- Volume 65, Issue 6, 2016

Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of Coxiella burnetii DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 104 bacteria ml−1) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for C. burnetii performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of C. burnetii DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium ml−1) than in non-lyophilized sera (102 bacteria ml−1). Among the 73 sera from patients with acute Q fever, 26 (36 %) were positive after lyophilization, demonstrating a sensitivity gain of 44 % for early negative sera and 30 % for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80 %) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples.

Triazole antifungal agents are the mainstay of aspergillosis treatment. As highlighted in numerous studies, the global increase in the prevalence of triazole resistance could hamper the management of aspergillosis. In the present three-year study, 513 samples (213 clinical and 300 environmental samples) from 10 provinces of Iran were processed and screened in terms of azole resistance (4 and 1 mg l−1 of itraconazole and voriconazole, respectively), using selective plates. Overall, 150 A . fumigatus isolates (71 clinical and 79 environmental isolates) were detected. The isolates were confirmed by partial sequencing of the β-tubulin gene. Afterwards, in vitro antifungal susceptibility tests against triazole agents were performed, based on the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The CYP51A gene was sequenced in order to detect mutations. The MIC of itraconazole against 10 (6.6 %) strains, including clinical (n=3, 4.2 %) and environmental (n=7, 8.8 %) strains, was higher than the breakpoint and epidemiological cut-off value. Based on the findings, the prevalence of azole-resistant A. fumigatus in Iran has increased remarkablyfrom 3.3 % to 6.6 % in comparison with earlier epidemiological research. Among resistant isolates, TR34/L98H mutations in the CYP51A gene were the most prevalent (n=8, 80 %), whereas other point mutations (F46Y, G54W, Y121F, G138C, M172V, F219C, M220I, D255E, T289F, G432C and G448S mutations) were not detected. Although the number of patients affected by azole-resistant A. fumigatus isolates was limited, strict supervision of clinical azole-resistant A. fumigatus isolates and persistent environmental screening of azole resistance are vital to the development of approaches for the management of azole resistance in human pathogenic fungi.

A study was undertaken to quantify the expression of prostaglandin (PG) receptors and find the effect of gestational age on expression of PG receptor genes in Chlamydia trachomatis-infected recurrent spontaneous aborters (RSA). Endometrial curettage tissue (ECT) was collected from 130 RSA (Group I) and 100 age-matched controls (Group II) at the Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi (India). PCR was performed for diagnosis of C. trachomatis cryptic plasmid; mRNA expression of PG receptor genes was assessed by real-time PCR (q-PCR), while serum progesterone/estrogen levels were determined by respective commercial kits. Data were evaluated statistically. A total of 15.4 % RSA (GroupI) were diagnosed as C. trachomatis-positive (200 bp), whereas controls were uninfected. q-PCR showed significant upregulation (P<0.0001) of PGE2 (EP-1, EP-2, EP-3, EP-4), PGF2α (FP) and PGI2 (IP) receptors in Group I versus Group II. The expression of PG receptors increased significantly with advanced gestational age (P<0.002); however, only contractile receptors, EP-1, EP-3 and FP, were positively correlated with gestational age in Group-I. In infected RSA, mean serum progesterone level was significantly low (P<0.0001) while serum oestrogen was high (P<0.0001). Overall, the data suggest that increased expression of PG receptors, particularly contractile gene receptors (EP-1, EP-3, FP), with advanced gestational age and altered steroid levels could be a possible risk factor for abortion in Chlamydia-infected RSA.

