Portable integrated digital PCR system for the point-of-care quantification of BK virus from urine samples.

This paper presents a portable integrated digital PCR (PI-dPCR) system with a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the quantitative analysis of BK virus (BKV) viral load directly from raw urine samples. Digital PCR is an accurate nucleic acid quantification method with single-molecule sensitivity, but the complexity of the instrument and the limited integration of the operation workflow greatly limit its application in clinical diagnostics, especially point-of-care testing (PoCT). Our PI-dPCR system has a small footprint, is lightweight, and is fully integrated with the thermal control and fluorescence imaging modules. Unlike the traditional SlipChip device, which requires the precise overlapping of microfeatures on the contacting surfaces to establish the fluidic loading path, this sp-SlipChip device utilizes microchannels with alternating depth and width for fluidic manipulation. This system can quantify BKV directly from raw urine samples with a simple "sample-to-digital-result" operation workflow without complex nucleic acid extraction and purification steps. The current design of the system provides a dynamic range of 3.0 × 104 to 1.5 × 108 copies/mL of BKV DNA in clinical urine samples within 2 h. We tested the system for the quantification of BKV viral load in thirty archived urine samples from kidney transplantation recipients and twelve additional samples from six patients before and after the adjustment of immunosuppressive treatment. This integrated system provides a promising method for both the detection and monitoring of viral infection in a point-of-care setting.

Intracellular Low Iron Exerts Anti-BK Polyomavirus Effect by Inhibiting the Protein Synthesis of Exogenous Genes.

BK polyomavirus (BKPyV) is a small double-stranded DNA virus and ubiquitous human pathogen that particularly affects immunocompromised individuals. Antiviral therapy for BKPyV is urgently needed. Intracellular irons have an important role in many viral infections, yet its contribution to BKPyV and replication has not been explored. In this study, we explored the interaction between BKPyV infection and intracellular iron and the inhibitory effect of iron depletion on BKPyV infection. By creating a low-intracellular-iron environment, we demonstrated that the iron-chelating-induced iron depletion inhibits BKPyV infection in primary renal tubular epithelial cells (RPTECs) and urinary bladder cancer cells (TCCSUP cells). Iron depletion exerts an inhibitory effect after BKPyV enters the nucleus, which might be due to the inhibition of the protein synthesis of exogenous genes in iron-depleted cells. Further exploration of the target proteins of iron-regulating viral infection could potentially be used to develop new strategies for urgently needed anti-BKPyV therapies. IMPORTANCE BKPyV poses a serious threat to the health of immunocompromised patients, and there are currently no curative drugs. Understanding the relationship between the virus and intracellular environment contributes to the discovery of antiviral targets. We demonstrate here that BKPyV is inhibited in cells with a low-iron environment. We also find that iron-chelating-induced iron depletion inhibits viral and exogenous protein synthesis. Further exploration of the target proteins of iron regulation could have great potential in developing new drugs against BKPyV and other viruses.

Heterogeneous Klebsiella pneumoniae Co-infections Complicate Personalized Bacteriophage Therapy.

Multidrug-resistant (MDR) organisms have increased worldwide, posing a major challenge for the clinical management of infection. Bacteriophage is expected as potential effective therapeutic agents for difficult-to-treat infections. When performing bacteriophage therapy, the susceptibility of lytic bacteriophage to the target bacteria is selected by laboratory isolate from patients. The presence of a subpopulation in a main population of tested cells, coupled with the rapid development of phage-resistant populations, will make bacteriophage therapy ineffective. We aimed to treat a man with multifocal urinary tract infections of MDR Klebsiella pneumoniae by phage therapy. However, the presence of polyclonal co-infectious cells in his renal pelvis and bladder led to the failure of three consecutive phage therapies. After analysis, the patient was performed with percutaneous nephrostomy (PCN). A cocktail of bacteriophages was selected for activity against all 21 heterogeneous isolates and irrigated simultaneously via the kidney and bladder to eradicate multifocal colonization, combined with antibiotic treatment. Finally, the patient recovered with an obviously improved bladder. The success of this case provides valuable treatment ideas and solutions for phage treatment of complex infections. Clinical Trial Registration: www.chictr.org.cn, identifier ChiCTR1900020989.