Pneumocystis jirovecii pneumonia (PCP) is one of the most common HIV-related opportunistic infections. The diagnosis of PCP is based on analyses from respiratory tract specimens which may require the invasive procedure of a diagnostic bronchoscopy. The objective of this study was to evaluate the diagnostic potential of Pneumocystis jirovecii PCR in serum combined with the 1,3-β-D-glucan (betaglucan) test for the diagnosis of PCP in HIV-infected patients. This was a retrospective case-control study including serum samples from 26 HIV-infected patients with PCP collected within 5 days prior to the start of PCP treatment, 21 HIV-infected control subjects matched by blood CD4+ cell counts, and 18 blood donors. The serum samples were analyzed for Pneumocystis jirovecii PCR and betaglucan. The reference standard for PCP was based on previously described microbiological and clinical criteria. All patients with PCP had detectabe Pneumocystis jirovecii DNA in serum yielding a sensitivity for the Pneumocystis jirovecii PCR assay in serum of 100%. All blood donors had negative Pneumocystis PCR in serum. The specificity when testing HIV-infected patients was 71%, but with a PCR Cycle threshold (Ct) value of 34 as cut-off the specificity was 90%. At a putative pretest probaility of 20%, the negative and positive predictive value for the Pneumocystis PCR assay in serum was 0.99 and 0.71, respectively. Betaglucan with cut-off level 200 pg/ml combined with a positive Pneumocystis jirovecii PCR result had sensitivity and specificity of 92 and 90%, respectively. The concentration of Pneumocystis jirovecii DNA in serum samples, expressed by the PCR Ct values, correlated inversely to the betaglucan levels in serum. In this case-control study including 70% of all HIV-infected patients with PCP treated at Sahlgrenska University Hospital during a time period of 13 years, Pneumocystis PCR analysis on serum samples had a very high sensitivity and negative predictive value for the diagnosis of PCP in HIV-infected patients. A serum-based diagnostic procedure either based on Pneumocystis jirovecii PCR alone or in combination with betaglucan analysis may thus be feasible and would facilitate the care of HIV-infected patients with suspected PCP.

Tick-borne ‘Neoehrlichia (N.) mikurensis’ is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding ‘N. mikurensis’ genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three ‘N. mikurensis’ strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10× Chromium technology, and sequencing by Illumina Hiseq-4500. ‘N. mikurensis’ was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that ‘N. mikurensis’ was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced ‘N. mikurensis’ strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium.

Diagnosis of Pneumocystis jirovecii pneumonia (PCP) relies on lower respiratory tract sampling, often requiring bronchoscopy. Given its invasive nature, non-invasive diagnostic methods are desirable. In this retrospective case–control study, we evaluated the diagnostic performance of serum P. jirovecii PCR and β-D-glucan for PCP diagnosis. Adult patients with hematologic malignancies and positive P. jirovecii PCR in lower respiratory tract samples were included as cases and classified as PCP or colonization. Controls had negative BAL PCR. Stored serum samples were analyzed by P. jirovecii PCR and β-D-glucan. Forty-one cases (29 PCP, 12 colonization) and 36 controls were included. Serum P. jirovecii PCR was positive in 20/41 cases (49%) and in 15/29 patients with PCP (52%), while no controls were serum PCR-positive. For PCP diagnosis, serum PCR had a sensitivity of 52% and a specificity of 89%. For detection of P. jirovecii in the lower respiratory tract, sensitivity was 49% and specificity 100%. Detectable serum P. jirovecii DNA was associated with higher P. jirovecii load in respiratory tract samples and higher β-D-glucan levels, but not with the presence or severity of pneumonia. Serum P. jirovecii PCR is a highly specific marker of P. jirovecii in the lower respiratory tract and may serve as a useful initial non-invasive test in patients with suspected PCP, prior to invasive respiratory sampling.