Effectiveness of PCR and Immunofluorescence Techniques for Detecting Human Cytomegalovirus in Blood and Bronchoalveolar Lavage Fluid.

Current diagnostic methods allow a rapid and reliable detection of active human cytomegalovirus (hCMV) infection by identifying the presence of pp65 CMV antigen or CMV DNA in peripheral blood and affected organs. The goal of this study was to evaluate the effectiveness of CMV detection in blood and organ-specific biological material, such as bronchoalveolar lavage fluid (BALF), by comparing two standard diagnostic methods, immunofluorescence (IF) and the real-time polymerase chain reaction (PCR). We evaluated 25 patients with concomitant respiratory disease who were referred to our hospital for diagnosis due to suspected acute CMV infection. The presence of hCMV was concomitantly evaluated by IF and PCR in 16 peripheral blood samples. In two patients, we observed positive results for both IF and PCR, and in two other patients the results were discordant. Of 11 patients, CMV DNA was detected in six BALF samples, and in one blood plasma sample. Real-time PCR detected CMV DNA in 54.6 % of BALF samples and 12.0 % of blood samples, while indirect IF testing confirmed antigenemia in 12.5 % of blood samples. The results from our study suggest that the IF method is as effective as PCR for detecting an ongoing CMV infection in blood samples. However, real-time PCR was much more effective at detecting CMV DNA in BALF compared to blood samples. Our results suggest that the biological material being tested during CMV diagnosis should be derived directly from the virally infected organ(s).

Cryptogenic Organizing Pneumonia: IL-1ß, IL-6, IL-8, and TGF- ß1 Serum Concentrations and Response to Clarithromycin Treatment.

Cryptogenic organizing pneumonia (COP) is a distinct clinicopathological entity with unknown etiology. Inflammatory cytokines play a role in the development of the disease. The present study was performed to assess the correlation between concentrations of IL-1ß, IL-6, IL-8, and TGF-ß1 in the serum with response to clarithromycin (CAM) treatment in patients with COP. A total of 39 patients with COP were enrolled in to this study. An oral dose of 500 mg CAM was administered to all of the patients twice daily for 3 months. A complete response was noticed in 31 (80 %) of patients, and 8 (20 %) patients failed to respond to treatment. The concentration of cytokines were assessed by ELISAs before and after treatment. CAM treatment was associated with decreases in serum IL-6 (3.8 pg/mL [IQR 0.9-11.8] vs. 1.1 pg/mL [IQR 0.2-3.1]; p = 0.004), IL-8 (13.6 pg/mL [IQR 9.8-17.5] vs. 8.1 pg/mL [IQR 6.2-13.2]; p = 0.004), and TGF-ß1 (37.1 ng/mL [IQR 31.7-46.2] vs. 25.7 ng/mL [IQR 22-41.7];p = 0.0001), which was particularly notable in the responders. We conclude that IL-6, IL-8, and TGF-ß1 may play a role in the pathogenesis of COP, as their decreased concentrations were associated with a positive response to CAM treatment.

Frequency of Rare Alpha-1 Antitrypsin Variants in Polish Patients with Chronic Respiratory Disorders.

The SERPINA1 gene encoding the alpha-1 antitrypsin (A1AT) protein is highly polymorphic. It is known that, apart from the most prevalent PI*S and PI*Z A1AT deficiency variants, other so-called rare variants also predispose individuals to severe chronic respiratory disorders such as emphysema and chronic obstructive pulmonary disease. Our aim was to assess the frequencies of common and rare SERPINA1 mutations in a group of 1033 Polish patients referred for A1AT deficiency diagnostics due to chronic respiratory disorders in the period of January 2014-September 2015. All blood samples were analyzed according to the routine diagnostic protocol, including A1AT serum concentration assessment by nephelometry and immune isoelectric focusing, followed by PCR genotyping and direct sequencing when necessary. A total of 890 out of the 1033 samples (86 %) carried the normal PI*MM genotype, whereas, in 143 samples (14 %), at least one A1AT deficiency variant was detected. In 132 subjects, PI*S (2.1 %) and PI*Z (10.8 %) common deficiency alleles were identified, yielding frequencies of 0.011 and 0.062, respectively. Rare SERPINA1 variants were detected in nine patients: PI*F (c.739C>T) (n = 5) and PI*I (c.187C>T) (n = 4). Samples from the patients with an A1AT serum concentration below 120 mg/dl and presenting a PI*MM-like phenotypic pattern were retrospectively analyzed by direct sequencing for rare SERPINA1 mutations, revealing a PI*M2Obernburg (c.514G>T) mutation in one patient and a non-pathogenic mutation (c.922G>T) in another. We conclude that the deficiency PI*Z A1AT allele is considerably more common in patients with chronic respiratory disorders than in the general Polish population. The prevalence of the PI*F allele seems higher than in other European studies.

Inflammatory markers in the exhaled breath condensate of patients with pulmonary sarcoidosis.

Pulmonary sarcoidosis may progress to fibrosis in some patients, so that close monitoring of its activity is essential for recommending clinical strategy. Examination of airway inflammatory markers in bronchoalveolar lavage (BAL) is one of the methods applied to assess the disease severity. Recently, the expired breath condensate (EBC) has become another source of cytokines and mediators. In sarcoidosis, except for NO and oxidative stress markers, no other mediators have yet been estimated in the exhaled air. In the present study we attempted to answer the question of whether airway inflammatory markers in pulmonary sarcoidosis patients might be assessable in EBC and to what extend these markers might reflect the disease activity in the lungs IL-6, TNF-alpha, PAI-1, and IGF-1 were measured by Elisa method in EBC and BALF samples from 9 patients with newly-diagnosed pulmonary sarcoidosis. TNF-alpha, IGF-1, and PAI-1 levels in EBC and BAL samples were comparable and closely positively correlated [TNF-alpha (r=0.79, P<0.001), IGF-1 (r=0.94, P<0.001), and PAI-1 (r=0.81, P<0.001)]. In contrast, IL-6 concentration in EBC was significantly lower compared with that in BALF, while the correlation between both materials was negative (r=-0.47, P<0.05). An important distinction in IL-6 performance, which might explain this inconsistency, is its tendency to form more complex molecular forms of a higher weight than that of other cytokines. Our study shows that EBC reflects cytokine production in the lung as effectively as BALF, providing that the characteristics of proteins evaluated allow their easy transfer into the exhaled air. Further studies are required before accepting EBC samples as an equivalent to BALF.