Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.

Human pathogenic microsporidia detection in agricultural samples: method development and assessment.

A detection method was developed and assessed for the sensitive recovery of microsporidia from livestock fecal and manure-impacted environmental samples. Sensitive recovery of microsporidia was achieved when samples were subjected to 1) purification by sucrose floatation, 2) DNA extraction using the Qiagen QIAamp DNA Stool Mini Kit, 3) polymerase chain reaction (PCR) analysis using generic primers for microsporidia, and 4) DNA sequence analysis to identify which microsporidia were present in each sample. Livestock fecal and wastewater samples were inoculated with 1,000 and 100 Encephalitozoon intestinalis spores/g or ml of feces or wastewater. For cattle wastewater, ten of ten replicates were positive by PCR at concentrations of 1,000 spores/ml, and two of ten replicates were positive at concentrations of 100 spores/ml. For swine wastewater, ten of ten replicates were positive at concentrations of 1,000 spores/ml, and four of ten replicates were positive at concentrations of 100 spores/ml. For cattle feces, three of ten replicates were positive at the concentration of 1,000 spores/g. Several environmental samples were screened using this method, with two of 34 samples positive for human pathogenic microsporidia. To our knowledge, this is the first report of Encephalitozoon cuniculi detection in swine feces and wastewater.

Microbial quality of runoff following land application of cattle manure and swine slurry.

Concentrations of human health-related microorganisms in runoff from agricultural plots (0.75 m x 2 m) treated with fresh and aged cattle manure, swine slurry and no manure (control) were determined. Three consecutive simulated rainfall events, producing 35 mm rainfall and separated by 24 h, were carried out for each plot. Fecal indicator (Escherichia coli, enterococci, Clostridium perfringens and coliphage) loads released in rainfall runoff from plots treated with fresh cattle manure, aged cattle manure and swine slurry treatments ranged from 5.52 x 10(5) to 4.36 x 10(9), 3.92 x 10(4) to 4.86 x 10(8), and 9.63 x 10(5) to 3.05 x 10(8), respectively. Plot runoff concentrations of protozoa (Cryptosporidium oocysts and Giardia cysts) ranged from 1.65 x 10(5) to 1.04 x 10(6), 2.93 x 10(3) to 2.75 x 10(5), and 9.12 x 10(4) to 3.58 x 10(6) for fresh cattle manure, aged cattle manure and swine slurry plot treatments, respectively. These results suggest that large microbial loads could be released via heavy precipitation events that produce runoff from livestock manure-applied agricultural fields, of even modest size, and could have a significant impact on water bodies within the watershed. Because of the lack of multiplication in the environment, highly elevated concentrations in manured land runoff, and correlation to protozoan parasite presence, Clostridium may be an alternative indicator for livestock manure contamination.

Inactivation of enteric adenovirus and feline calicivirus by ozone.

Little information is available regarding the effectiveness of ozone on the inactivation of caliciviruses and enteric adenoviruses. Inactivation experiments were conducted with feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, and adenovirus type 40 (AD40). Experiments were carried out in buffered disinfectant demand free water at pH 7 and 5 degrees C. Ct values; concentration of ozone multiplied by contact time with virus; were determined from application of the efficiency factor hom (EFH) model. Ct values for 4-log (99.99%) ozone inactivation at 5 degrees C and pH 7 ranged from 0.07 to 0.60 mg/l min for AD40 and <0.01 to 0.03 mg/l min for FCV. Ct values listed in the US environmental protection agency "Guidance Manual for Compliance with Filtration and Disinfection Requirements for Public Water Systems Using Surface Water Sources" were higher than Ct values generated by this study. Very low ozone residuals (<0.01(mg/l) substantially inactivated FCV and AD40 under the studied conditions.

Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide.

Chlorine dioxide (ClO2) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO2 multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct99.99%) at 5 degrees C were >0.77 to <1.53 mg/liter x min and >0.80 to <1.59 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C AD40 experiments, >0.49 to <0.74 mg/liter x min and <0.12 mg/liter x min Ct99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct99.99% ranges for 5 degrees C experiments were >20.20 to <30.30 mg/liter x min and >0.68 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C FCV experiments, Ct99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter x min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15 degrees C than at 5 degrees C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct99.99% ranges and EFH model Ct99.99% values demonstrated that FCV is more resistant to ClO2 than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct99.99% values are higher than Ct99.99% values calculated from bench-scale experiments and from EFH model application.

Chlorine inactivation of adenovirus type 40 and feline calicivirus.

Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5 degrees C than at 15 degrees C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15 degrees C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5 degrees C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation.

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.

Detection of protozoan parasites and microsporidia in irrigation waters used for crop production.

The occurrence of human pathogenic parasites in irrigation waters used for food crops traditionally eaten raw was investigated. The polymerase chain reaction was used to detect human pathogenic microsporidia in irrigation waters from the United States and several Central American countries. In addition, the occurrence of both Cryptosporidium oocysts and Giardia cysts was determined by immunofluorescent techniques. Twenty-eight percent of the irrigation water samples tested positive for microsporidia, 60% tested positive for Giardia cysts, and 36% tested positive for Cryptosporidium oocysts. The average concentrations in samples from Central America containing Giardia cysts and Cryptosporidium oocysts were 559 cysts and 227 oocysts per 100 liters. In samples from the United States, averages of 25 Giardia cysts per 100 liters and <19 (average detection limit) Cryptosporidium oocysts per 100 liters were detected. Two of the samples that were positive for microsporidia were sequenced, and subsequent database homology comparisons allowed the presumptive identification of two human pathogenic species, Encephalitozoon intestinalis (94% homology) and Pleistophora spp. (89% homology). The presence of human pathogenic parasites in irrigation waters used in the production of crops traditionally consumed raw suggests that there may be a risk of infection to consumers who come in contact with or eat these products.