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EBioMedicine 81, 104098 (2022). doi:10.1016/j.ebiom.2022.104098, Published by Elsevier, Amsterdam [u.a.]

Background.We describe our experience using a real-time polymerase chain reaction (PCR) assay for methicillin-resistant Staphylococcus aureus (MRSA) during a period of active surveillance in the neonatal intensive care unit (NICU) from March 2007 until November 2007.Objective.TO compare PCR with bacterial culture methods and find the screening algorithm that most successfully ensures appropriate isolation of colonized patients.Methods.Patients in the NICU were screened for MRSA on admission and weekly thereafter until discharge. Healthcare workers (HCWs) were also screened as part of an outbreak investigation. A total of 599 individuals were screened for MRSA with both a PCR assay and selective bacterial culture. Strain typing was performed on all MRSA isolates to determine clonal relatedness.Results.Twenty-one of 435 infants (4.8%) screened positive for MRSA with the PCR assay. Only 11 patients (52.4%) had concomitant bacterial cultures positive for MRSA. Compared to bacterial culture, the PCR assay had a sensitivity of 100% and a specificity of 97.6%, with a positive predictive value (PPV) of 52.4%. Infants that tested positive for MRSA by both culture and PCR were more likely to have a positive PCR assay result when retested than were those who tested positive by PCR alone (80% vs 20%; P = .02). Strain typing of MRSA isolates identified a common clone in only 2 colonized infants.Conclusion.Our data show that, in our neonatal population, the reproducibility of PCR assay results for culture-negative patients was low compared with the reproducibility of results for culture-positive Patients. Furthermore, the low PPV suggests that for nearly half of individuals who were PCR-positive, the result was falsely positive, which argues against the use of PCR assays alone for MRSA screening in the NICU.

The hypermetabolic response characteristic of thermal injury results in marked negative nitrogen balance, loss of body weight, and depletion of energy reserves. The magnitude of the catabolic process is directly related to the extent of injury and its impact on the patient is related to pre-burn nutrition and condition. Left unabated, protein breakdown results in loss of lean body mass at a rate of 30 to 35 g of nitrogen per day with impairment of wound healing, diminution of immunological competence, and lowered resistance to infection. Although provision of adequate nitrogen and calories will not return metabolism to normal levels, it will reduce nitrogen loss, promote healing, and hasten the recovery of the patient.

It is now well established that folic acid, when taken periconceptionally, can prevent many neural tube defects. It is also becoming clear that folic acid does not work by correcting a nutritional deficiency in pregnant women. Rather, it appears that a metabolic defect is responsible for these neural tube defects and that this defect or defects can be corrected by a sufficiently large dose of folic acid. Our recent work demonstrates that homocysteine metabolism is likely to be the critical pathway affected by folic acid. We have demonstrated significantly higher homocysteine levels in women carrying affected fetuses than in control women. These findings indicate that one of the enzymes responsible for homocysteine metabolism is likely to be abnormal in affected pregnancies. Animal studies suggest that the conversion of homocysteine to methionine could be the critical step. Rat embryos in culture require methionine for neural tube closure. Methionine synthase, cystathionine synthase, and 5,10 methylene tetrahydrofolate reductase are all important in the metabolism of homocysteine in humans. If methionine synthase is the critical enzyme, it would raise the interesting public health issue that vitamin B-12 might be able to stimulate the abnormal enzyme as folic acid does. Adding vitamin B-12 might make it possible to reduce the dose of folic acid required in fortified food, thus allaying concerns about overexposure to folic acid.

Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal diet that was very low in Co (12.9-17.6 micrograms Co per kg), or the same diet supplemented with cobalt, for a total of 64 weeks. Vitamin B12 deficiency was induced, as judged by hepatic concentrations of vitamin B12 and plasma concentrations of MMA. However, the activity of holo-methylmalonyl CoA mutase was significantly reduced only in brain. This was reflected in very minor alterations in the tissue concentrations of branched chain- and odd numbered-fatty acids. The activity of holo-methionine synthase was significantly reduced in liver and brain, but there were no consequent alterations in the concentrations of phosphatidyl choline and phosphatidyl ethanolamine. This study confirms that cattle are less susceptible to the effects of cobalt deficiency than sheep, and concludes that prolonged cobalt deficiency had little significant effect on tissue metabolism.

