A 68-year-old man with end-stage renal disease of unknown etiology underwent a KTx from a 32-year old deceased female donor, who died of head trauma (cytomegalovirus D-/R+, Epstein-Barr virus D+/R+). He had a past history of type 2 diabetes, hypertension, coronary artery disease and squamous cell carcinoma of the tibial area resected 8 years before the KTx. His past medical history also included pulmonary tuberculosis. He had been maintained on haemodialysis for the preceding seven years. At the time of KTx he was in a good general condition. HLA mismatching was 0 : 1 : 0 (A, B, DR), respectively. Anesthesia and surgery were uneventful, with good initial kidney perfusion. He underwent basiliximab induction therapy and commenced EVR, cyclosporine (CsA) and prednisone maintenance therapy. EVR was introduced because of the history of squamous cell carcinoma. CsA was chosen over tacrolimus in order to prevent significant deterioration of glycemic control in a patient with type 2 diabetes. The doses of EVR and cyclosporine were adjusted to maintain standard target trough levels. The chest X-ray performed on the day of KTx showed no obvious abnormality. Three months later the patient presented with fever, progressive dyspnea, and a productive cough for two weeks. On general examination, the patient was conscious and well-oriented. He had a pulse rate of 108/min, lood pressure of 100/60 mmHg, respiratory rate of 28 cycles/min and there was evident cyanosis. Room air oxygen saturation was around 90%. Other systemic examinations were normal. Admission chest radiography showed patchy infiltrates suggestive of bacterial pneumonia in the left inferior segments corresponding to crackles, rhonchi and wheezes on auscultation. His laboratory testing revealed a white blood cell (WBC) count of 15.8 x 109/l, hemoglobin 11 g/dl, platelet count 216 x 109/l, absolute neutrophil count 14.9 x 109/l, absolute lymphocyte count 0.32 x 109/l, and C-reactive protein (CRP) 281 mg/l. Kidney function was stable with serum creatinine of 0.92 mg/dl and MDRD eGFR on admission > 60 ml/min/1.73 m2. The EVR level was 7.1 ng/ml and cyclosporine level 150 ng/ml (Table 1).
He initially received amoxicillin/clavulanic acid. Bacterial cultures of blood and urine were negative. The symptoms improved, and fever resolved, with a decrease in both WBC and CRP levels over the next few days with empiric antibiotic treatment. There was clinical improvement, but without a radiological resolution. An HRCT scan (Fig. 1A) revealed a disseminated nodular pattern in both lungs, accompanied by peribronchial consolidations and lung parenchyma retraction dominating in the lower lobes. No pleural effusion was seen. Bronchoscopy was unremarkable. A transbronchial biopsy revealed mild nonspecific inflammation with areas of fibrosis in the bronchial walls. Bronchoalveolar lavage was sent for cell count, bacterial and viral culture, and fungal and acid-fast bacillus analysis. Both sputum and bronchoalveolar lavage cultures were positive for Achromobacter denitrificans. After a complete diagnostic workup including repeated bacterial and mycobacterial cultures, QuantiFERON1-TB Gold test, blood CMV-PCR, blood Aspergillus galactomannan and Candida mannan, immunofluorescent staining of bronchoalveolar fluid for Pneumocystis jiroveci, other pulmonary opportunistic infections were ruled out, and a diagnosis of A. denitrificans pneumonia and EVR pulmonary toxicity was made. A 17-day course of piperacillin/tazobactam chosen according to the susceptibility profile (Table 2), and a reduction of the EVR dose to the target trough levels of 2.8-3.2 ng/ml resulted only in partial resolution of radiological abnormalities confirmed by HRCT (Fig. 1B). After three weeks of antibiotic treatment EVR was withheld. EVR discontinuation with no additional antibiotic treatment resulted in complete recovery and a complete resolution of pulmonary infiltrates in an HRCT performed at a further 10-week follow-up (Fig. 1C). The immunosuppressive regimen on discharge was restricted to cyclosporine and glucocorticosteroids.
