Continuous flow nonthermal CO2 processing: the lethal effects of subcritical and supercritical CO2 on total microbial populations and bacterial spores in raw milk.

The effect of pressurized (<50 MPa) CO2 as a nonthermal process for bacterial reduction in raw skim milk was examined using a unique pressurized continuous flow system. The lethal effects of subcritical and super-critical CO2 applied at different temperatures and pressures toward total native psychrotrophic microbial populations, total inoculated Pseudomonas fluorescens, and total inoculated spore populations were studied and compared. Pressures between 10.3 and 48.3 MPa; temperatures of 15, 30, 35, and 40 degrees C; and CO2 concentrations of 0, 3, 66, and 132 g/kg of milk were studied. For both native populations and inoculated P. fluorescens, greater total microbial lethality was observed under supercritical CO2 conditions than under subcritical CO2 conditions. At 30 degrees C, there was no effect on total microbial lethality of increasing pressure up to 20.7 MPa with either 66 or 132 g/kg of CO2; at 35 degrees C, there was a positive relationship between pressure and lethality at CO2 levels of 132 g/kg, but no relationship at 66 g/kg of CO2. For total microbial populations and P. fluorescens, CO2 applied at 132 g/kg at 30 degrees C and pressures of 10.3 to 20.7 MPa resulted in an average standard plate count reduction of 3.81 and 2.93 log, respectively; at 35 degrees C and 20.7 MPa, maximum reductions achieved were 5.36 and 5.02 log, respectively. For both total microbial populations and inoculated P. fluorescens, CO2 exhibited a greater overall lethal effect at 132 g/kg than at 66 g/kg and a greater effect at 35 degrees C than at 30 degrees C. At 24.1 and 48.3 MPa and 40 degrees C, microbial lethality in raw aged milk treated with 3 g/kg of CO2 was not significantly different than that observed for uncarbonated milk; lethality achieved in milk treated with 132 g/kg of CO2 was significantly higher than that achieved in these 2 low-level CO2 treatments. No treatment studied had any significant impact on spore populations. Our work shows that, using the studied system, pressurized CO2 results in greater microbial lethality in milk above critical temperatures than below and suggests that a critical concentration threshold level of CO2 is required for lethal effects. Our work also suggests that supercritical CO2 processing in a continuous flow system can achieve reductions in some microbial populations equal to or better than that typically achieved during high-temperature, short-time pasteurization.

A crystalline lipid phase in a dry biological system: evidence from X-ray diffraction analysis of Typha latifolia pollen.

The temperature limits for germination in Typha latifolia pollen lie within the range 4-40 degrees C. These limits correlate at the low-temperature end with the 'crystallization' of endogenous triacylglycerols and on the high-temperature end with the 'melting' of a gel-like lipid component in intact pollen. X-ray diffraction analysis was used to structurally characterize and to trace the latter gel-like lipid from the intact pollen through a range of pollen lipid fractions. We tentatively identify this component as a fatty acyl sterol ester and present evidence that it resides in the exine of the pollen grain. Its thermotropic behavior is insensitive to pollen hydration. The possibility of interpreting a crystalline lipid phase as being membrane-derived when in fact it originates from contaminating non-membranous neutral lipid is discussed. The total lipid content of T. latifolia pollen is 123 mg/g dry weight, of which 37% is polar lipid. The neutral lipid consists primarily of triacylglycerols and of the aforementioned sterol ester, which represents 0.34% (w/w) of pollen dry weight. The polar lipid fraction has phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid as major components with lesser amounts of phosphatidylglycerol and phosphatidylinositol. Palmitic (16:0) and linoleic (18:2) acids, in a 1:2 molar ratio, constitute the major fatty acids of both polar and neutral lipid fractions with lesser amounts of linolenic (18:3), oleic (18:1) and stearic (18:0) acid in evidence.

Low pressure CO2 storage of raw milk: microbiological effects.

The effects of holding raw milk under carbon dioxide pressures of 68 to 689 kPa at temperatures of 5, 6.1, 10, and 20 degrees C on the indigenous microbiota were investigated. These pressure-temperature combinations did not cause precipitation of proteins from the milk. Standard plate counts from treated milks demonstrated significantly lower growth rate compared with untreated controls at all temperatures, and in some cases, the treatment was microcidal. Raw milk treated with CO2 and held at 6.1 degrees C for 4 d exhibited reduced bacterial growth rates at pressures of 68, 172, 344, and 516 kPa; and at 689 kPa, demonstrated a significant loss of viability in standard plate count assays. The 689-kPa treatment also reduced gram-negative bacteria and total Lactobacillus spp. The time required for raw milk treated at 689 kPa and held at 4 degrees C to reach 4.30 log10 cfu/mL increased by 4 d compared with untreated controls. Total coliform counts in the treated milk were maintained at 1.95 log10 cfu/mL by d 9 of treatment, whereas counts in the control significantly increased to 2.61 log10 cfu/mL by d 4 and 2.89 log10 cfu/mL by d 9. At d 8, Escherichia coli counts had not significantly changed in treated milk, but significantly increased in the control milk. Thermoduric bacteria counts after 8 d were 1.32 log10 cfu/mL in treated milk and 1.98 log10 cfu/mL in control milk. These data indicated that holding raw milk at low CO2 pressure reduces bacterial growth rates without causing milk protein precipitation. Combining low CO2 pressure and refrigeration would improve the microbiological quality and safety of raw milk and may be an effective strategy for shipping raw single strength or concentrated milk over long distances.

