Determination of tacrolimus in human whole blood in kidney transplant recipients using a rapid and specific LC-MS/MS method.

OBJECTIVE: To develop and validate an LC-M/SMS method for the determination of tacrolimus in human whole blood. METHOD: The LC-MS/MS method for the determination of tacrolimus in whole blood was developed and validated according to the guidelines. Concentrations of TAC in 100 kidney transplant patients measured by LC-MS/MS were compared with CMIA using correlation analysis and Bland-Altman plots. RESULTS: The method had a total chromatographic run time of 5 min. The calibration curves were linear over the range of 0.5-100.0 ng/mL with a lower limit of quantification of 1 ng/mL. The intra- and interday accuracy was within the range of 93.3%-109.2% and 96.0%-108.4%, respectively, with precision ranging from 0.8 to 9.4%. The mean extraction recoveries of TAC ranged from 102.6 to 107.8%. The mean concentrations of TAC in whole blood of kidney transplant patients measured by the two assays were different at 1, 3 months and all time points (p < 0.001), but no significant difference was observed at 6 months (p = 0.094). The correlation of data was good with the correlation coefficients (r2 ) of 0.7581, 0.8811, 0.8777, and 0.8077, respectively. Passing-Bablok regression analysis demonstrated good correlations with r2 values higher than 0.88 between TAC levels measured by LC-MS/MS and CMIA. Using Bland-Altman plots yielded average biases of 1.29, 0.79, 0.11, and 0.65 ng/mL at 1, 3, and 6 months and all time points. CONCLUSION: The LC-MS/MS method was validated for the accurate determination of TAC in human whole blood. The comparison of tacrolimus concentrations measured by the LC-MS/MS with CMIA showed a good correlation and agreement of two methods, suggesting LC-MS/MS should be used routinely to monitor TAC concentrations in kidney transplant patients.

Stepwise Reduction of Mycophenolate Mofetil with Conversion to Everolimus for the Treatment of Active BKV in Kidney Transplant Recipients: A Single-Center Experience in Vietnam.

Background: No specific antiviral drug can effectively treat BKV reactivation after kidney transplantation. Thus, we evaluated stepwise-reduced immunosuppression to treat BKV reactivation. Methods: 341 kidney-transplant recipients were monitored for BKV infection (BKV-viremia, BKV-viruria). Positive samples with a significant virus load were nested PCR-genotyped in the VP1 region. In 97/211 patients presenting BKV viremia ≥104 copies/mL and/or BKV viruria ≥107 copies/mL, or BKV-nephropathy immunosuppression (i.e., mycophenolate mofetil [MMF]) was reduced by 50%. If viral load did not decrease within 28 days, MMF dose was further reduced by 25%, although calcineurin-inhibitor (CNI) therapy remained unchanged. If BKV viral load did not decrease within another 28 days, MMF was withdrawn and replaced by everolimus combined with reduced CNIs. Results: Only 41/97 BKV (+) cases completed the 6-month follow-up. Among these, 29 (71%) were in the BKV-I group and 12 (29%) were in BKV-IV. BKV viruria and BKV viremia were significantly decreased from 9.32 to 6.09 log10 copies/mL, and from 3.59 to 2.45 log10 copies/mL (p < 0.001 and p = 0.024, respectively). 11/32 (34.4%) patients were cleared of BKV viremia; 2/32 (6.3%) patients were cleared of BKV in both serum and urine, and 9/9 (100%) only had BKV viruria but did not develop BKV viremia. eGFR remained stable. No patient with BKV-related nephropathy had graft loss. There was a significant inverse relationship between changes in eGFR and serum BKV load (r = −0.314, p = 0.04). Conclusions: This stepwise immunosuppressive strategy proved effective at reducing BKV viral load in kidney transplant recipients that had high BKV loads in serum and/or urine. Renal function remained stable without rejection.

Frequencies and Association of CYP3A5 Polymorphism With Tacrolimus Concentration Among Renal Transplant Recipients in Vietnam.

