- Volume 64, Issue 7, 2015

Delay of active antimicrobial therapy in haematological patients with Gram-negative bacilli bacteraemia during profound neutropenia exposes them to increased morbidity and mortality. The digestive tract is the main source of enterobacteria causing bacteraemia in these patients. We thus evaluated the usefulness of broad-spectrum beta-lactam resistant enterobacteria (BSBL-RE) faecal shedding assessment in forecasting the susceptibility to BSBLs of the strains isolated from blood cultures. From 2002 to 2011, neutropenic haematological patients with bacteraemia caused by enterobacteria who had a stool culture during the previous 7 days were retrospectively included. BSBL-RE intestinal carriers were compared with non-carriers in terms of clinical and microbiological criteria. One hundred and four patients were included and 16 of them (15.4 %) were BSBL-RE carriers. Multivariate analysis showed that BSBL-RE carriage was independently associated with BSBL-RE identified in blood cultures (P < 0.001) and the use of carbapenems as empirical treatment of the bacteraemia (P = 0.008). Sensitivity, specificity, and the positive and negative predictive values of the test were 80 %, 91 %, 50 % and 98 %, respectively. Among the carriers, those with the highest level of BSBL-RE carriage were also those with the highest risk of bacteraemia due to BSBL-RE (P < 0.001). Close monitoring of BSBL-RE intestinal carriage may help to choose the most appropriate initial antimicrobial treatment for neutropenic haematological patients with bacteraemia.

Acanthamoeba species are responsible for causing the potentially sight-threatening condition, Acanthamoeba keratitis, which is commonly associated with contact lens use. In this report, we highlight the challenges faced using conventional laboratory identification methods to identify this often under-reported pathogen, and discuss the reasons for introducing the first national service in Scotland for the rapid and sensitive molecular identification of Acanthamoeba species. By comparing culture and molecular testing data from a total of 63 patients (n = 80 samples) throughout Scotland presenting with ocular eye disease, we describe the improvement in detection rates where an additional four positive cases were identified using a molecular assay versus culture. The testing of a further ten patients by confocal imaging is also presented. This report emphasizes the importance of continuing to improve clinical laboratory services to ensure a prompt, correct diagnosis and better prognosis, in addition to raising awareness of this potentially debilitating opportunistic pathogen.

To assess the level of practice consistent with UK national standards for Cryptosporidium testing, an audit was performed of 156 publicly funded clinical microbiology laboratories in England and Wales between August 2013 and April 2014. Responses were received from 85 (54 %) laboratories. First line diagnostic methods used were mainly microscopy with modified Ziehl–Neelsen (mZN) or auramine phenol (AP) staining (68/85, 80 %), enzyme immunoassays (EIAs) (16/85, 19 %) or in-house PCR (1/85, 1 %). The use of EIAs was more widespread than reported previously. Various methods were used for confirmation of positive EIA reactions and laboratories frequently resorted to sending samples to the national reference laboratory for this purpose, indicating that guidance is required for performance monitoring and confirmation of positive reactions. Laboratory positivity rates were related to the diagnostic test used, with highest median rates reported by those using PCR, EIAs or AP microscopy, and the lowest by those using mZN microscopy. One-third of responding laboratories (28/85, 33 %) routinely tested all stools for Cryptosporidium. However, 16 (19 %) laboratories used stool consistency to decide whether to test for this parasite. Other selection criteria included patient age (n = 18; 21 % laboratories), history or clinical details (n = 40; 47 %), duration of hospitalization (n = 18; 21 %) or clinician requests (n = 25; 29 %). To encourage laboratories to test all stools submitted for the investigation of diarrhoeal illness for Cryptosporidium, revision of the guidance in the national standards is under way. This will enable improved assessment of the burden of illness and ability to monitor outbreaks, and measure changes in reported cases.

This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.

A novel lateral-flow device (LFD) has been invented for use as a diagnostic tool for invasive aspergillosis (IA). We conducted a meta-analysis to assess the diagnostic accuracy of the device. Published studies that used the European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria and provided sufficient data were included. Two reviewers independently collected the data from each study and assessed the risk bias using the Quality Assessment of Diagnostic Accuracy Studies-2. The pooled sensitivity, specificity and diagnostic odds ratio (DOR) were computed and reported with a 95  % confidence interval (CI). Seven studies published between 2008 and March 2015 were included. The pooled sensitivity, specificity and DOR for the proven/probable versus no IA cases were 0.86 (95  % CI, 0.76–0.93), 0.93 (95  % CI, 0.89–0.96) and 65.94 (95  % CI, 27.21–159.81) in the LFD test using bronchoalveolar lavage (BAL) fluid, and 0.68 (95  % CI, 0.52–0.81), 0.87 (95  % CI, 0.80–0.92) and 11.90 (95  % CI, 3.54–39.96) in the LFD test using serum. We concluded that the Aspergillus LFD had a good diagnostic value in immunocompromised patients at risk of IA. The BAL LFD might have a better performance than the serum LFD test.