Seroprevalence of hepatitis B virus and delta agent in parenteral drug abusers. Immunogenicity of hepatitis B vaccine.

The seroprevalence of hepatitis B virus (HBV) markers and antibody to the delta agent (anti-delta) was determined for 112 parenteral drug abusers entering a methadone maintenance program. Markers of HBV infection were found in 87.5 percent of the group, and seropositivity was significantly associated with duration of drug abuse (p = 0.02). Antibody to the hepatitis B core antigen (anti-HBc) was present in all seropositive subjects; three (2.7 percent) were hepatitis B surface antigen-positive, and 16 (14.2 percent) had only anti-HBc. Five (10.6 percent) of 47 subjects with HBV markers had anti-delta. Anti-delta was more common in subjects who reported multiple symptomatic episodes of hepatitis (p = 0.02) and fewer than three daily drug injections (p = 0.05). Ten susceptible subjects received hepatitis B vaccine, and seroconversion rates at one, three, and six months were 20.0, 88.8, and 100 percent, respectively. The data indicate that hepatitis B vaccine is immunogenic in this population, and that anti-HBc is the optimal prevaccination screening test. Recent outbreaks of fulminant HBV and delta co-infection among drug abusers emphasize the need for early immunization in this group.

Hepatitis B exposure in emergency medical personnel. Prevalence of serologic markers and need for immunization.

To assess the occupational risk of hepatitis B infection in emergency medical personnel, a seroepidemiologic survey of 87 emergency medical technicians and paramedics was conducted. Serologic markers indicating exposure to hepatitis B virus were detected in 18 percent. The prevalence of markers was associated with race (p = 0.006), with a relative risk of 3.5 (95 percent confidence interval 1.42 to 8.63) for nonwhites. Seropositivity was not associated with age, sex, previous clinical hepatitis, or blood transfusion. There was a suggestion that duration of employment as an emergency medical technician was related to the prevalence of hepatitis B markers (p = 0.11). Efforts to control the risk of hepatitis B infection in this profession are complicated by unique problems with post-exposure prophylaxis and uncontrolled exposure to blood. Immunization with hepatitis B vaccine would be the optimal strategy to reduce infection in this high-risk occupation.

The independent role of cytomegalovirus as a risk factor for invasive fungal disease in orthotopic liver transplant recipients. Boston Center for Liver Transplantation CMVIG-Study Group. Cytogam, MedImmune, Inc. Gaithersburg, Maryland.

PURPOSE: To assess impact of cytomegalovirus (CMV) donor-recipient serostatus, infection, or disease on development of invasive fungal infection in orthotopic liver transplant recipients. PATIENTS AND METHODS: An analysis of prospectively collected data in 146 liver transplant recipients (intention to treat cohort) from 4 tertiary care, university-affiliated transplant centers in Boston (Boston Center for Liver Transplantation). Patients were observed for 1 year after transplantation for the development of CMV infection, CMV disease, CMV pneumonia, as well as for the development of opportunistic fungal infections, graft survival, and mortality. Weekly cultures were taken of urine and throat and every other week of buffy coat for CMV for 2 months, then monthly for 6 months, at 1 year, and at the time of any clinical illness. Pre- and posttransplant variables including CMV-serostatus of donor and recipient, fungal isolation from sterile body sites, fungemia, bacteremia, antibiotic use, immunosuppression, treatment for rejection, and volumes of blood products were measured. RESULTS: Survival analysis demonstrated that 36% of patients with CMV disease developed invasive fungal disease within the first year post-transplant compared with 8% of those without CMV disease (P < 0.0001). One-year mortality in patients with invasive fungal disease was 15 of 22 (68%) compared with 23 of 124 (19%) in those without invasive fungal disease (P < 0.001). A multivariable, time-dependent analysis demonstrated that being a CMV-seronegative recipient of a CMV-seropositive donor organ (P < 0.001), having bacteremia (P = 0.001), UNOS (United Network for Organ Sharing) status 4 (need for life support measures) at transplant (P = 0.002), and volume of platelets (P = 0.002) were independently associated with invasive fungal disease. Restriction of cases of invasive fungal disease to those that occurred more than 2 weeks after transplant demonstrated an association with CMV disease (P = 0.003), bacteremia (P = 0.003), need for life support (P = 0.03), and volume of blood products transfused (P = 0.02). CONCLUSION: CMV disease or being a CMV-seronegative recipient of a CMV-seropositive donor organ is an important predictor for invasive fungal disease following orthotopic liver transplantation.

