Longitudinal study of rotavirus infection in child-care centres

To describe the epidemiology of symptomatic and asymptomatic rotavirus infection among young children attending Sydney child-care centres during the 1994 rotavirus season.

Methods:

Children aged 0–36 months in 11 child-care centres participated in the study. A weekly stool specimen was collected from each subject and tested for rotavirus antigen by commercial enzyme immunoassay.

Results:

One hundred and seventy-eight children (76 girls and 102 boys) with a mean age of 18.2 ± 6.5 (SD) months were enrolled for a total of 2249 child-weeks. Of 1653 weekly faecal specimens, 59 (3.6%) were positive for rotavirus antigen. Positivity for rotavirus antigen peaked at 8.0% and 7.4% of specimens in weeks 6 and 7, respectively. The 59 positive specimens were obtained from 44 children in eight of the 11 study centres. One child appeared to suffer a second episode. Eighty-two per cent of episodes were associated with symptoms of gastroenteritis. Overall, 32% of the children in the eight affected centres were infected; 52% of those <12 months were infected compared to 26% of older children. Secondary spread to household contacts was also documented.

Conclusions:

Rotavirus infection poses a significant health problem in under-3-year-old children attending child-care centres in Sydney. We believe that these results are applicable to all Australian children of this age attending group child care.     

Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase

The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a "high-ATP syndrome" by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased.     

Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after in-vitro fertilization

A retrospective analysis of clinical and laboratory data was made of all in-vitro fertilization (IVF) patients with tubal pathology who had their first ever embryo transfer cycle between January 1st, 1992 and September 1st, 1996. The aim of the study was to determine the effect of the presence of a hydrosalpinx, whether or not visible by ultrasound, on pregnancy, multiple pregnancy and implantation rates in our patient population. The IVF success rate was also analysed by calculating cumulative ongoing pregnancy rates of the same patient group using the lifetime table approach. In the presence of an ultrasound-visible hydrosalpinx, rates of pregnancy and multiple pregnancy appeared reduced, but the differences were not significant. The rates of implantation, clinical implantation and ongoing implantation were significantly lower in the presence of an ultrasound- visible hydrosalpinx (odds ratios 0.33-0.46, C.I. 0.21-0.96). The cumulative chance of achieving an ongoing pregnancy after one or more IVF cycles was significantly reduced in the presence of an ultrasound- visible hydrosalpinx (relative hazard 0.36, C.I. 0.22-0.59). In the presence of a hydrosalpinx not visible by ultrasound the IVF outcome was not reduced. This retrospective study confirms that patients with hydrosalpinges have an impaired IVF outcome. Unique to this study and previously unobserved is the finding that there is a subgroup of patients with hydrosalpinges, those with ultrasound-visible hydrosalpinges, which is exclusively responsible for this impaired outcome.      

Prognostic factors for an unsatisfactory primary methotrexate treatment of cervical pregnancy: a quantitative review

To determine the risks when the primary methotrexate (MTX) treatment of cervical pregnancy has an unsatisfactory outcome, we conducted a Medline search on relevant literature published from January 1983 to June 1997. The search yielded 28 publications of 48 cases of cervical pregnancy. These and four new cases from our institutions were used in our study. A cervical pregnancy that presented with a serum beta-human chorionic gonadotrophin concentration of > or = 10,000 mIU/ml [odds ratio (OR) 10.82, 95% confidence interval (CI) 2.59, 45.14], gestational age at > or = 9 weeks (OR 6.44, 95% CI 1.46, 28.52), embryonic cardiac activity (OR 14.29, 95% CI 2.95, 76.92), and crown- rump length of >10 mm (OR 13.33, 95% CI 1.46, 120.48) was considered to be associated with a higher unsatisfactory rate of primary MTX treatment. A concomitant feticide was found to enhance the therapeutic effect of MTX treatment if embryonic cardiac activity was evident (OR 0.13, 95% CI 0.02, 0.68). Administration of a high dose of MTX did not seem to be more effective than a lower one. Our findings supported some previous observations and, more importantly, provided useful clinical information in selecting appropriate candidates for MTX treatment in cases of cervical pregnancy.      

Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria

Mutation analysis of genomic DNA samples obtained from 17 unrelated South African patients with variegate porphyria (VP) revealed three novel missense mutations in the protoporphyrinogen oxidase (PPOX) gene. A common C to T transition at nucleotide position 452 (R59W) was identified in 15 of the patients analysed, while base changes at positions 336 (H20P) and 779 (R168C) were identified in the remaining two patients. Using protein analysis software we were able to predict that all three mutations have a similar biophysical effect on the protein, being the disturbance of amphiphatic regions within the protein, which might result in misfolding of the protein. Mutation R59W, identified in the majority of South African VP families, was shown to create a Styl restriction site, while mutation R168C would abolish a Dsal restriction site in genomic DNA of affected individuals. As 100% of the index patients analysed were molecularly characterized, the combined use of restriction enzyme and single-strand conformation polymorphism (SSCP) analysis now allows a rapid and accurate diagnosis of VP in South Africa. Mutation R59W was furthermore shown to be in association with one of four potential haplotypes defined by two newly described polymorphisms in exon 1 of the PPOX gene. Our molecular data thus strongly support the founder hypothesis for VP in South Africa.      

Molecular basis of the brindled mouse mutant (Mo(br)): a murine model of Menkes disease

The brindled mouse mutant (Mo(br)) is the closest animal model of the human genetic copper deficiency, Menkes disease, which is presumed to be due to a mutation at the X-linked mottled locus (Mo). The mutant mice are hypopigmented and die at around 15 days after birth, but can be saved by treatment with copper before the 10th postnatal day. Menkes disease has been shown to be due to mutations of the gene ATP7A which encodes P-type ATPase (referred to here as MNK). MNK is likely to function in copper efflux from cells, but the full range of its biological activity is not fully understood. The nature of the mutation in the brindled mouse is of importance in our understanding of the role of MNK and for devising treatment strategies for Menkes disease. Here we show that the brindled mouse has a deletion of two amino acids in a highly conserved, but functionally uncharacterized, region of Mnk. Comparison with the Ca ATPases suggests this region may be involved in conformational changes associated with the E1/E2 transition fundamental to the action of P-type ATPases. We also describe the first Western blot data for Mnk in tissues, and these show normal levels of Mnk in mutant and brindled kidneys but none in liver. In the kidney, immunohistochemistry demonstrated Mnk in the proximal and distal tubules, the distribution is identical in mutant and normal. This distribution is consistent with Mnk being involved in copper resorption from the urine.      

Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects

It is now well recognized that periconceptional folic acid or folic acid containing multivitamin supplementation reduces the risk of neural tube defects (NTDs). Recently we were able to show that homozygosity for a thermolabile variant of the enzyme methylenetetrahydrofolate reductase is associated with an increased risk for spina bifida in patients recruited from the Dutch population. However, this genetic risk factor could not account for all folic acid preventable NTDs. In an attempt to identify additional folate related enzymes that contribute to NTD etiology we now studied the methylenetetrahydrofolate dehydrogenase gene on chromosome 14q24 which encodes a single protein with three catalytic properties important in the folate metabolism. The cDNA sequence of 38 familial and 79 sporadic patients was screened for the presence of mutations by single strand conformation polymorphism (SSCP) analysis followed by sequencing. Two amino acid substitutions were identified. The first one (R293H) was detected in a patient with familial spina bifida and not in 300 control individuals. The mutation was inherited from the unaffected maternal grandmother and was also present in two younger brothers of the index patient, one of them displaying spina bifida occulta and the other being unaffected. The second change turned out to be an amino acid polymorphism (R653Q) that was present in both patients and controls with similar frequencies. Our results so far provide no evidence for a major role of the methylenetetrahydrofolate-dehydrogenase (MTHFD) gene in NTD etiology. However, the identification of a mutation in one family suggests that this gene can act as a risk factor for human NTD.     

Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state

The heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was investigated in five women aged 29.4 +/- 3.2 years (mean +/- SD) throughout their menstrual cycles and in five post- menopausal women aged 53.8 +/- 5.6 years. Chromatofocusing (pH range 7- 4) revealed menstrual cycle stage- and postmenopausal-related differences in the serum gonadotrophin charge. There were differences in the proportion of FSH with an isoelectric point (pl) > 4.3 across phases of the menstrual cycle (P = 0.019): midcycle (MC) 50%; early to mid-follicular (EMF) 36%; late follicular (LF) 37%, luteal (L) 29% and following the menopause (PM) 17%. There was no significant difference in the proportion of LH with pl > 6.55 between midcycle (53%) and EMF, LF or L phases (36, 43 and 32% respectively); although all were greater than that found in the menopause (13%). Concanavalin A chromatography revealed less (P < 0.005) complex FSH and LH glycoforms at midcycle (63 and 13%) than in the EMF, LF and L phases (90 and 18; 90 and 20 and 93 and 24% respectively). Menopausal gonadotrophins were least complex (FSH 34%, LH 4%). There was a direct relationship between serum FSH and FSH pl/complexity, and less acidic FSH was associated with reduced FSH complexity. Increased oestradiol was associated with basic FSH isoforms during the menstrual cycle and reduced follicular phase FSH complexity. We conclude that changes in gonadotrophin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively simple glycoforms.      

Endometrial perfusion across the normal human menstrual cycle assessed by laser Doppler fluxmetry

This study investigated variations in microvascular perfusion of human endometrium across the menstrual cycle, using a laser Doppler technique to assess red blood cell (RBC) flux. Endometrial RBC flux was monitored by laser Doppler fluxmetry via a fibre optic probe inserted transvaginally into the uteri of 19 conscious normal volunteer women, on four occasions at weekly intervals over one menstrual cycle. Regional variation in RBC flux was investigated in 16 surgical patients under general anaesthesia and in five excised uteri. Endometrial perfusion exhibited short-term temporal variations consistent with the cardiac cycle and often also showed vasomotion (5-12 cycles/min). Mean endometrial perfusion differed between phases of the menstrual cycle in conscious women, being highest during early proliferative and early follicular phases. There were no significant regional differences in local mean endometrial perfusion in anaesthetized patients. No evidence of endometrial ischaemia/reperfusion episodes was found in any subject using this technique. This study provides benchmark data of variations in RBC flux per unit volume of tissue in the luminal approximately 1 mm of endometrium, across the normal human menstrual cycle. Flux values were highest at times associated with endometrial growth and preparation for implantation, indicating that RBC flux may be a useful parameter for assessment of endometrial physiology.      

Do we truly see what we think we see? The role of cognitive bias in pathological interpretation

In the histomorphological grading of prostate carcinoma, pathologists have regularly assigned comparable scores for the architectural Gleason and the now‐obsolete nuclear World Health Organization (WHO) grading systems. Although both systems demonstrate good correspondence between grade and survival, they are based on fundamentally different biological criteria. We tested the hypothesis that this apparent concurrence between the two grading systems originates from an interpretation bias in the minds of diagnostic pathologists, rather than reflecting a biological reality. Three pathologists graded 178 prostatectomy specimens, assigning Gleason and WHO scores on glass slides and on digital images of nuclei isolated out of their architectural context. The results were analysed with respect to interdependencies among the grading systems, to tumour recurrence (PSA relapse > 0.1 ng/ml at 48 months) and robust nuclear morphometry, as assessed by computer‐assisted image analysis. WHO and Gleason grades were strongly correlated (r = 0.82) and demonstrated identical prognostic power. However, WHO grades correlated poorly with nuclear morphology (r = 0.19). Grading of nuclei isolated out of their architectural context significantly improved accuracy for nuclear morphology (r = 0.55), but the prognostic power was virtually lost. In conclusion, the architectural organization of a tumour, which the pathologist cannot avoid noticing during initial slide viewing at low magnification, unwittingly influences the subsequent nuclear grade assignment. In our study, the prognostic power of the WHO grading system was dependent on visual assessment of tumour growth pattern. We demonstrate for the first time the influence a cognitive bias can have in the generation of an error in diagnostic pathology and highlight a considerable problem in histopathological tumour grading. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.