Circulating microbial small RNAs are altered in patients with rheumatoid arthritis.

OBJECTIVES: To determine if plasma microbial small RNAs (sRNAs) are altered in patients with rheumatoid arthritis (RA) compared with control subjects, associated with RA disease-related features, and altered by disease-modifying antirheumatic drugs (DMARDs). METHODS: sRNA sequencing was performed on plasma from 165 patients with RA and 90 matched controls and a separate cohort of 70 patients with RA before and after starting a DMARD. Genome alignments for RA-associated bacteria, representative bacterial and fungal human microbiome genomes and environmental bacteria were performed. Microbial genome counts and individual sRNAs were compared across groups and correlated with disease features. False discovery rate was set at 0.05. RESULTS: Genome counts of Lactobacillus salivarius, Anaerobaculum hydrogeniformans, Staphylococcus epidermidis, Staphylococcus aureus, Paenisporosarcina spp, Facklamia hominis, Sphingobacterium spiritivorum, Lentibacillus amyloliquefaciens, Geobacillus spp, and Pseudomonas fluorescens were significantly decreased in the plasma of RA compared with control subjects. Three microbial transfer RNA-derived sRNAs were increased in RA versus controls and inversely associated with disease activity. Higher total microbial sRNA reads were associated with lower disease activity in RA. Baseline total microbial sRNAs were threefold higher among patients who improved with DMARD versus those who did not but did not change significantly after 6 months of treatment. CONCLUSION: Plasma microbial sRNA composition is altered in RA versus control subjects and associated with some measures of RA disease activity. DMARD treatment does not alter microbial sRNA abundance or composition, but increased abundance of microbial sRNAs at baseline was associated with disease activity improvement at 6 months.

FGF21 is a biomarker for mitochondrial translation and mtDNA maintenance disorders.

OBJECTIVE: To validate new mitochondrial myopathy serum biomarkers for diagnostic use. METHODS: We analyzed serum FGF21 (S-FGF21) and GDF15 from patients with (1) mitochondrial diseases and (2) nonmitochondrial disorders partially overlapping with mitochondrial disorder phenotypes. We (3) did a meta-analysis of S-FGF21 in mitochondrial disease and (4) analyzed S-Fgf21 and skeletal muscle Fgf21 expression in 6 mouse models with different muscle-manifesting mitochondrial dysfunctions. RESULTS: We report that S-FGF21 consistently increases in primary mitochondrial myopathy, especially in patients with mitochondrial translation defects or mitochondrial DNA (mtDNA) deletions (675 and 347 pg/mL, respectively; controls: 66 pg/mL, p < 0.0001 for both). This is corroborated in mice (mtDNA deletions 1,163 vs 379 pg/mL, p < 0.0001). However, patients and mice with structural respiratory chain subunit or assembly factor defects showed low induction (human 335 pg/mL, p < 0.05; mice 335 pg/mL, not significant). Overall specificities of FGF21 and GDF15 to find patients with mitochondrial myopathy were 89.3% vs 86.4%, and sensitivities 67.3% and 76.0%, respectively. However, GDF15 was increased also in a wide range of nonmitochondrial conditions. CONCLUSIONS: S-FGF21 is a specific biomarker for muscle-manifesting defects of mitochondrial translation, including mitochondrial transfer-RNA mutations and primary and secondary mtDNA deletions, the most common causes of mitochondrial disease. However, normal S-FGF21 does not exclude structural respiratory chain complex or assembly factor defects, important to acknowledge in diagnostics. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that elevated S-FGF21 accurately distinguishes patients with mitochondrial myopathies from patients with other conditions, and FGF21 and GDF15 mitochondrial myopathy from other myopathies.

Blood Aspergillus RNA is a promising alternative biomarker for invasive aspergillosis.

A critical challenge for the successful application of antifungal therapies for invasive aspergillosis (IA) is a lack of reliable biomarkers to assess early treatment response. Patients with proven or probable IA were prospectively enrolled, and serial blood samples were collected at 8 specified time points during 12-week antifungal therapy. Total nucleic acid was extracted from 2.5 ml blood and tested for Aspergillus-specific RNA by a pan-Aspergillus real-time nucleic acid sequence-based amplification (NASBA) assay. Serum 1, 3-ß-D-glucan (BG) and galactomannan (GM) were measured in parallel. Clinical outcome was evaluated at 6 and 12 weeks. Overall, 48/328 (14.6%) blood samples from 29/46 (63%) patients had positive NASBA detection at baseline and/or some point during the study. Positive NASBA results during the first 4 and 6 weeks of treatment are significantly associated with the 12-week outcome. Blood RNA load change during weeks 4-6 may be informative to predict outcome at 12 weeks. While independent of serum GM, the kinetic change of circulating Aspergillus RNA appears to be well correlated with that of BG on some patient individuals. Monitoring blood Aspergillus RNA during the first 4-6 weeks of antifungal treatment may help assess therapeutic response. Combination of circulating Aspergillus RNA and BG may be a useful adjunct to assess response.