BACKGROUND: This study aims to investigate the frequencies and association of CYP3A5 polymorphism with tacrolimus concentration among renal transplant recipients in Vietnam. METHODS: Sixty-eight kidney transplant recipients were included in this study from the department of nephrology and dialysis, Military Hospital 103. Blood samples were collected for monitoring of tacrolimus levels and determination of CYP3A5 genetic polymorphism. RESULTS: A total of 68 patients studied. The CYP3A5*3*3, CYP3A5*1*3, and CYP3A5*1*1 genotypes were detected in 48 (70.6%), 16 (23.5%), and 4 (5.9%), respectively. Tacrolimus concentrations were much lower in CYP3A5 expressors than in CYP3A5 nonexpressors on the first day, month 1, 3, 6, and 12 (5.98 ± 1.05 vs 6.57 ± 1.03, P = .03; 5.79 ± 1.13 vs 6.82 ± 1.05, P < .001; 4.76 ± 1.48 vs 6.73 ± 1.09, P < .001; 4.29 ± 1.64 vs 6.46 ± 1.23, P < .001; 4.20 ± 1.36 vs 6.04 ± 1.26, P < .001), respectively. Notably, the concentration/dose ratio in the CYP3A5 expressors was lower than in CYP3A5 nonexpressors at time points of follow up (P < .001). However, there were no significant differences in the age, sex, HLA mismatch, type of donors, acute rejection, and creatinine levels at time points between group of CYP3A5 expressors and those of CYP3A5 nonexpressors. CONCLUSION: In conclusion, this research indicated the significant association of CYP3A5 genetic polymorphism with daily dose and tacrolimus concentrations in renal transplant recipients. This study provided a closer step to individualize the dose of tacrolimus in renal transplant patients in Vietnam.

Genomic Mutations of BK Polyomavirus in Patients after Kidney Transplantation: A Cross-Sectional Study in Vietnam.

OBJECTIVES: The purpose of this study was to identify the SNP sites and determine the BKV genotype circulating in kidney-transplant Vietnamese recipients based on the VP1 gene region. METHODS: 344 samples were collected from post-kidney-transplant recipients at the 103 Vietnam Military Hospital to investigate the number of BKV infections. Positive samples with a sufficient virus concentration were analyzed by nested PCR in the VP1 region, sequencing detected genotyping and single-nucleotide polymorphism. RESULTS: BKV infection was determined in 214 patients (62.2%), of whom 11 (5.1%) were diagnosed with BKV-associated nephropathy. Among the 90 BKV-I strains sequenced, 89 (98.88%) were strains of I/b-1 and 1 (1.12%) was strain I/b-2. The 60 BKV-IV strains had a greater diversity of subgroups, including 40% IV/a-1, 1.66% IV/a-2, 56.68% IV/c-1, and 1.16% IV/c-2. Additionally, of 11 cases diagnosed with BKVN, seven belonged to subgroup I/b-1 (63.6%) and four to subgroup IV/c-1 (36.4%). Moreover, 22 specific SNPs that were genotype I or IV were determined in this Vietnamese population. Specifically, at position 1745, for the Vietnamese BKV-IV strains, the SNP position (A→G) appeared in 57/60 samples (95%). This causes transformation of the amino acid N→S. This SNP site can enable detection of genotype IV in Vietnam. It represents a unique evolution pattern and mutation that has not been found in other international strains. CONCLUSION: The BKV-I genotype was more common than BKV-IV; however, mutations that occur on the VP1 typing region of BKV-IV strains were more frequent than in BKV-I strains.

Species Distribution and Antifungal Susceptibility Pattern of Candida Recovered from Intensive Care Unit Patients, Vietnam National Hospital of Burn (2017-2019).

