Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Diversity of community acquired MRSA carrying the PVL gene in Queensland and New South Wales, Australia

Emergence and dissemination of community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains are being reported with increasing frequency in Australia and worldwide. These strains of CA-MRSA are genetically diverse and distinct in Australia. Genotyping of CA-MRSA using eight highly-discriminatory single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring the dissemination of these strains in the community. In this study, a SNP genotyping method was used to investigate the molecular epidemiology of 249 community acquired non-multiresistant MRSA (nm-MRSA) isolates over a 12-month period from routine diagnostic specimens. A real-time PCR for the presence of Panton-Valentine leukocidin (PVL) was also performed on these isolates. The CA-MRSA isolates were sourced from a large private laboratory in Brisbane, Australia that serves a wide geographic region encompassing Queensland and Northern New South Wales. This study identified 16 different STs and 98% of the CA-MRSA isolates were positive for the PVL gene. The most common ST was ST93 with 41% of isolates testing positive for this clone.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients’ isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ≥2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

Markers of systemic inflammation in diagnostics and in prediction of outcome of community-acquired infection

Sepsis is associated with a systemic inflammatory response. It is characterised by an early proinflammatory response and followed by a state of immunosuppression. In order to improve the outcome of patients with infection and sepsis, novel therapies that influence the systemic inflammatory response are being developed and utilised. Thus, an accurate and early diagnosis of infection and evaluation of immune state are crucial. In this thesis, various markers of systemic inflammation were studied with respect to enhancing the diagnostics of infection and of predicting outcome in patients with suspected community-acquired infection. A total of 1092 acutely ill patients admitted to a university hospital medical emergency department were evaluated, and 531 patients with a suspicion of community-acquired infection were included for the analysis. Markers of systemic inflammation were determined from a blood sample obtained simultaneously with a blood culture sample on admission to hospital. Levels of phagocyte CD11b/CD18 and CD14 expression were measured by whole blood flow cytometry. Concentrations of soluble CD14, interleukin (IL)-8, and soluble IL-2 receptor α (sIL-2Rα) were determined by ELISA, those of sIL-2R, IL-6, and IL-8 by a chemiluminescent immunoassay, that of procalcitonin by immunoluminometric assay, and that of C-reactive protein by immunoturbidimetric assay. Clinical data were collected retrospectively from the medical records. No marker of systemic inflammation, neither CRP, PCT, IL-6, IL-8, nor sIL-2R predicted bacteraemia better than did the clinical signs of infection, i.e., the presence of infectious focus or fever or both. IL-6 and PCT had the highest positive likelihood ratios to identify patients with hidden community-acquired infection. However, the use of a single marker failed to detect all patients with infection. A combination of markers including a fast-responding reactant (CD11b expression), a later-peaking reactant (CRP), and a reactant originating from inflamed tissues (IL-8) detected all patients with infection. The majority of patients (86.5%) with possible but not verified infection showed levels exceeding at least one cut-off limit of combination, supporting the view that infection was the cause of their acute illness. The 28-day mortality of patients with community-acquired infection was low (3.4%). On admission to hospital, the low expression of cell-associated lipopolysaccharide receptor CD14 (mCD14) was predictive for 28-day mortality. In the patients with severe forms of community-acquired infection, namely pneumonia and sepsis, high levels of soluble CD14 alone did not predict mortality, but a high sCD14 level measured simultaneously with a low mCD14 raised the possibility of poor prognosis. In conclusion, to further enhance the diagnostics of hidden community-acquired infection, a combination of inflammatory markers is useful; 28-day mortality is associated with low levels of mCD14 expression at an early phase of the disease.

Evaluation of a WHO-Validated Serotype-Specific Serological Assay for the Diagnosis of Pneumococcal Etiology in Children with Community-Acquired Pneumonia.

BACKGROUND: The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. METHODS: In this study, pneumococcal aetiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalised in Belgium with X-ray-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of Streptococcus pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for nine serotypes using World Health Organization validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with non proven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal aetiology in 55% of patients with non-proven P-CAP. Serotypes 1 (51.6%), 7F (19%), and 5 (15.7%) were the most frequent according to IgG ELISA. CONCLUSIONS: In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.

Pneumococcal etiology in hospitalised children with community-acquired penumonia in Belgium :evaluation by culture, RT-PCR and serotype-specific IgG and IgA serology.

info:eu-repo/semantics/nonPublished

Pneumococcal aetiology and serotype distribution in paediatric community-acquired pneumonia.

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0-14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology.

Community-acquired infections and their association with myeloid malignancies

Introduction: Antigenic stimulation is a proposed aetiologic mechanism for many haematological malignancies. Limited evidence suggests that community-acquired infections may increase the risk of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). However, associations with other myeloid malignancies including chronic myeloid leukaemia (CML) and myeloproliferative neoplasms (MPNs) are unknown.