Short Communication: Diagnosis of Pneumocystis jirovecii Pneumonia by Detection of DNA in Blood and Oropharyngeal Wash, Compared with Sputum.

Molecular diagnostic methods on lower respiratory specimens for Pneumocystis pneumonia (PCP) are recommended, but specimens can be difficult to obtain. This study examined the diagnostic use of PCP polymerase chain reaction (PCR) on oropharyngeal wash (OPW) and blood versus sputum (spontaneous and induced) to find faster, simpler, and less invasive diagnostic methods. We prospectively recruited consenting adults with symptoms consistent with PCP. Real-time PCR targeted the Pneumocystis mitochondrial large subunit ribosomal RNA gene, using the aforementioned specimens. Clinical data were collected from routine records. Forty-five participants provided 45 sputa, 31 OPW, and 41 blood samples. Median age was 39 years and 41 (91%) were male, with median CD4 count being 64 cells/µL. Sputum PCR was positive in 27/45 (60%) participants. Comparative sensitivity of OPW was 9/19 (47%, 95% confidence interval [CI] 23-71) and blood 12/24 (50%, 95% CI 29-71) participants, both with specificity 100%. Including only samples obtained ≤2 days after start of treatment, sensitivity of OPW was 80% (8/10, 95% CI 51-100), that of blood was 57% (8/14, 95% CI 29-86), and that of combined tests was 88% (14/16, 95% CI 70-100). In 14/16 individuals with PCP and specimens obtained ≤2 days after start of treatment, diagnosis was possible using nonrespiratory samples. Despite moderate sensitivity of individual tests, combined PCP PCR on early blood and OPW specimens had high sensitivity and could reduce the need for invasive procedures. There were no false-positive results on nonrespiratory samples. Sampling and laboratory methods use routine technology and so require few additional resources.

RT-qPCR detection of Aspergillus fumigatus RNA in vitro and in a murine model of invasive aspergillosis utilizing the PAXgene® and Tempus™ RNA stabilization systems.

Molecular diagnosis of invasive aspergillosis (IA) is a potentially life-saving tool in the care of at-risk individuals. To date, the development of PCR-based diagnostic tests has been hampered by the lack of standardization in the methods for such critical activities. In this study, we used both spiked volunteer blood samples and a murine model of IA to test the utility of the PAXgene and Tempus systems for stabilization and isolation of fungal RNA from blood as part of an evaluation of a new diagnostic strategy. In spiking experiments, RNA isolation followed by RT-qPCR that targeted the 18S gene was compared to a standard DNA isolation and qPCR assay that targeted the ITS ribosomal region. We demonstrated that both PAXgene and Tempus RNA stabilization and extraction systems followed by RT-qPCR had similar performance in detecting fungal RNA in blood samples from Aspergillus fumigates-infected mice. In spiked samples, the Tempus system performed better than the PAXgene system as it detected 100% of all samples spiked with 10 or 20 germinated Aspergillus conidia/ml blood sample as compared to the PAXgene system which detected 33% and 56% of the samples spiked with 10 or 20 conidia/ml, respectively. The stabilization of fungal nucleic acids in blood samples and its efficient isolation by a commercial method is an important step in the development of standardized molecular diagnostic tools that are needed to improve the outcomes for individuals with IA.

Pneumocystis murina colonization in immunocompetent surfactant protein A deficient mice following environmental exposure.