INTRODUCTION: Candida species is the most common cause of invasive fungal infection. With the wide variation in species distribution and antifungal susceptibility of causative agents, local epidemiological profiles are needed to provide effective guidelines for the treatment of invasive candidiasis. OBJECTIVE: To find out the species distribution and antifungal susceptibilities of Candida strains isolated from patients in an intensive care unit (ICU) of Vietnam. METHODS: All patients in ICU of Vietnam National Hospital of Burn with Candida isolation reported from January 2017 to December 2019 were retrospectively studied. Species identification and antifungal susceptibility testing were performed using VITEK 2 Compact. The identification was reconfirmed by sequencing of the internal transcribed spacer regions when needed. RESULTS: A total of 186 yeasts belonging to ten species were collected. The most common agent was C. tropicalis (45.7%), followed by C. albicans (42.4%), and C. parapsilosis (7.53%). The isolated yeasts showed less susceptibility to fluconazole (resistant rate R 10.7%) than to micafungin, caspofungin, flucytosine and amphotericin B (R 0%, 0.6%, 2.3% and 3.4%, respectively, p < 0.05). C. albicans isolates were more susceptible to fluconazole (R 5.2%) than C. tropicalis (R 15.7%). Resistance to voriconazole was seen only among C. albicans (3.9%) and C. tropicalis isolates (9.9%). CONCLUSION: Non-albicans species (especially C. tropicalis) is the predominant species, and there is a significant proportion of isolates with reduced susceptibility to azole but not to echinocandin.

A simple method for detection of a novel coronavirus (SARS-CoV-2) using one-step RT-PCR followed by restriction fragment length polymorphism.

A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. Early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. We aimed to establish a simple method for the detection of SARS-CoV-2 in differentiating with SARS-CoV. Primers of our in-house reverse transcription polymerase chain reaction (RT-PCR) assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I, and AluI to distinguish between SARS-CoV-2 and SARS-CoV. In this report, a 396-bp fragment of the RdRp gene and 345-bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP-amplified product of SARS-CoV-2 generating three fragments of 45, 154, and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating two fragments of 248 and 97 bp without cutting to SARS-CoV. The accuracy of the assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, in the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV.

Identification of BK Virus Genotypes in Recipients of Renal Transplant in Vietnam.

OBJECTIVES: This study aims to investigate the prevalence and distribution of BK polyomavirus (BKV) genotypes in recipients of renal transplant in Vietnam. METHODS: One hundred six patients who underwent renal transplantation were included in this study. These patients were from the Department of Nephrology, Military Hospital 103, Vietnam Military Medical University. Quantification and genotyping of the BK virus was performed using in-house molecular methods from urine and plasma samples. RESULTS: BKV infection was detected in 82 patients (77.4%), including 58 patients who had the presence of both BK viremia and BK viruria, and 24 patients (22.60%) with BKV positive findings in the urine alone. Particularly, 16 patients (15.09%) had high BK viremia >104 copies/mL and 20 patients (18.9%) had BK viruria >107 copies/mL. BK virus nephropathy (BKVN) was confirmed in 6 patients (5.66%) by immunohistochemistry examination. Genotyping of BKV was performed successfully in 50 out of the 82 patients, with 36 out of 50 (72%) belonging to the BKV-I subtype and 14 out of 50 (28%) belonging to the BKV-IV subtype; no cases of genotypes II or III were observed. Using phylogenetic analysis of the subgroups, the BKV-I/b-1 subtype was found the predominant subgroup (100%), whereas BKV-IV included 21.43% of IV/a-1, 6.67% of IV/a-2, and 50% of IV/c-1, respectively. There remain 3 cases of BKV-IV (21.43%) that could not be categorized into any subgroups. In the 6 patients diagnosed BKVN, 5 of them were infected with subgroup I/b-1 (83.3%) and 1 patient was infected with subgroup IV/c-1 (16.7%). No significant difference between BKV genotypes was observed in relation to age, sex, HLA mismatch, viral load, BKVN, and immunosuppressive therapy. CONCLUSIONS: This research indicated a high prevalence of BKV infection and BKV-I was predominant, followed by BKV-IV among recipients of renal transplant in Vietnam.

Dengue Virus IgM Serotyping by ELISA with Recombinant Mutant Envelope Proteins.

We developed an IgM-based ELISA that identifies the dengue virus serotype of recent infections. Dominant serotypes were detectable in 91.1% of samples from travelers and 86.5% of samples from residents of endemic regions; 97.1% corresponded to the serotype identified by PCR. This ELISA enables more accurate reporting of epidemiologic findings.