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Background: Valganciclovir prophylaxis against cytomegalovirus (CMV) is recommended for solid organ transplant recipients, but is associated with drawbacks, including expense and leukopenia. Our center adopted a strategy of serial assessment with a CMV‐specific T cell immunity panel (CMV‐TCIP) and cessation of valganciclovir prophylaxis upon demonstration of adequate CD4+ responses in kidney transplant patients at high risk of CMV disease. Methods: We retrospectively reviewed adult recipients of a kidney or pancreas transplant between August 2019 and July 2021 undergoing serial CMV‐TCIP monitoring. Included patients were considered high risk for CMV, defined by donor positive (D+)/recipient negative (R−) CMV IgG serostatus, or recipient positive (R+) patients who received induction with a lymphocyte‐depleting agent. Prophylaxis was discontinued after a patient's first CMV‐specific CD4+ T cell value of ≥0.20%. Risk of clinically significant CMV infection (csCMVi) in those who underwent early discontinuation of CMV prophylaxis and predictors of CMV T cell immunity were analyzed. Results: Of 54 included patients, 22 stopped prophylaxis early due to CMV‐specific CD4+ T cell immunity at a median of 4.7 (IQR: 3.8–5.4) months after transplant. No instances of csCMVi were observed in the 22 patients who had prophylaxis discontinued early, of whom 19/22 were CMV R+ and 3/22 were CMV D+/R−. Donor/recipient CMV serostatus was predictive of immunity (p <.001). Conclusion: Early discontinuation of valganciclovir prophylaxis in patients with CMV CD4+ T cellular immunity appears safe and potentially beneficial in this preliminary series, especially in R+ patients. Further study is warranted, given that truncated prophylaxis may yield patient‐level benefits. [ABSTRACT FROM AUTHOR]

A novel on-chip inexpensive platform to perform DNA amplification has been fabricated by optimizing the design and microfabrication processes using polydimethyl siloxane (PDMS) and silicon. The silicon base contains a set of microfabricated platinum heater structures on the bottom with a 140-nm-thick spin-on-glass (SOG) layer on the top and a 3 [micro]1 replica molded PDMS chamber with feed channels and inlet-outlet ports bonded to this film. The plasma exposed SOG surface is found to undergo recovery of hydrophobicity with time as indicated by an increase in advancing contact angle and bonds very well to another plasma exposed PDMS piece. The bonding protocol developed can be used for a diverse range of substrates, which may form a basis for integration of fluidic assays with microelectronics. The hydrophobic recovery of the microchamber and channels also eliminate the need for various surface passivation techniques for polymerase chain reaction (PCR) chips. A thermal cycler with flexible PCR cycle control is designed and implemented by using a sensing thermocouple. The amplification has been tested using picogram-level template DNA concentration. We have further been able to show negligible nonspecific binding of the template DNA to the hydrophobic interiors of our device by fluorescence measurements and have been able to successfully demonstrate the possibility of multiple usage of this chip without cross-contamination from the previous run. [1708] Index Terms--DNA, infectious bovine rhinetracheitis (IBR), polydimethyl siloxane (PDMS), polymerase chain reaction (PCR) spin-on-glass (SOG), surface recovery.

Abstract Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG). UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the CT for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 μl reaction with a 20 min, 30°C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the CT for RNA detection was increased by 1.2 cycles.

Background. The sensitivity of blood cultures for diagnosing invasive candidiasis (IC) is poor. Methods. We performed a validated Candida real-time polymerase chain reaction (PCR) and the Fungitell 1,3-β-D-glucan (BDG) assay on blood samples collected from prospectively identified patients with IC (n = 55) and hospitalized controls (n = 73). Patients with IC had candidemia (n = 17), deep-seated candidiasis (n = 33), or both (n = 5). Controls had mucosal candidiasis (n = 5), Candida colonization (n = 48), or no known Candida colonization (n = 20). Results. PCR using plasma or sera was more sensitive than whole blood for diagnosing IC (P 5 = .008). Plasma or sera PCR was more sensitive than BDG in diagnosing IC (80% vs 56%; P = .03), with comparable specificity (70% vs 73%; P = .31). The tests were similar in diagnosing candidemia (59% vs 68%; P = .77), but PCR was more sensitive for deep-seated candidiasis (89% vs 53%; P = .004). PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% vs 17%; P = .0005 and .003, respectively). PCR and culture identified the same Candida species in 82% of patients. The sensitivity of blood cultures combined with PCR or BDG among patients with IC was 98% and 79%, respectively. Conclusions. Candida PCR and, to a lesser extent, BDG testing significantly enhanced the ability of blood cultures to diagnose IC. [ABSTRACT FROM AUTHOR]