We performed a functional analysis of peripheral blood neutrophils and monocytes using Phagotest and Phagoburst tests (both Glycotope Biotechnology, Germany).
Phagotest measures the ability to perform phagocytosis, or specifically the ability of the phagocytes to take up the bacteria, by assessing the level of fluorescence emitted by phagocytes - both monocytes and neutrophils. The fluorescence comes from fluorescently stained bacteria engulfed by the phagocytes. Phagotest was performed in accordance with the manufacturer's protocol and with modifications. The original protocol involves the use of Escherichia coli stained with FITC (fluorescein) only (included in kit). Phagotest was also used for assessing the phagocytosis of A. denitrificans stained with CFDA (carboxyfluorescein diacetate succinimidyl ester). This method was established in our laboratory and described elsewhere. The fluorescence emitted by phagocytes reflects the intensity of the engulfment of bacteria. Apart from using different bacteria, all stages of the manufacturer's manual were performed in the same manner for E. coli and A. denitrificans phagocytosis assessment. Phagoburst measures the ability of phagocytes to effectively kill the bacteria by the oxidative burst. The intensity of oxidative burst is estimated by assessing the conversion of non-fluorescent DHR123 (dihydrorhodamine 123) into green fluorescence-emitting R123 (rhodamine 123) in the presence of reactive oxidants. The higher the fluorescence is, the more intense is the oxidative burst. As in case of Phagotest, the assessment with E. coli (already in the manufacturer's kit) and A. denitrificans isolated from the patient's sputum and bronchoalveolar lavage was performed according to the manufacturer's protocol. The fluorescence intensity was assessed using a FACScan cytometer (BD, USA).
Flow cytometry techniques have been extensively used to evaluate phagocyte function. Both Phagotest and Phagoburst have been shown to be useful tests for the evaluation of phagocytosis and oxidative burst activity in different research contexts, providing repeatable and credible results.
The ex vivo phagocytic activity (Fig. 2) and intensity of oxidative burst (Fig. 3) measured by the mean fluorescent intensity (MFI) of neutrophils and monocytes against E. coli and A. denitrificans were assessed using patients' cells collected on EVR (2.8 ng/ml) and using patient cells collected 3 weeks after EVR discontinuation, and using cells from a healthy control.
The phagocytic activity against A. denitrificans and E. coli of patient neutrophils and monocytes collected during EVR treatment was notably lower compared to neutrophils and monocytes from a healthy control, especially in the case of E. coli. The phagocytic activity of patient neutrophils and monocytes after 3 weeks of EVR absence was higher than patient cell activity in the presence of EVR. The phagocytic activity of patient cells off EVR against A. denitrificans was similar to the activity of healthy controls; however, the activity of patient cells off EVR against E. coli was still below the activity of control cells (Fig. 2).
The level of oxidative burst from neutrophils and monocytes, even more so, obtained from the patient on EVR treatment was significantly lower than the oxidative burst from the healthy control cells. After 3 weeks of EVR absence, the oxidative burst of patient neutrophils engulfing E. coli and A. denitrificans was restored to a level similar to the healthy control, even though the patient continued to receive prednisone and CsA. The level of oxidative burst carried out by patient monocytes was also improved with EVR absence, but still lower by half compared to monocytes of a healthy control (Fig. 3).
To confirm the aforementioned EVR properties we performed a similar assay in vitro using peripheral blood neutrophils and monocytes from a healthy donor. These cells were exposed to four different concentrations of EVR, ranging from 2 to 15 ng/ml (2 ng/ml, 5 ng/ml, 8 ng/ml and 15 ng/ml). The processes of phagocytosis and oxidative burst generation against both A. denitrificans and E. coli were again significantly affected by increasing doses of EVR in a dose-dependent manner (Figs. 4 and 5).
Initial computed tomography (CT) of the lungs shows poorly defined bilateral patchy consolidations without pleural effusion,.