Developing managed aquifer recharge (MAR) to augment irrigation water resources in the sand and gravel (Crag) aquifer of coastal Suffolk, UK.

Managed aquifer recharge (MAR) offers a potential innovative solution for addressing groundwater resource issues, enabling excess surface water to be stored underground for later abstraction. Given its favourable hydrogeological properties, the Pliocene sand and gravel (Crag) aquifer in Suffolk, UK, was selected for a demonstration MAR scheme, with the goal of supplying additional summer irrigation water. The recharge source was a 4.6 km drainage channel that discharges to the River Deben estuary. Trialling the scheme in June 2022, 12,262 m3 of source water were recharged to the aquifer over 12 days via a lagoon and an array of 565 m of buried slotted pipes. Groundwater levels were raised by 0.3 m at the centre of the recharge mound with an approximate radius of 250 m, with no detrimental impact on local water features observed. The source water quality remained stable during the trial with a mean chloride concentration (133 mg L-1) below the regulatory requirement (165 mg L-1). The fraction of recharge water mixing with the groundwater ranged from 69% close to the centre and 5% at the boundary of the recharge mound, leading to a reduction in nitrate-N concentration of 23.6 mg L-1 at the centre of the mound. During July-September 2022, 12,301 m3 of recharge water were abstracted from two, 18 m boreholes to supplement surface irrigation reservoirs during drought conditions. However, the hydraulic conductivity of the Crag aquifer (∼10 m day-1) restricted the yield and thereby reduced the economic viability of the scheme. Construction costs for the MAR system were comparatively low but the high costs of data collection and securing regulatory permits brought the overall capital costs to within 18% of an equivalent surface storage reservoir, demonstrating that market-based mechanisms and more streamlined regulatory processes are required to incentivise similar MAR schemes.

Headteachers' and chairs of governors' perspectives on adolescent obesity and its prevention in English secondary school settings.

BACKGROUND: Secondary schools are an important setting for preventing obesity in adolescence. Headteachers and chairs of governors are identified in national guidance as crucial stakeholders for school-based preventative action. Despite this, their views remain unexplored and unrepresented. METHODS: A sequential mixed method study was conducted. Semi-structured interviews were undertaken with a purposive sample of 22 secondary school headteachers and chairs of governors in England. Data were thematically analysed and informed the development of a descriptive cross-sectional survey, completed by 127 participants from the same population. RESULTS: Unhealthy dietary and sedentary behaviours were viewed as a more significant problem than adolescent obesity. Obesity was perceived as complex and multi-causal, and a range of stakeholders were deemed to have responsibility for its prevention, most notably parents. Support was identified for the role of secondary schools, although this was not an explicit priority and extensive internal and external barriers exist, which hinder preventative action. CONCLUSIONS: Whilst secondary school settings in England remain an important setting for the prevention of adolescent obesity, it is crucial for policy makers and public health professionals to recognize the factors affecting school leaders' ability and willingness to contribute to this agenda.

Search for Neutrinoless Double-ß Decay in

The Majorana Collaboration is operating an array of high purity Ge detectors to search for neutrinoless double-ß decay in ^{76}Ge. The Majorana Demonstrator comprises 44.1 kg of Ge detectors (29.7 kg enriched in ^{76}Ge) split between two modules contained in a low background shield at the Sanford Underground Research Facility in Lead, South Dakota. Here we present results from data taken during construction, commissioning, and the start of full operations. We achieve unprecedented energy resolution of 2.5 keV FWHM at Q_{ßß} and a very low background with no observed candidate events in 9.95 kg yr of enriched Ge exposure, resulting in a lower limit on the half-life of 1.9×10^{25} yr (90% C.L.). This result constrains the effective Majorana neutrino mass to below 240-520 meV, depending on the matrix elements used. In our experimental configuration with the lowest background, the background is 4.0_{-2.5}^{+3.1} counts/(FWHM t yr).

Dietary patterns of university students in the UK: a cross-sectional study.