Vulvovaginal candidiasis, a superficial infection caused predominantly by the pathogenic fungus Candida albicans, is frequently treated with clotrimazole. Some drug formulations contain lactate for improved solubility. Lactate may modify C. albicans physiology and drug sensitivity by serving as a carbon source for the fungus and/or affecting local pH. Here, we explored the effects of lactate, in combination with pH changes, on C. albicans proliferation, morphology and clotrimazole sensitivity. Moreover, we determined the influence of growth phase and morphology per se on drug sensitivity. We showed that utilization of lactate as a carbon source did not promote fast fungal proliferation or filamentation. Lactate had no influence on clotrimazole-mediated killing of C. albicans in standard fungal cultivation medium but had an additive effect on the fungicidal clotrimazole action under in vitro vagina-simulative conditions. Moreover, clotrimazole-mediated killing was growth-phase and morphology dependent. Post-exponential cells were resistant to the fungicidal action of clotrimazole, whilst logarithmic cells were sensitive, and hyphae showed the highest susceptibility. Finally, we showed that treatment of pre-formed C. albicans hyphae with sublethal concentrations of clotrimazole induced a reversion to yeast-phase growth. As C. albicans hyphae are considered the pathogenic morphology during mucosal infections, these data suggest that elevated fungicidal activity of clotrimazole against hyphae plus clotrimazole-induced hyphae-to-yeast reversion may help to dampen acute vaginal infections by reducing the relative proportion of hyphae and thus shifting to a non-invasive commensal-like population. In addition, lactate as an ingredient of clotrimazole formulations may potentiate clotrimazole killing of C. albicans in the vaginal microenvironment.

Recent reports have hypothesized that colonization of the maternal genital tract with non-capsulated Haemophilus influenzae could result in neonatal invasive disease. In this study, genital carriage of the genus Haemophilus was investigated in 510 pregnant women attending an Italian hospital for routine controls. Overall, vaginal carriage of the genus Haemophilus was 9.0 % (46/510). A high colonization rate with Haemophilus parainfluenzae (37/510, 7.3 %) was found; other species, such as Haemophilus pittmaniae (7/510, 1.4 %) and Haemophilus haemolyticus (2/510, 0.4 %), were detected for the first time in the genital flora by 16S rRNA gene sequencing. Notably, no H. influenzae was identified, in agreement with previous investigations indicating that this species is rarely isolated from the genito-urinary tract of pregnant women. No antibiotic resistance was detected in H. pittmaniae and H. haemolyticus, but quite a high degree of ampicillin (10/37, 27 %) and ciprofloxacin (3/37, 8.1 %) resistance was observed in H. parainfluenzae. Five ampicillin-resistant isolates were β-lactamase producers, whereas five isolates exhibited a β-lactamase-negative ampicillin-resistant (BLNAR) phenotype. Sequencing of penicillin-binding protein 3 revealed that Val511Ala, Asn526Ser, Ala530Ser and Thr574Ala changes were associated with BLNAR phenotypes. Two ciprofloxacin-resistant isolates carried substitutions in both GyrA (Ser84Phe and Asp88Tyr) and ParC (Ser84Tyr and Met198Leu); the other ciprofloxacin-resistant isolate had substitutions in ParC, only (Ser138Thr and Met198Leu). In conclusion, ∼10 % of pregnant women carried a species of Haemophilus in their genital tract. The emergence of non-β-lactamase-mediated resistance in genital H. parainfluenzae is a matter of concern because of the risk of mother-to-baby transmission.

A total of 1085 strains of Acinetobacter sp. obtained from 280 medical institutions in the Chugoku and Shikoku areas of Japan were investigated between 2011 and 2013. Among these strains, 20 (1.84 %) showing meropenem or imipenem resistance with a MIC of >4 μg ml− 1 were detected. Of these 20 strains, the bla OXA-23 gene was detected in 17 strains isolated from the same institution. The PFGE patterns of the 17 strains were separated into two clusters, and multi-locus sequence typing showed the sequence types (STs) to be ST2 and ST246. This investigation demonstrated that A. baumannii ST2 of international clone II, which has rarely been isolated in Japan, has not yet spread nationwide.