BACKGROUND: Pneumocystis spp. are opportunistic pathogens that cause pneumonia in immunocompromised humans and animals. Pneumocystis colonization has also been detected in immunocompetent hosts and may exacerbate other pulmonary diseases. Surfactant protein A (SP-A) is an innate host defense molecule and plays a role in the host response to Pneumocystis. METHODS: To analyze the role of SP-A in protecting the immunocompetent host from Pneumocystis colonization, the susceptibility of immunocompetent mice deficient in SP-A (KO) and wild-type (WT) mice to P. murina colonization was analyzed by reverse-transcriptase quantitative PCR (qPCR) and serum antibodies were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Detection of P. murina specific serum antibodies in immunocompetent WT and KO mice indicated that the both strains of mice had been exposed to P. murina within the animal facility. However, P. murina mRNA was only detected by qPCR in the lungs of the KO mice. The incidence and level of the mRNA expression peaked at 8-10 weeks and declined to undetectable levels by 16-18 weeks. When the mice were immunosuppressed, P. murina cyst forms were also only detected in KO mice. P. murina mRNA was detected in SCID mice that had been exposed to KO mice, demonstrating that the immunocompetent KO mice are capable of transmitting the infection to immunodeficient mice. The pulmonary cellular response appeared to be responsible for the clearance of the colonization. More CD4+ and CD8+ T-cells were recovered from the lungs of immunocompetent KO mice than from WT mice, and the colonization in KO mice depleted CD4+ cells was not cleared. CONCLUSION: These data support an important role for SP-A in protecting the immunocompetent host from P. murina colonization, and provide a model to study Pneumocystis colonization acquired via environmental exposure in humans. The results also illustrate the difficulties in keeping mice from exposure to P. murina even when housed under barrier conditions.

[Detection of Aspergillus infection in blood of malignant hematopoietic tumor patients by two-step polymerase chain reaction].

OBJECTIVE: To investigate the sensitivity and specificity of two-step PCR in detection of Aspergillus species in blood from malignant hematopoietic tumor patients. METHODS: Forty-one blood samples from high-risk patients were detected with two-step PCR. The clinical applicability was assessed with analyzing computed tomography, Aspergillus culture, neutropenia and outcome after anti-fungal therapy. RESULTS: Specific band of PCR was obtained from Aspergillus strains and products of PCR had a high homogeneous in sequence with Aspergillus species. Sixteen of 41 patients were PCR positive and controls were all PCR negative. Among PCR positive patients, 4 patients had positive cultures. The lowest WBC count was (0.30 +/- 0.14) x 10(9)/L and (0.50 +/- 0.26) x 10(9)/L respectively and duration with WBC count less than 1.0 x 10(9)/L was (19.00 +/- 8.31) days and (12.69 +/- 6.95) days respectively in the two groups. After antifungal therapy, one of the patients with PCR positive died of chemotherapy-related early death. CONCLUSIONS: The two-step PCR assay allows for highly sensitive and specific detection of Aspergillus pathogens in vitro and in vivo. The detection with PCR has some value in the early diagnosis and helps us to make decision for antifungal therapy.

Polyadenylation of ribosomal RNA by Candida albicans also involves the small subunit.

BACKGROUND: Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes. RESULTS: We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha. CONCLUSIONS: The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process.

Clinical evaluation of a NASBA-based assay for detection of Candida spp. in blood and blood cultures.

The number of life-threatening opportunistic fungal infections has shown a dramatic increase. However, the diagnosis of candidemia remains difficult. Nucleic acid amplification assays may improve the detection rate and decrease the time needed for detection and identification of Candida spp. Whole blood samples of patients suspected of having candidemia were analyzed using Nucleic Acid Sequence-Based Amplification (NASBA). Furthermore, aliquots of blood cultures of the patients after 2 days of culturing were tested. Eleven data sets from ten patients in two hospitals were generated. None of the whole blood samples was positive in the NASBA assay. Eight samples were positive in the NASBA assay after two days of culturing, whereas only two additional positive samples were found after longer incubation periods. Thus, a two-day culture step is sufficient to greatly improve the sensitivity of the NASBA assay. The NASBA assay detected Candida RNA in three patients. In one patient, the yeast was not detected by automated blood culturing, in another patient the NASBA assay detected the infection two days earlier than the blood culture system.

Detection of Candida spp. in blood cultures using nucleic acid sequence-based amplification (NASBA).

Candida spp. are the main causes of fungal infections in immunocompromised patients. It is known, that the routinely used automated blood culture systems may fail to detect yeasts. We therefore investigated, whether Nucleic Acid Sequence-Based Amplification (NASBA) can be used to improve the detection rate of Candida spp. in blood cultures. Culture-positive as well as negative blood cultures from patients with a proven candidaemia were analyzed, and the results of BacT/Alert monitoring were compared with the results of NASBA-based detection of yeast RNA. With the NASBA-assay, the number of positive blood cultures increased from 21% to 34%. The NASBA-assay may confirm the diagnosis and demonstrate the need for prolonged treatment. In addition it may shorten the time to detection. In summary, using NASBA for the detection of yeast RNA in blood cultures, we have shown for the first time that it is possible to improve the detection rate of yeasts in blood cultures by using amplification technology.

Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.