BACKGROUND: University represents a key transition into adulthood for many adolescents but there are associated concerns about health and behaviours. One important aspect relates to diet and there is emerging evidence that university students may consume poor quality diets, with potential implications for body weight and long-term health. This research aimed to characterise dietary patterns of university students in the UK and their sociodemographic and lifestyle antecedents. METHODS: An online, cross-sectional survey was undertaken with a convenience sample of 1448 university students from five UK universities (King's College London, Universities of St Andrews, Southampton and Sheffield, and Ulster University). The survey comprised a validated food frequency questionnaire alongside lifestyle and sociodemographic questions. Dietary patterns were generated from food frequency intake data using principal components analysis. Nutrient intakes were estimated to characterise the nutrient profile of each dietary pattern. Associations with sociodemographic variables were assessed through general linear modelling. RESULTS: Dietary analyses revealed four major dietary patterns: 'vegetarian'; 'snacking'; 'health-conscious'; and 'convenience, red meat & alcohol'. The 'health-conscious' pattern had the most favourable micronutrient profile. Students' gender, age, year of study, geographical location and cooking ability were associated with differences in pattern behaviour. Female students favoured the 'vegetarian' pattern, whilst male students preferred the 'convenience, red meat & alcohol' pattern. Less healthful dietary patterns were positively associated with lifestyle risk factors such as smoking, low physical activity and take-away consumption. The health-conscious pattern had greatest nutrient density. The 'convenience, red meat & alcohol' pattern was associated with higher weekly food spending; this pattern was also identified most consistently across universities. Students reporting greater cooking ability tended towards the 'vegetarian' and 'health-conscious' patterns. CONCLUSIONS: Food intake varied amongst university students. A substantial proportion of students followed health-promoting diets, which had good nutrient profiles obviating a need for dietary intervention. However, some students consumed poor diets, incurred greater food costs and practised unfavourable lifestyle behaviours, which may have long-term health effects. University policy to improve students' diets should incorporate efforts to promote student engagement in cooking and food preparation, and increased availability of low cost healthier food items.

The utility of near-infrared spectroscopy in the regression of low-frequency physiological noise from functional magnetic resonance imaging data.

Near-infrared spectroscopy (NIRS) signals have been shown to correlate with resting-state BOLD-fMRI data across the whole brain volume, particularly at frequencies below 0.1Hz. While the physiological origins of this correlation remain unclear, its existence may have a practical application in minimizing the background physiological noise present in BOLD-fMRI recordings. We performed simultaneous, resting-state fMRI and 28-channel NIRS in seven adult subjects in order to assess the utility of NIRS signals in the regression of physiological noise from fMRI data. We calculated the variance of the residual error in a general linear model of the baseline fMRI signal, and the reduction of this variance achieved by including NIRS signals in the model. In addition, we introduced a sequence of simulated hemodynamic response functions (HRFs) into the resting-state fMRI data of each subject in order to quantify the effectiveness of NIRS signals in optimizing the recovery of that HRF. For comparison, these calculations were also performed using a pulse and respiration RETROICOR model. Our results show that the use of 10 or more NIRS channels can reduce variance in the residual error by as much as 36% on average across the whole cortex. However the same number of low-pass filtered white noise regressors is shown to produce a reduction of 19%. The RETROICOR model obtained a variance reduction of 6.4%. Our HRF simulation showed that the mean-squared error (MSE) between the recovered and true HRFs is reduced by 21% on average when 10 NIRS channels are applied and by introducing an optimized time lag between the NIRS and fMRI time series, a single NIRS channel can provide an average MSE reduction of 14%. The RETROICOR model did not provide a significant change in MSE. By each of the metrics calculated, NIRS recording is shown to be of significant benefit to the regression of low-frequency physiological noise from fMRI data.

Transient haemodynamic events in neurologically compromised infants: a simultaneous EEG and diffuse optical imaging study.

We describe a series of novel simultaneous EEG and diffuse optical imaging studies of newborn infants. These experiments provide evidence of large, transient haemodynamic events which occur repeatedly and consistently within and across several infants with neurological damage, all of whom were diagnosed with seizures. A simple but independent process of rejecting artifacts and identifying events within diffuse optical imaging data is described, and this process is applied to data from 4 neurologically damaged neonates and from 19 healthy, age-matched controls. This method results in the consistent identification of events in three out of four of the neurologically damaged infant group which are dominated by a slow (>30s) and significant increase in oxyhaemoglobin concentration, followed by a rapid and significant decrease before a slow return to baseline. No comparable events are found in any of our control data sets. The importance and physiological implications of our findings are discussed, as is the suitability of a combined EEG and diffuse optical imaging approach to the study and monitoring of neonatal brain injury.

A tissue-like optically turbid and electrically conducting phantom for simultaneous EEG and near-infrared imaging.

We describe a phantom for simultaneous electroencephalography (EEG) and near-infrared imaging which consists of a solid, optically turbid and electrically conducting interface enclosing a tissue-mimicking aqueous scattering solution. The interface provides an electrical contact impedance comparable to that of the human scalp while the phantom as a whole has optical properties and electrical conductivity equivalent to that of head tissue. The construction is described and our design is evaluated experimentally using an optically absorbing target which also provides an EEG-equivalent electric field source. The results of this simultaneous EEG and near-infrared imaging experiment are presented.

Design and evaluation of a probe for simultaneous EEG and near-infrared imaging of cortical activation.