Materials and methods: Using the Surveillance, Epidemiology and End Result (SEER)-Medicare database, fourteen community-acquired infections were compared between myeloid malignancy patients [AML (n=8489), CML (n=3626) diagnosed 1992-2005; MDS (n=3072) and MPNs (n=2001) diagnosed 2001-2005; and controls (200,000 for AML/CML and 97,681 for MDS/MPN]. Odds ratios (ORs) and 95% confidence intervals were adjusted for gender, age and year of selection excluding infections diagnosed in the 13-month period prior to selection to reduce reverse causality.

Results: Risk of AML and MDS respectively, were significantly associated with respiratory tract infections, bronchitis (ORs 1.20 [95% CI: 1.14-1.26], 1.25 [95% CI: 1.16-1.36]), influenza (ORs 1.16 [95% CI: 1.07-1.25], 1.29 [95% CI: 1.16-1.44]), pharyngitis (ORs 1.13 [95% CI: 1.06-1.21], 1.22 [95% CI: 1.11-1.35]), pneumonia (ORs 1.28 [95% CI: 1.21-1.36], 1.52 [95% CI: 1.40-1.66]), sinusitis (ORs 1.23 [95% CI: 1.16-1.30], 1.25 [95% CI: 1.15-1.36]) as was cystitis (ORs 1.13 [95% CI: 1.07-1.18], 1.26 [95% CI: 1.17-1.36]). Cellulitis (OR 1.51 [95% CI: 1.39-1.64]), herpes zoster (OR 1.31 [95% CI: 1.14-1.50]) and gastroenteritis (OR 1.38 [95% CI: 1.17-1.64]) were more common in MDS patients than controls. For CML, associations were limited to bronchitis (OR 1.21 [95% CI: 1.12-1.31]), pneumonia (OR 1.49 [95% CI: 1.37-1.62]), sinusitis (OR 1.19 [95% CI: 1.09-1.29]) and cellulitis (OR 1.43 [95% CI: 1.32-1.55]) following Bonferroni correction. Only cellulitis (OR 1.34 [95% CI: 1.21-1.49]) remained significant in MPN patients. Many infections remained elevated when more than 6 years of preceding claims data were excluded.

Discussion: Common community-acquired infections may be important in the malignant transformation of the myeloid lineage. Differences in the aetiology of classic MPNs and other myeloid malignancies require further exploration.

Common community-acquired infections and subsequent risk of multiple myeloma: a population-based study

The role of bacteria and viruses as aetiological agents in the pathogenesis of cancer has been well established for several sites, including a number of haematological malignancies. Less clear is the impact of such exposures on the subsequent development of multiple myeloma (MM). Using the population-based U.S. Surveillance Epidemiology and End Results-Medicare dataset, 15,318 elderly MM and 200,000 controls were identified to investigate the impact of 14 common community-acquired infections and risk of MM. Odds ratios (ORs) and associated 95% confidence intervals (CIs) were adjusted for sex, age and calendar year of selection. The 13-month period prior to diagnosis/selection was excluded. Risk of MM was increased by 5-39% following Medicare claims for eight of the investigated infections. Positive associations were observed for several infections including bronchitis (adjusted OR 1.14, 95% CI 1.09-1.18), sinusitis (OR 1.15, 95% CI 1.10-1.20) pneumonia (OR 1.27, 95% CI 1.21-1.33), herpes zoster (OR 1.39, 95% CI 1.29-1.49) and cystitis (OR 1.09, 95% CI 1.05-1.14). Each of these infections remained significantly elevated following the exclusion of more than 6 years of claims data. Exposure to infectious antigens may therefore play a role in the development of MM. Alternatively, the observed associations may be a manifestation of an underlying immune disturbance present several years prior to MM diagnosis and thereby part of the natural history of disease progression.

Community-acquired infections associated with increased risk of lymphoplasmacytic lymphoma/Waldenström macroglobulinaemia

Emerging evidence supports the role of immune stimulation in the development of lymphoplasmacytic lymphoma/Waldenström Macroglobulinaemia (LPL/WM). Using the population-based Surveillance, Epidemiology End Results-Medicare database we investigated the exposure to 14 common community-acquired infections and subsequent risk of LPL/WM in 693 LPL/WM cases and 200 000 controls. Respiratory tract infections, bronchitis [odds ratio (OR) 1·56], pharyngitis (OR 1·43), pneumonia (OR 1·42) and sinusitis (OR 1·33) and skin infection, herpes zoster (OR 1·51) were all significantly associated with subsequent increased risk of LPL/WM. For each of these infections, the findings remained significantly elevated following the exclusion of more than 6 years of Medicare claims data prior to LPL/WM diagnosis. Our findings may support a role for infections in the development of LPL/WM or could reflect an underlying immune disturbance that is present several years prior to diagnosis and thereby part of the natural history of disease progression.