Legionella pneumophila is the leading cause of Legionnaires’ disease, a severe pneumonia that can occur as sporadic cases or point-source outbreaks affecting multiple patients. The infection is acquired by inhalation of aerosols from contaminated water systems. In order to identify the probable source and prevent further cases, clinical and environmental isolates are compared using phenotypic and genotypic methods. Typically up to 10 days are required to isolate L. pneumophila prior to the application of standard typing protocols. A rapid protocol using a real-time PCR specific for L. pneumophila and serogroup 1, combined with nested direct molecular typing, was adopted by Public Health England in 2012 to reduce reporting time for preliminary typing results. This rapid protocol was first used to investigate an outbreak that occurred in July/August 2012 and due to the positive feedback from that investigation, it was subsequently applied to other incidents in England and Wales where faster typing results would have aided incident investigation. We present here results from seven incidents that occurred between July 2012 and June 2015 where the use of this rapid approach provided preliminary characterization of the infecting strain in an average 1.58 days (SD 1.01) after sample receipt in contrast to 9.53 days (SD 3.73) when standard protocols were applied.

Nutritionally variant streptococci, now classified as Abiotrophia defectivaor Granulicatella spp., are thought to account for 2 % of all infective endocarditis cases but estimates of their frequency are complicated by changes in nomenclature and difficulties in obtaining positive microbiology cultures. Their growth characteristics and difficulty undertaking antibiotic susceptibility testing may impede optimal antibiotic treatment decisions. We describe three patients with definite infective endocarditis due to these organisms seen at our hospital between 2005 and 2010, all of whom presented with neurological symptoms due to infectious intracranial cerebral aneurysms. We recommend that, for patients with left-sided infective endocarditis due to A. defictiva and Granulicatella spp., clinicians should consider imaging the central nervous system.

Earlier targeted therapy for bacteraemia optimizes patient outcomes and reduces broad spectrum antibiotic use. Standardized susceptibility testing results are available at 36–48 h. Direct disc susceptibility testing from blood culture broth reduces time to results but the inoculum is not standardized. No studies have looked at the clinical utility of direct susceptibility results. This retrospective cohort study aimed to assess the correlation between direct and formal testing methods as well as the clinical utility of direct susceptibility results. 160 episodes of bacteraemia with paired direct and formal susceptibility testing were studied. Direct disc testing was performed on blood culture broth. Formal testing was performed on isolates, using automated broth microdilution or Etests. The rate of error was 9.0 % (95 % CI 7.0–11.6 %). In 10 cases (6.3 %, 95 % CI 3.0–11.2 %), inappropriate antibiotics were used due to direct susceptibility results, including two cases with ineffective (as opposed to too broad) antibiotics being used. Antibiotics were changed in 28.1 % of cases once direct susceptibility data was available. There was a decreased time to effective antibiotics in 9.3 % (95 % CI 5.3–15.0 %), and a decreased time to a targeted antibiotics in 14.3 % (95 % CI 9.3–20.8 %) of cases. Despite the error rate, the advantages of earlier times to effective and targeted antibiotics justifies continuing direct testing in bacteraemia episodes with Gram-negative rods. In the Gram-positive group, given the contamination rate, the availability of adjunctive PCR, and the fact that early identification of the isolate could equally influence antibiotic choices, direct susceptibility testing may no longer be warranted.

The aim of this study was to assess Chlamydia trachomatis (CT) infection prevalence and serovar distribution in a high-density urban area in the north of Italy, by comparing different groups of subjects divided on the basis of the type of care provider they referred to (STI Clinic, gynaecologists or general practitioners). From January 2011 to May 2014, all the specimens submitted to the Microbiology Laboratory of St Orsola Hospital in Bologna for CT detection were tested by PCR assay. For positive specimens, molecular genotyping based on RFLP analysis was performed. Total prevalence of CT infection was 8.1 %, with significant differences between subgroups (P<0.01) but stable during the study period. The STI Clinic was mainly responsible for CT diagnosis, whereas the lowest infection prevalence was detected in gynaecological clinics, despite the high number of tests performed. Extra-genital samples were almost exclusively collected from males at the STI Clinic. Interestingly, 13.3 % of patients providing extra-genital specimens were positive for CT on rectal and/or pharyngeal swabs, and 4.4 % of cases would have been missed if extra-genital sites had not been tested. The most common serovar was E, and serovar distribution was influenced by gender (P<0.01), age (P<0.01), care provider (P=0.01) and anatomical site (P<0.01). The L2 serovar was detected only in extra-genital samples from males at the STI Clinic. Knowledge about care providers’ contributions in CT testing and diagnosis is essential for infection control. CT typing is crucial for appropriate management of specific infections, such as lymphogranuloma venereum in extra-genital samples of high-risk populations.

Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis. It affects mainly small ruminants and causes significant economic losses worldwide. Because symptoms are not immediately noticeable, CLA clinical diagnosis is not effective. Numerous serological tests are being developed to detect the disease in asymptomatic animals, but currently available immunoassays have problems with sensitivity. Current ELISA formats use native bacterial antigens, and recombinant proteins could be useful for improving the immunoassay parameters. The C. pseudotuberculosis proteins CP0126a, CP0369 and CP1957 were identified from 2097 candidate proteins by mature epitope density (MED) analysis, expressed in Escherichia coli and evaluated in an indirect immunoenzymic system. The CP0126a, CP0369 and CP1957 ELISAs showed 77.5 %, 92.5 % and 92.5 % specificity and 95 %, 90 % and 85 % sensitivity, respectively. Receiver operating characteristic (ROC) curve analysis showed an area under the curve of 0.874, 0.951 and 0.881, respectively. The proteins identified in silico were recognized by antibodies in the sera from infected animals without being recognized in negative samples. The ELISA assay using the rCP0369 protein as antigen had the greatest specificity and sensitivity values, followed by rCP1957. This is an interesting strategy for seroepidemiological investigations in sheep flocks due to its significant specificity and sensitivity.

Many isolates of Escherichia coli carrying bla OXA-48 referred to Public Health England’s national reference laboratory during 2014 and 2015 shared similar pulsed-field gel electrophoresis (PFGE) profiles, despite coming from patients in multiple different hospitals and regions. Whole genome sequencing on an Illumina platform revealed that these belonged to sequence type (ST) 38. The OXA-48 gene is usually carried on a 62 kb IncL/M plasmid (pOXA48a), but those belonging to this ST appeared either to lack plasmid elements or to have only a partial complement. Two isolates, one belonging to a main cluster sharing identical PFGE profiles and the other having a distinct profile, were further sequenced on a minION. The long reads provided by the nanopore sequencing technology facilitated assembly of a much larger contig around the bla OXA-48 region, showing that both isolates shared a similar arrangement, with a plasmid fragment containing bla OXA-48 flanked by IS1R elements integrated into the chromosome, although the length of the plasmid fragment and the insertion site differed between the two isolates. That belonging to the main cluster contained a 21.9 kb Tn6237 insert, as previously described in E. coli EC-15 from Lebanon, but in a different insertion site. PCR mapping indicated that a further 14/31 representatives of this cluster also contained this insert in the same insertion site, with most of the remainder differing only by having additional E. coli sequence on one side of the insertion. This sub-cluster of ST38 was found from 25 different hospital laboratories, suggesting widespread distribution of a successful type.

This study describes the molecular characteristics and risk factors associated with carbapenem-resistant Klebsiella pneumoniae strains. Risk factors associated with KPC-producing K. pneumoniae strains were investigated in this case-control study from May 2011 to May 2013. Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT) and ertapenem hydrolysis using MALDI-TOF MS. The presence of β-lactamase-encoding genes was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE. Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K. pneumoniae strains and by MALDI-TOF MS in 57. The presence of the bla KPC-2 gene was identified in 57 strains. The bla KPC-2 gene was not found in 11 carbapenem-resistant K. pneumoniae; instead, the bla CTX–M-1-like, bla CTX–M-2-like, bla CTX-M-8 like, bla CTX-M-14-like and bla SHV- like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three KPC-producing K. pneumoniae strains were clonally related, and patients infected with these strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K. pneumoniae was associated with several healthcare‐related risk factors, including recent surgery.