Folate acts as a cofactor for enzymes involved in DNA and RNA biosynthesis. Folate is also involved in the supply of methyl groups to the so-called methylation cycle, which uses methionine and makes homocysteine. The folate cofactor, N5-methyltetrahydrofolate, donates its methyl group to a vitamin B12-dependent enzyme, methionine synthase, which recycles homocysteine back to methionine. The cell's ability to methylate important compounds such as proteins, lipids and myelin will be compromised by deficiency of folate or vitamin B12, resulting in impaired cellular function. Methionine synthase plays another role: it converts circulating N5-methyltetrahydrofolate into tetrahydrofolate. The latter but not the former can act as a substrate for polyglutamate synthase, thereby becoming retained in the cell as polyglutamate. Interruption of DNA biosynthesis or methylation reactions could prevent the proper closure of the neural tube. Such inhibition could be caused by simple deficiency of either folic acid or vitamin B12. Studies comparing serum folate and vitamin B12 status in women who have had an affected pregnancy to those in control women indicate no difference between the two groups and show that most cases are not clinically deficient in either vitamin. A small number of studies using the level of folate in red blood cells, which is a better reflection of tissue stores, confirm this, suggesting instead a metabolic impairment in the biochemical functions of one of these vitamins. The trials using folic acid to prevent neural tube defects thus seem to be effectively overcoming a metabolic block rather than treating folate deficiency.

Blood was taken at the first antenatal clinic from 56,049 pregnant women. Neural tube defect (NTD) pregnancies (81) were compared to controls (247) for plasma vitamin B12 (B12) (ng/l), plasma folate (microgram/l), and red cell folate (RCF) (microgram/l). Median values were significantly different and were, respectively, 243 and 296 (p = 0.001); 3.47 and 4.59 (p = 0.002); and 269 and 338 (p0.001). There was a significant correlation between plasma B12 and RCF in cases (r = 0.31, p = 0.004) but not in controls (r = 0.02, p = 0.725). In cases only, multiple regression showed that both plasma B12 and plasma folate influenced the maternal RCF (multiple r = 0.68, p0.001). Plasma folate and plasma B12 were independent risk factors for NTDs, suggesting that the enzyme methionine synthase is involved directly or indirectly in the aetiology. The levels of folate and B12 where increased risk occurred were not those usually associated with deficiency, calling for a re-evaluation of their recommended daily allowances. Whether the aetiology is purely nutritional or a metabolic defect, this study suggests that consideration should be given to including B12 as well as folic acid in any programme of supplementation or food fortification to prevent NTDs.

Activity of the vitamin B12-dependent enzyme, methylmalonyl CoA mutase, was measured in the tissues of pigs, fed a diet which was low in cobalt and vitamin B12, and which were intermittently exposed to nitrous oxide until they displayed marked ataxia. Methylmalonyl CoA mutase activity was reduced in liver, kidney and brain. However, the methylmalonic acid concentration was reduced in liver and heart, in marked contrast to the expected increase which was only observed in brain. Liver and kidney also showed an unexpected reduction in the concentration of C17 odd-numbered fatty acids, possibly as a consequence of reduced propionate availability. Brain however, which had elevated methylmalonic acid concentrations showed no change in either odd-numbered or branched-chain fatty acids. These results suggest that nitrous oxide-induced neuropathy does not occur as a result of misincorporation of odd-numbered/branched-chain fatty acids in brain.

A disease, known as ovine white liver disease (OWLD) was experimentally reproduced in lambs by feeding a diet depleted of cobalt. At necropsy, affected animals had pale, swollen, friable fatty livers, and showed marked accumulation of lipofuscin. Control animals, fed the same diet to which adequate amounts of cobalt had been added, were clinically normal. In animals with OWLD, liver triglyceride and free fatty acid concentrations were increased. A decrease in the ratio of phosphatidyl choline to phosphatidyl ethanolamine in the liver may result in a reduced ability to export triglycerides as very low density lipoprotein. This may cause the lipid accumulation characteristic of OWLD. Lipofuscin accumulation, another feature of OWLD, is a consequence of lipid peroxidation. Evidence for a peroxidative challenge was provided by the finding of reduced concentrations of alpha-tocopherol, elevated concentrations of induced 4-hydroxynonenal, and decreased amounts of the most readily peroxidizable fatty acids in the liver of animals with OWLD, by comparison with controls. The initiator of the peroxidative challenge is unknown, but may be related to the finding of increased concentrations of homocysteine in the plasma of animals with OWLD.

It is now well-established that folic acid, taken peri-conceptionally, can reduce the risk of neural tube defects (NTDs). Recent work has demonstrated that an abnormality of homocysteine metabolism is a critical factor. The gene for 5,10 methylenetetrahydrofolate reductase, an enzyme important in homocysteine metabolism, was studied in relation to NTDs. To determine the frequency of the allele for the thermolabile form of the reductase, DNA samples were collected from people with NTDs, parents of people with NTDs, and normal controls. Of 82 people with NTDs, 15 (18.3%) were homozygous for the abnormal, thermolabile allele. This was significantly higher (p = 0.01) than the rate of 6.1% in the control population (odds ratio 3.47, 95% CI 1.28-9.41). This is the first specific genetic abnormality to be identified in NTDs. It explains the association between some NTDs and elevated homocysteine, given that the reductase is important in homocysteine metabolism. It also explains how folic acid supplementation prevents some NTDs, by overcoming a partial block in the conversion of 5,10 methylenetetrahydrofolate to 5 methyltetrahydrofolate. Genetic screening could identify women who will require folic acid supplements to reduce their risk of having a child with an NTD.