S. aureus infection because they offer an advantage in terms of cost and are fast and easy to use compared to other diagnostic tests.

Gap statement. The main challenge of the immunoassays is to identify antigens or serological markers that allow accurate discrimination between infected and uninfected cows with S. aureus , since this bacterium can naturally colonize different areas of the animal body.

Aim. To evaluate three S. aureus proteins (IsdA, ClfA, SdrD) involved in the adhesion process as antigens to detect indicator antibodies of bovine intramammary infections.

Methodology. Ninety-six cows in lactation and not vaccinated against S. aureus were included. Forty-eight of these cows were infected with S. aureus , while the rest (n=48 cows) were uninfected. Blood and milk samples were collected from each animal to recover serum and whey. IgG titres against the three proteins individually and combined (Mix) were measured in each sample using an enzyme-linked immunosorbent assay (ELISA) test.

Results. Significant differences in the IgG response against the proteins evaluated were observed, highlighting the antigenic potential of IsdA and demonstrating that some antigens can detect specific antibodies of infection better than others. According to receiver operating characteristic (ROC) curve analysis, the combined proteins showed the most remarkable capacity (sensitivity of 79 % and specificity of 77 %) to differentiate between infected and uninfected cows when blood samples were used. In addition, the combined proteins also showed the highest specificity (94 %) when using milk samples.

Conclusion. Our findings provide information on the usefulness of three adhesion-associated S. aureus proteins in detecting serological markers of intramammary infections in bovines.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.

Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.

Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.

Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).

Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcu s spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.

Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Introduction. Wild animals are one of the putative reservoirs of antimicrobial-resistant bacteria, but the significance of raccoon dogs remains to be investigated.

Hypothesis. Raccoon dogs can be a reservoir of antimicrobial-resistant bacteria.

Aim. This study aimed to explore the prevalence of antimicrobial resistance, mainly extended-spectrum cephalosporins resistance, in Escherichia coli isolates from faeces of 80 Japanese raccoon dogs in Kanagawa Prefecture, Japan.

Methodology. All of the 80 faecal samples were streaked onto deoxycholate-hydrogen sulfate-lactose (DHL) and cefotaxime (CTX)-supplemented DHL (DHL-CTX) agars. Susceptibilities to ten antimicrobials were determined using the agar dilution method. Additionally, extended-spectrum β-lactamases (ESBLs) and AmpC-type β-lactamases (ABLs) were identified in addition to sequence types (STs), in ESC-resistant isolates by a polymerase chain reaction and sequencing.

Results. Out of all the samples, 75 (93.8 %) and 20 (25.0 %) E. coli isolates were isolated by DHL and DHL-CTX agars, respectively. Significantly higher resistance rates to most of the drugs were found in DHL-CTX-derived isolates than DHL-derived isolates (P<0.01). Genetic analysis identified CTX-M-14 (n=6), CTX-M-2 (n=2), CTX-M-1 (n=1) and CTX-M-55 (n=1) as ESBLs, and CMY-2 (n=8) and DHA-1 (n=1) as ABLs in 20 DHL-CTX-derived isolates. Most of the detected STs were related to Japanese humans (i.e. ST10, ST58, ST69, ST131, ST357, ST648 and ST4038). Notably, this is the first report on ST69, ST131, ST155 and ST648, which are well-known international high-risk clones in Japanese raccoon dogs.

Conclusion. Our findings underscore the need to understand the significance of raccoon dogs as an antimicrobial-resistant bacteria reservoir using one health approach.