We present a novel probe design which enables simultaneous electroencephalography (EEG) and near-infrared (NIR) optical imaging to be performed in a manner which is easy to apply, allows for optimum co-registration of the two forms of data and maximizes the number of sensors which can be applied to a given area. Our probe design is evaluated using a dual-modality, tissue-mimicking phantom and by performing a simple functional activation study of the human motor cortex. We successfully acquired NIR optical and EEG data simultaneously for both our phantom and our human motor cortex experiments, clearly demonstrating the effectiveness and suitability of our 'opto-electrode'.

Solo doctors and ethical isolation.

This paper uses the case of solo doctors to explore whether working in relative isolation from one's peers may be detrimental to ethical decision-making. Drawing upon the relevance of communication and interaction for ethical decision-making in the ethical theories of Habermas, Mead and Gadamer, it is argued that doctors benefit from ethical discussion with their peers and that solo practice may make this more difficult. The paper identifies a paucity of empirical research related to solo practice and ethics but draws upon more general medical ethics research and a study that identified ethical isolation among community pharmacists to support the theoretical claims made. The paper concludes by using the literary analogy of Soderberg's Doctor Glas to illustrate the issues raised and how ethical decision-making in relative isolation may be problematical.

'Islands' and 'doctor's tool': the ethical significance of isolation and subordination in UK community pharmacy.

Empirical ethics research is increasingly valued in offering insights into how ethical problems and decision-making occur in healthcare. In this article, the findings of a qualitative study of the ethical problems and decision-making of UK community pharmacists are presented, and it is argued that the identified themes of pharmacists' relative isolation from others and their subordination to doctors are ethically significant. Semi-structured interviews were conducted with 23 community pharmacists in England, UK. Analysis of interviews revealed that isolation involved separation of pharmacists from their peers, other healthcare professionals, patients and customers. Such isolation is argued to be inimical to ethical practice - impeding ethical discourse as understood by Habermas, resulting in a form of anomie that inhibits the transmission of professional values, leading to a lack of proximity between pharmacist and patient or customer that may impede ethical relationships and resulting, psychologically, in less ethical concern for those who are less close. Pharmacists' subordination to doctors not only precipitated some ethical problems but also allowed some pharmacists to shift ethical responsibility to a prescribing doctor, as in the case of emergency hormonal contraception. The emergence of atrocity stories further supports a culture of subordination that may cause ethical problems. The study has implications for community pharmacy practice in terms of supervision issues, developments such as prescribing responsibilities and how ethical values can be taught and communicated. The potential for isolation and subordination in other healthcare professions, and resultant ethical problems, may also need to be addressed and researched.

Ethical decision-making, passivity and pharmacy.

BACKGROUND: Increasing interest in empirical ethics has enhanced understanding of healthcare professionals' ethical problems and attendant decision-making. A four-stage decision-making model involving ethical attention, reasoning, intention and action offers further insights into how more than reasoning alone may contribute to decision-making. AIMS: To explore how the four-stage model can increase understanding of decision-making in healthcare and describe the decision-making of an under-researched professional group. METHODS: 23 purposively sampled UK community pharmacists were asked, in semi-structured interviews, to describe ethical problems in their work and how they were resolved. Framework analysis of transcribed interviews utilised the four decision-making stages, together with constant comparative methods and deviant-case analysis. RESULTS: Pharmacists were often inattentive and constructed problems in legal terms. Ethical reasoning was limited, but examples of appeals to consequences, the golden rule, religious faith and common-sense experience emerged. Ethical intention was compromised by frequent concern about legal prosecution. Ethical inaction was common, typified by pharmacists' failure to report healthcare professionals' bad practices, and ethical passivity emerged to describe these negative examples of the four decision-making stages. Pharmacists occasionally described more ethically active decision-making, but this often involved ethical uncertainty. DISCUSSION: The four decision-making stages are a useful tool in considering how healthcare professionals try to resolve ethical problems in practice. They reveal processes often ignored in normative theories, and their recognition and the emergence of ethical passivity indicates the complexity of decision-making in practice. Ethical passivity may be deleterious to patients' welfare, and concerns emerge about improving pharmacists' ethical training and promoting ethical awareness and responsibility.

Observation of coherent elastic neutrino-nucleus scattering.

The coherent elastic scattering of neutrinos off nuclei has eluded detection for four decades, even though its predicted cross section is by far the largest of all low-energy neutrino couplings. This mode of interaction offers new opportunities to study neutrino properties and leads to a miniaturization of detector size, with potential technological applications. We observed this process at a 6.7σ confidence level, using a low-background, 14.6-kilogram CsI[Na] scintillator exposed to the neutrino emissions from the Spallation Neutron Source at Oak Ridge National Laboratory. Characteristic signatures in energy and time, predicted by the standard model for this process, were observed in high signal-to-background conditions. Improved constraints on nonstandard neutrino interactions with quarks are derived from this initial data set.