Which renal transplant patients should receive cytomegalovirus immune globulin? A cost-effectiveness analysis.

Our study objective was to determine the cost-effectiveness of prophylaxis with cytomegalovirus immune globulin for preventing CMV-associated disease in renal transplant patients. We used a decision analytic model applied to 5 groups of renal transplant recipients at varying risks of developing CMV-associated disease. We obtained the rates of developing CMV-associated disease, graft rejection, and mortality, and the effectiveness of CMV-IG from the published literature. We used actual variable costs of CMV-IG, hospitalization, dialysis, and outpatient follow-up. The incremental cost of administering CMV-IG compared with withholding it ranged from $29,800 per life saved for the highest risk group, CMV-seronegative recipients of kidneys from CMV-seropositive cadaveric donors, to $1.68 million per additional life saved for the lowest risk group, CMV-seronegative recipients of grafts from CMV-seronegative donors. The outcome was somewhat sensitive to the effectiveness of CMV-IG in preventing CMV-associated disease but not sensitive to wide variations in CMV-IG costs, dialysis costs, outpatient costs, and the probability of graft rejection. Our conclusion is that prophylaxis with CMV-IG is very worthwhile for renal transplant recipients at high risk of CMV-associated disease and is possibly worthwhile for some patients at lower risk. The cost-effectiveness of other strategies for preventing CMV-associated disease, such as prophylaxis with acyclovir, CMV vaccine, or unselected immune globulin, should be compared with CMV-IG.

Cytomegalovirus disease is associated with increased cost and hospital length of stay among orthotopic liver transplant recipients.

Cytomegalovirus (CMV) is a cause of considerable morbidity and mortality among orthotopic liver transplant (OLT) recipients. To study the impact of CMV on cost and hospital length of stay in this population, we undertook an analysis of a cohort of OLT recipients from four transplant centers in Boston who participated in a CMV prophylaxis trial. First posttransplant year hospital length of stay (including the hospital stay after transplantation and readmissions within 1 year after transplantation) was available for all 141 patients included in the study. In a multiple linear regression model bacteremia (P=0.0001), CMV disease (P=0.0007), abdominal reexploration (excluding retransplantation) (P=0.0070), recipient age < or = 16 years (P=0.0352), and the number of units of blood products (red blood cells, platelets, or fresh frozen plasma) administered during transplantation (P=0.0523) were shown to be independently associated with longer first posttransplant year hospital length of stay. Cost data for in-hospital care for the year beginning with admission for liver transplantation were available for 66 OLT recipients. Using a multiple linear regression model, development of CMV disease (P=0.0001), the number of units of blood products administered during transplantation (P=0.0001), bacteremia (P=0.0002), decreased pretransplant renal function (estimated by creatinine clearance) (P=0.0109), and need for retransplantation (P=0.0619) were shown to be independently associated with higher cost. These data strongly suggest that CMV disease has a direct impact on cost and hospital length of stay in liver transplantation.

Significance of cytomegalovirus for long-term survival after orthotopic liver transplantation: a prospective derivation and validation cohort analysis.

BACKGROUND: Cytomegalovirus (CMV) infection and disease has been found to be associated with decreased graft and patient survival among heart transplant recipients. We sought to explore the effect of CMV infection and disease on long-term survival in orthotopic liver transplant (OLT) recipients using a derivation and validation cohort. METHODS: For derivation-validation modeling, we used data collected from two prospectively followed cohorts as the basis for multivariate analyses: 167 OLT recipients from the Boston Center for Liver Transplantation (the derivation set; median follow-up: 5.5 years, mortality: 40%) and an independent cohort of 294 OLT recipients from the Mayo Clinic (the validation set; median follow-up: 4.8 years, mortality: 27%). RESULTS: Underlying liver disease other than primary biliary cirrhosis or sclerosing cholangitis, number of units of red blood cells administered during transplantation, and donor CMV seropositivity were the pre- and intratransplant variables independently associated (P<0.01) with decreased long-term survival in the derivation cohort. For variables collected up to 1 year after transplantation, the need for retransplan. tation, CMV pneumonia, invasive fungal disease, and underlying liver disease other than primary biliary cirrhosis or sclerosing cholangitis were independently associated (P<0.01) with decreased long-term survival in the derivation cohort. The magnitude of the relationship of each pre-, intra-, and posttransplant factor with survival, as measured by the relative risk, did not significantly differ between the derivation and validation cohorts. The derivation model, incorporating pre-, intra-, and posttransplant factors, had receiver operating characteristic areas of 73% and 74% for 5-year mortality in the derivation and validation cohorts, respectively. CONCLUSIONS: Data from a derivation and an independent validation cohort demonstrate that CMV factors (reflected by either donor CMV seropositivity at transplantation, CMV pneumonia, or CMV disease within the first posttransplant year) are independently associated with decreased long-term survival in OLT recipients.

