Pneumocystis jiroveci

For PCR evaluation of respiratory specimens, we used the Fast-track Diagnostics Respiratory Pathogens 33 multiplex PCR kit (FTD Resp-33 kit) (Fast-track Diagnostics, Sliema, Malta). NP/OP specimens were collected in viral transport medium (universal transport medium [UTM], Copan Diagnostics, Bresica, Italy) and refrigerated at 2°C–8°C for a maximum of 8 hours, or frozen at –80°C prior to nucleic acid extraction. Induced sputum, pleural fluid, and lung aspirate specimens were collected in saline in universal containers and either refrigerated at 2°C–8°C for a maximum of 24 hours, or frozen at –80°C prior to nucleic acid extraction.
Total nucleic acid extraction was performed on respiratory specimens using the NucliSENS easyMAG platform (bioMérieux, Marcy l’Etoile, France). Four hundred microliters of each respiratory specimen (NP specimen in UTM, induced sputum aliquot in normal saline, pleural fluid aliquot, or lung aspirate aliquot) was eluted to a final volume of 60–110 μL nucleic acid. Prior to extraction, induced sputum specimens were digested with 1:1 dithiothreitol and incubated at ambient temperature until any mucus was broken down.
The FTD Resp-33 kit is a real-time PCR arranged in 8 multiplex groups for the detection of the following 33 viruses, bacteria, and fungi: influenza A, B, and C; parainfluenza viruses 1, 2, 3, and 4; coronaviruses NL63, 229E, OC43, and HKU1; human metapneumovirus A/B; human rhinovirus; respiratory syncytial virus A/B; adenovirus; enterovirus, parechovirus; bocavirus; cytomegalovirus; Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Streptococcus pneumoniae; Haemophilus influenzae type b; Staphylococcus aureus; Moraxella catarrhalis; Bordetella pertussis; Klebsiella pneumoniae; Legionella species; Salmonella species; and Haemophilus influenzae species. The K. pneumoniae target was not used in any of the final analyses because of difficulties with assay specificity, as has been found elsewhere [9 (link)]. Positive, negative, and internal extraction controls were included in each run.
Quantitative PCR (qPCR) data were generated through the creation of standard curves using 10-fold serial dilutions of plasmid standards provided by FTD on an approximately quarterly basis at each study site, with calculation of pathogen density (copies/milliliter) from the sample cycle threshold (Ct) values. Because the results for the known standards were highly consistent across laboratories, standard curve data from all sites were pooled to create “standardized” standard curves for each pathogen target; data points beyond 2 standard deviations of the mean were excluded. Quantitative PCR was performed at each site using an Applied Biosystems 7500 (ABI-7500) platform (Applied Biosystems, Foster City, California). Cycling conditions were 50°C for 15 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 8 seconds followed by 60°C for 34 seconds.

Before the administration of ATG (1.5 mg/kg/day intravenously), chlorpheniramine and acetaminophen were given intravenously as a premedication. The dose of ATG was reduced by 50% in patients with thrombocytopenia (platelet count 50,000–75,000 per cubic millimeter) or neutropenia (absolute neutrophil count 2000–3000 per cubic millimeter). ATG was discontinued when the patiet developed severe thrombocytopenia (platelet count < 50,000 per cubic millimeter) or severe neutropenia (absolute neutrophil count < 2000 per cubic millimeter).
The maintenance immunosuppressants consisted of tacrolimus, mycophenolate mofetil, and seven-day methylprednisolone taper. Tacrolimus was initiated two days before kidney transplantation at a dose of 0.05 mg/kg twice a day, and the target trough level was 6–8ng/ml until one year post-transplant. Mycophenolate mofetil was given 750 mg twice a day in both groups. Methylprednisolone was administered at a dose of 500 mg intravenously on day 0, 250 mg on day 1, and 125 mg on day 2 and 3. Thereafter, a fast taper was carried out with oral prednisone in the first week post-transplant.
All recipients received oral doses of trimethoprim 80 mg-sulfamethoxazole 400 mg daily for six months for bacterial and Pneumocystis jiroveci prophylaxis. Valganciclovir was administered for cytomegalovirus (CMV) prophylaxis for six months when a seronegative recipient had kidney transplantation from a seropositive donor. For low-to-intermediate risk patients, CMV monitoring was performed on a weekly basis using CMV-PCR assay for preemptive treatment. If the viral load of CMV was more than 4.0 log in the CMV-PCR assay, intravenous ganciclovir or oral valganciclovir was administered until CMV viremia was eliminated. In addition, BKV-PCR assay was also done on a monthly basis for BKV monitoring.