Diagnosis of external ventricular drainage related infections with real-time 16S PCR and third-generation 16S sequencing.

OBJECTIVE: Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). METHODS: Subjects with suspected EVDRI were prospectively included at Uppsala University Hospital. Subjects were included into three groups: subjects with negative CSF culture with and without antibiotic treatment and subjects with positive CSF culture, respectively. CSF was analysed with real-time 16S PCR and third-generation 16S sequencing. Real-time 16S PCR positivity/negativity and number of 16S sequence reads were compared between groups. For culture positive subjects, species identification in third-generation sequencing and routine culture was compared. RESULTS: 84 subjects were included. There were 18, 44 and 22 subjects in the three groups. Real-time PCR was positive in 17 of 22 subjects in the culture positive group and negative in 61 of the 62 subjects in the two culture negative groups. The sensitivity and specificity for real-time 16S PCR compared to culture was estimated to 77% and 98%, respectively. Species identification in 16S sequencing and culture was concordant in 20 of 22 subjects. The number of 16S sequence reads were significantly higher in the culture positive group than in both culture negative groups (p < 0.001). There was no significant difference in number of 16S sequences between the two culture negative groups. CONCLUSIONS: Real-time 16S PCR predict culture results with sufficient reliability. Third-generation 16S sequencing could enhance sensitivity and species identification in diagnostics of EVD-related infections. False negative culture results appear to be uncommon in patients with suspected EVDRI.

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans.

BACKGROUND: A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country. OBJECTIVE: To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS). METHODS: WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates. RESULTS: The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST. CONCLUSION: No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.

Comparative distribution of extended-spectrum beta-lactamase-producing Escherichia coli from urine infections and environmental waters.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli have been reported in natural environments, and may be released through wastewater. In this study, the genetic relationship between ESBL-producing E. coli collected from patient urine samples (n = 45, both hospitalized patients and out-patients) and from environmental water (n = 82, from five locations), during the same time period, was investigated. Three independent water samples were collected from the municipal wastewater treatment plant, both incoming water and treated effluent water; the receiving river and lake; and a bird sanctuary near the lake, on two different occasions. The water was filtered and cultured on selective chromID ESBL agar plates in order to detect and isolate ESBL-producing E. coli. Illumina whole genome sequencing was performed on all bacterial isolates (n = 127). Phylogenetic group B2 was more common among the clinical isolates than the environmental isolates (44.4% vs. 17.1%, p < 0.01) due to a significantly higher prevalence of sequence type (ST) 131 (33.3% vs. 13.4%, p < 0.01). ST131 was, however, one of the most prevalent STs among the environmental isolates. There was no significant difference in diversity between the clinical isolates (DI 0.872 (0.790-0.953)) and the environmental isolates (DI 0.947 (0.920-0.969)). The distribution of ESBL genes was similar: blaCTX-M-15 dominated, followed by blaCTX-M-14 and blaCTX-M-27 in both the clinical (60.0%, 8.9%, and 6.7%) and the environmental isolates (62.2%, 12.2%, and 8.5%). Core genome multi-locus sequence typing showed that five environmental isolates, from incoming wastewater, treated wastewater, Svartån river and Hjälmaren lake, were indistinguishable or closely related (≤10 allele differences) to clinical isolates. Isolates of ST131, serotype O25:H4 and fimtype H30, from the environment were as closely related to the clinical isolates as the isolates from different patients were. This study confirms that ESBL-producing E. coli are common in the aquatic environment even in low-endemic regions and suggests that wastewater discharge is an important route for the release of ESBL-producing E. coli into the aquatic environment.

Evaluation of a Commercial Multiplex PCR Assay for Detection of Pathogen DNA in Blood from Patients with Suspected Sepsis.

The Magicplex Sepsis Real-time Test (MST) is a commercial multiplex PCR that can detect more than 90 different pathogens in blood, with an analysis time of six hours. The aim of the present study was to evaluate this method for the detection of bloodstream infection (BSI). An EDTA whole blood sample for MST was collected together with blood cultures (BC) from patients with suspected sepsis at the Emergency Department of a university hospital. Among 696 study patients, 322 (46%) patients were positive with at least one method; 128 (18%) were BC positive and 268 (38%) were MST positive. Considering BC to be the gold standard, MST had an overall sensitivity of 47%, specificity of 66%, positive predictive value (PPV) of 23%, and a negative predictive value of 87%. Among the MST positive samples with a negative BC, coagulase-negative staphylococci (CoNS) and species that rarely cause community-acquired BSI were frequently noted. However, the quantification cycle (Cq) values of the MST+/BC- results were often high. We thus hypothesized that the performance of the MST test could be improved if the Cq cut-off level was adjusted downwards. With a lower Cq cut-off value, i.e. 6.0 for Staphylococcus species and 9.0 for all other species, the number of MST positive cases decreased to 83 (12%) and the overall sensitivity decreased to 38%. However, the PPV increased to 59% and the specificity increased to 96%, as many MST positive results for CoNS and bacteria that rarely cause community-acquired BSI turned MST negative. In conclusion, our study shows that with a lower Cq cut-off value, the MST will detect less contaminants and findings with unclear relevance, but to the cost of a lower sensitivity. Consequently, we consider that a positive MST results with a Cq value above the adjusted cut-off should be interpreted with caution, as the result might be clinically irrelevant. In a correspondent way, quantitative results could probably be useful in the interpretation of positive results from other molecular assays for the detection of BSI.

Irreversible Kidney Damage due to Multicentric Castleman's Disease.

Castleman's Disease (CD) is a rare lymphoproliferative disorder accompanied by marked systemic inflammatory response. Morphological diagnosis of CD requires biopsy of the whole of the involved lymph node tissue. Three histologic variants have already been described in CD morphology (hyaline vascular, plasma-cell, and mixed). In this study, we report a case of a multicentric Castleman's disease of the plasma cell variant type with negative Herpes Virus 8. The clinical presentation of this patient was of systemic amyloidosis as a result of both a delayed diagnosis and medical management. Previously described cases of CD with secondary amyloidosis have been of the localized type. Regardless, long-standing clinical remission of CD by cytotoxic drugs and anti-CD20 antibody therapy was achieved, but the nephrotic syndrome remained irreversible.