Use of cytomegalovirus immunoglobulin in multiply transfused premature neonates.

We undertook a randomized, placebo-controlled, double blind trial of cytomegalovirus (CMV) immunoglobulin (CMVIG) for prevention of CMV-associated disease in 183 multiply transfused, premature neonates. CMVIG (150 mg/kg) or placebo was given within 24 hours of the first transfusion and at Day 10. If an intravenous catheter was still in place an additional dose was given between Days 20 and 30. The globulin and placebo groups were well-matched with respect to birth weight, gestational age, Apgar score, birth to a CMV-seropositive mother, requirement for assisted ventilation and exposure to CMV-positive, unscreened blood products. Among infants followed for more than 10 days, 18 (10.5%) developed CMV infection; 9 had symptomatic CMV disease (5 placebo; 4 CMVIG). Among infants born to a CMV-seropositive mother, CMVIG use was associated with a CMV syndrome rate of 3.2% (95% confidence interval, 0.2 to 18.5%) compared to 12.5% (95% confidence interval, 4.5 to 27.6%) among placebo recipients (P = 0.163). Among placebo recipients infants born to CMV-seropositive mothers were more likely to have a virologically confirmed CMV syndrome than those born to a CMV-seronegative mother, despite receipt of blood not screened for CMV antibody (P = 0.012). Multivariate analysis demonstrated that two factors were independently associated with CMV acquisition: the volume of CMV-seropositive blood products transfused (P = 0.005); and birth to a CMV-seropositive mother (P = 0.006). Infusions of CMVIG were well-tolerated. This study reaffirms that perinatally acquired CMV disease is more common among infants born to CMV-seropositive mothers than CMV-seronegative mothers, even without use of CMV-screened blood products.

New method for detection of Mycobacterium tuberculosis Direct Test inhibitors in clinical specimens.

Existing protocols for the detection of Mycobacterium tuberculosis Direct Test (MDT) inhibitors require substantial quantities of specimen and cannot distinguish Mycobacterium tuberculosis complex from other mycobacteria if inhibitors are present. We describe a preliminary evaluation of a simple and practical protocol for MTD inhibitor testing that circumvents these difficulties.

e Antigen in hepatitis B virus infected dialysis patients: assessment of its prognostic value.

Many investigations consider the presence of e antigen (HBeAg) valuable in predicting which patients with acute hepatitis B are at risk of remaining infected and developing chronic liver disease. We tested this hypothesis in a retrospective study of serial samples from patients undergoing long-term hemodialysis. We found HBeAg in the early phase of all hepatitis B virus (HBV) infections. There was no significant difference between transiently and persistently hepatitis B surface antigen (HBsAg)-positive persons with regard to the frequency of HBeAg during the first 3 months of HBs antigenemia. Thus during the early period of viral activity, the presence of HBeAg is of no prognostic values in determining chronicity of HBV infection. We believe the disagreement on the prognostic value of HBeAg is a reflection of variations in time and frequency of sampling. The HBeAg remains, however, a useful indicator of potential infectivity of HBsAg(+)persons.

Effect of carbon dioxide on the growth of Bacillus cereus spores in milk during storage.

The effects of the addition of 11.9 mM CO2 on the growth of Bacillus cereus spores inoculated into sterile homogenized whole milk at 101 and 106 spores/ml and stored at 6.1 degrees C, was examined weekly for 35 d. Colony-forming units from CO2 treated inoculated milks decreased over 35 d at a rate similar to that of untreated inoculated milk, as defined by linear regression. Plate counts for treated and control milks inoculated at 10(1) cfu/ml were not significantly different on sampling d 0, 14, 21, and 28. Plate counts at d 7 were significantly different and counts at d 35 were at undetectable levels for both treated and control milks. Plate counts for milk inoculated at 10(6) cfu/ml were not significantly different on d 0, 28, and 35; they were significantly different on d 7, 14, and 21. There was no consistency as to whether the control or test milks were higher in counts on days when the differences were significant. Added CO2 reduced the pH of the milk from an average value of 6.61 to an average value of 6.31; however, this drop did not correlate with changes in any other parameter measured. These data suggest that moderate levels of CO2 do not enhance the outgrowth of B. cereus spores over long-term storage and do not increase the risk of foodborne illness due to the organism.