The prevalence of community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) strains has become a serious problem worldwide. The aim of this study was to investigate the annual transitions of MRSA strains with the CA-MRSA feature, which were identified as SCCmec type IV or V, in a hospital setting in Japan. Between 2005 and 2012, MRSA strains were collected from a tertiary-care hospital in Tokyo, Japan, and SCCmec typing, detection of the virulence factors and antimicrobial susceptibility testing were conducted. The rate of detection of type II SCCmec, which is found mainly in healthcare-associated MRSA, significantly decreased from 90.0 (2005–2006) to 74.3 % (2011–2012) (P < 0.01). In contrast, the rate of detection of type IV SCCmec, which is mainly found in CA-MRSA, significantly increased from 5.8 (2005–2006) to 16.3 % (2011–2012) (P < 0.01). The rate of detection of the toxic shock syndrome toxin-1 gene significantly decreased from 66.7 (2005–2006) to 51.6 % (2011–2012) (P < 0.01), whilst that of the Panton-Valentine leukocidin gene significantly increased from 0.1 (2005–2006) to 2.1 % (2011–2012) (P < 0.01). The resistance rates of cefotaxime, levofloxacin, clarithromycin and minocycline decreased every year. The resistance rates of these antimicrobial agents for the SCCmec type IV or V strains were significantly lower than those for the SCCmec type I or II strains (P < 0.01, respectively). Therefore, these results suggest that the annual transitions of the virulence factors and antibiograms in MRSA are closely related to the increase of SCCmec type IV/V strains.

In recent years, there has been an increase in the number of reported hepatitis E virus (HEV) infections from developed countries. To describe recent trends in notification and potential risk groups and risk factors in Japan, HEV infection cases and demographic, food consumption, clinical and laboratory data reported during 2007–2013 were analysed. In total, 530 HEV infections were reported during 2007–2013. Amongst 462 domestic cases, the mean age was 56.5 years (sd 13.9) and 80.1 % were male. Forty-three cases (9.3 %) were asymptomatic, amongst which 11 were detected from blood donations. Whilst ∼50 cases were reported annually during 2007–2011, the number of reported cases increased to 121 in 2012 and 126 in 2013. The increase was characterized by a rise in the number of domestic, symptomatic cases (P = 0.05) and cases confirmed by anti-HEV IgA detection (P < 0.01). HEV genotypes G3 and G4 were consistently dominant. The major suspected source of infection was food-borne, and the major suspected foods were pig, wild boar and deer meat. The observed increase during 2012–2013 was most likely due to the coverage of the anti-HEV IgA assay by the National Health Insurance system in Japan in October 2011 and its acceptance for surveillance purposes. However, the increase was not associated with detection of asymptomatic cases. Moreover, males aged 50–69 years remained as the high-risk group, and pork and other meats continued to be the most suspected items. Our findings indicated that HEV infection is an emerging and important public health concern in Japan.

This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

This study determined the mechanisms and patterns of antimicrobial resistance among the isolates obtained from different wards of a teaching hospital in the city of Mashhad in north-east Iran. Between January 2012 and the end of June 2012, 36 isolates of Acinetobacter baumannii were collected from different wards of Ghaem Hospital. Antimicrobial susceptibility testing and epsilometer testing (E-test) were performed. The genetic resistance determinants of A, B and D classes of β-lactamases, aminoglycoside modifying enzymes (AMEs), efflux pumps and ISAba1 elements were assessed by PCR. Repetitive extragenic palindromic element (REP)-PCR was performed to find the genetic relatedness of the isolates. Colistin was the most effective antibiotic of those tested, where all isolates were susceptible. E-test results revealed high rates of resistance to imipenem, ceftazidime and ciprofloxacin. The majority of isolates (97  %) were multidrug-resistant. OXA-51, OXA-23 and tetB genes were detected in all isolates, but OXA-58, IMP and tetA were not detected. The prevalence of OXA-24, bla TEM, bla ADC, bla VIM and adeB were 64, 95, 61, 64 and 86  %, respectively. ISAba1 was found to be inserted into the 5′ end of OXA-23 in 35 isolates (97  %). Of the AMEs, aadA1 (89  %) was the most prevalent, followed by aphA1 (75  %). The band patterns reproduced by REP-PCR showed that 34 out of 36 isolates belonged to one clone and two singletons were identified. The results confirmed that refractory A. baumannii isolates were widely distributed and warned the hospital infection control team to exert strict measures to control the infection. An urgent surveillance system should be implemented.

We evaluated 3122 children with E. coli urinary isolates over a 10 year period in order to assess the emergence of fluoroquinolone resistance. Susceptibilities remained stable; however, hospitalized children had a statistically higher risk of developing fluoroquinolone-resistant isolates when compared with outpatients. Stewardship monitoring of fluoroquinolone use amongst hospitalized children is warranted.