The Use of CHROMID® Colistin R for the Detection of Colistin-Resistant Gram-Negative Bacteria in Positive Blood Cultures.

The aim of this study was to assess the utility of CHROMID® Colistin R for direct detection of colistin-resistant Gram-negative bacteria from positive blood cultures. A total of 390 blood cultures from hospitalised patients containing Gram-negative bacteria were included in this study. These blood cultures were referred to clinical laboratories in the United Kingdom and Türkiye. A further 16 simulated positive blood culture bottles were included that contained a range of colistin-resistant strains as well as susceptible control strains. Fluid from each positive blood culture was diluted 1/200 in saline and 10 µL aliquots cultured onto cystine-lactose-electrolyte-deficient agar and CHROMID® Colistin R. All recovered bacteria were identified, and for Gram-negative bacteria, their minimum inhibitory concentration of colistin was measured using the broth microdilution method. From a total of 443 Gram-negative isolates, 57 colistin-resistant isolates were recovered, of which 53 (93%) grew on CHROMID® Colistin R within 18 h. Of the 377 isolates determined to be colistin-susceptible, only 9 isolates were able to grow, including 6 isolates of Pseudomonas aeruginosa. For positive blood cultures that are shown to contain Gram-negative bacteria, culture on CHROMID® Colistin R is a useful diagnostic tool to detect susceptibility or resistance to colistin within 18 h.

Aetiology of paediatric pneumonia after the introduction of pneumococcal conjugate vaccine.

We describe the aetiology of community-acquired pneumonia in children before and after the introduction of the pneumococcal conjugate vaccination (PCV) programme in 2006. Prospective studies were conducted in 2001-2002 (pre-vaccine) and 2009-2011 (post-vaccine) of children aged 0-16 years with radiologically confirmed pneumonia seen in hospital. Investigations included culture, serology, immunofluorescence antibody and urine antigen testing, with an increased use of PCR assays and expanded panels of pathogens in the post-vaccine study. 241 and 160 children were enrolled in the pre- and post-vaccine studies, respectively (73% aged <5 years). Identification of a causative pathogen was higher post-vaccination (61%) than pre-vaccination (48.5%) (p=0.019). Rates of bacterial infections were not different between post- and pre-vaccine studies (17.5% versus 24%, p=0.258). Viral (31%) and mixed (12.5%) infections were found more often post-vaccination (19.5%, p=0.021) than pre-vaccination (5%, p=0.015). Rates of identified pneumococcal infections were comparable between pre- and post-vaccine studies (14.7% versus 17.4%, p=0.557). Diagnosis of pneumococcal infection post-vaccination improved when PCR was used compared to culture (21.6% versus 6%, p=0.0004). Serotypes included in PCV13 but not PCV7 were identified in 75% (18 out of 24) post-vaccination. Infection with nonvaccine pneumococcal serotypes continues to be a significant cause of pneumonia in children in the UK.

Pneumococcal diagnosis and serotypes in childhood community-acquired pneumonia.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced routinely in the UK from September 2006 and replaced by PCV13 in 2010. In a prospective study from 2009 to 2011 of 160 children aged ≤16 years with radiologically confirmed pneumonia, likely pneumococcal infections were identified in 26%. Detection of pneumococci was improved with polymerase chain reaction compared to culture (21.6% versus 6% of children tested, P = 0.0004). Where serotyping was possible, all (n = 23) were non-PCV7 but PCV13 serotypes; 1 (43.5%), 3 (21.7%), 7A/F, and 19A (17.4% each).

Rapid detection and quantification of CMV DNA in urine using LightCycler-based real-time PCR.

A real-time quantitative PCR-hybridisation assay was developed for the detection of human cytomegalovirus DNA in clinical material. The assay is based on a LightCycler (LC) and provides both rapid results (<1 h) and quantification over a broad dynamic range (2 x 10(3)-5 x 10(8) CMV DNA copies/ml). Given that the assay showed a 3-fold increase in sensitivity compared to detection of early antigen fluorescent foci (DEAFF) testing of urine samples, we investigated the practicality of testing surveillance such specimens from immunocompromised patients at risk of CMV disease. Over a 12-month period, CMV DNA was detected in 81 (7%) of 1154 urine samples examined. A total of 28 patients tested positive; urine viral loads were higher in 13 infants being investigated for suspected congenital infection (median 1.6 x 10(5) copies/ml) compared with 15 transplant recipients (median 9 x 10(3) copies/ml). Urine samples could be tested directly without processing such that results were available in <1h. Real-time PCR provided information on the quantification of CMV DNA in urine and proved a reliable method for the surveillance of immunocompromised patients at risk of CMV disease. This approach should facilitate a better understanding of the epidemiology and natural history of CMV disease. Moreover, LC-based quantitative PCR is a potentially valuable tool for the management of CMV disease; assisting with the prompt initiation of treatment and assessing therapeutic response.

Detection of pneumolysin in sputum.

Western blot detection of the species-specific pneumococcal product, pneumolysin (SPN), was shown to be almost as sensitive as PCR for the non-cultural detection of pneumococci in 27 Streptococcus pneumoniae culture-positive sputa from patients stated to have chest infections. Both techniques were considerably more sensitive than counter-current immuno-electrophoresis for pneumococcal capsular polysaccharide antigens (CPS-CIE) on the same specimens. Sensitivities for PCR, SPN-immunoblotting and CPS-CIE were 100%, 85% and 67%, respectively. In 11 S. pneumoniae culture-negative sputa taken from patients receiving antibiotics, but with proven recent pneumococcal infection, PCR and SPN-blot were positive in six (in two of which CPS-CIE was also positive), PCR alone was positive in one and SPN-blot alone was positive in one. In 11 S. pneumoniae culture-negative samples from patients not receiving antibiotics, all three tests were negative in eight, PCR was positive in three (in one of which CPS-CIE was also positive), but SPN-blot was negative in all 11. In 16 S. pneumoniae culture-negative samples from patients receiving antibiotics and with no known recent pneumococcal infections, one or more non-cultural test was positive in 11. Although further evaluation is required to assess the significance of pneumolysin detection in relation to carriage and infection and to devise a more suitable test format, these preliminary studies suggest that pneumolysin detection is a promising new approach to the non-cultural diagnosis of pneumococcal chest infection.