Recombinant GM-CSF/IL-3 fusion protein: its effect on in vitro human megakaryocytopoiesis.

An evaluation of the effectiveness of a genetically engineered recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein (FP) as a means of delivering cytokine combinations to megakaryocyte (MK) progenitor cells was performed, utilizing a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the FP, alone and in combination with a variety of other cytokines, on the primitive MK progenitor cell, the megakaryocyte burst-forming unit (BFU-MK), and the more differentiated megakaryocyte colony-forming unit (CFU-MK) were assessed. Subpopulations of bone marrow cells (CD34+ DR- for BFU-MK and CD34+ DR+ for CFU-MK) served as sources of these two classes of MK progenitor cells. The FP was equivalent to a combination of optimal concentrations of GM-CSF and IL-3 in promoting both the number and size of BFU-MK-derived colonies. The GM-CSF/IL-3 combination, however, promoted the formation of far greater CFU-MK-derived colonies than did the FP alone. The size of MK colonies formed in the presence of the FP or GM-CSF/IL-3 was similar. The ability of the FP to stimulate BFU-MK- but not CFU-MK-derived colony formation was also further augmented by the addition of interleukin 1 alpha (IL-1 alpha). The addition of c-kit ligand (KL) increased both FP-stimulated CFU-MK- and BFU-MK-derived colony numbers but only BFU-MK-derived colony size. In addition, the FP alone sustained long-term megakaryocytopoiesis in vitro to a level equivalent to that of the GM-CSF/IL-3 combination and was superior in this regard to either GM-CSF or IL-3 alone. These data indicate that FP is capable of supporting various stages of human megakaryocytopoiesis. We conclude that such genetically engineered molecules as the FP may prove to be effective means of pharmacologically delivering the biological effects of specific cytokine combinations.

Effects of recombinant human interleukin-11 (Neumega rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow.

We examined the effects of recombinant human interleukin 11 (rhIL-11) on in vivo human hematopoiesis. Twelve women with advanced breast cancer and no evidence of bone marrow (BM) involvement were treated with 10, 25, 50, or 75 micrograms/kg/day of rhIL-11 administered subcutaneously for 14 consecutive days. Examination of bone marrow trephine biopsies obtained before and after rhIL-11 treatment revealed unchanged BM cellularity at all doses, and a statistically significant increase in megakaryocyte (MK) frequencies (from 0.5 +/- 0.1% to 1.0 +/- 0.3%) following administration of the two highest doses (p < 0.001). The BM biopsies also showed an increased proportion of immature myeloid and erythroid precursors following 14 days of treatment in all cases. The mean proportion of marrow cells stained with PC10, a monoclonal antibody (mAb) that recognizes the proliferating cell nuclear antigen (PCNA), increased from 16.3 +/- 5.7% to 45.8 +/- 17.1% (p < 0.001) following the two highest treatment doses. Most of the PC10+ cells were promyelocytes and proerythroblasts. In this same group, the proportion of PC10+ MKs increased from 28.3 +/- 11.5% to 56.8 +/- 24.3% (p < 0.01) after treatment, while megakaryocyte ploidy analysis revealed a greater number of higher ploidy (64N) megakaryocytes following rhIL-11 treatment (p < 0.012). Numbers of BM and peripheral blood (PB) CD34+, CD34+DR+, and CD34+DR- cells did not change following rhIL-11 treatment. Following rhIL-11 therapy at the highest dose studied, a 3- and 10-fold increase in the number of committed BM MK progenitor cells (CFU-MK) was observed in two of three patients, while no change was seen in the number of the other BM or PB progenitor cells examined. rhIL-11 administration was also associated with an increase in BM reticulin content (fibrosis grade 1-2) in 7 patients. These results indicate that the administration of rhIL-11 to patients with normal hematopoiesis stimulates MK endoreduplication, PCNA expression, and, at high doses, increases MK and CFU-MK progenitor cell numbers. In addition, rhIL-11 was able to stimulate precursor cells of different marrow lineages without affecting the number of assayable progenitor cells.

Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

Differential transduction efficiency of SCID-repopulating cells derived from umbilical cord blood and granulocyte colony-stimulating factor-mobilized peripheral blood.

The gene transfer efficiency into nonobese diabetic/severe combined immunodeficient (NOD/SCID)-repopulating cells (SRCs) derived from umbilical cord blood (UCB) (n = 11 NOD/SCID mice) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) (n = 64 NOD/SCID mice) was compared using a clinically relevant protocol and a retrovirus vector expressing the enhanced green fluorescent protein (EGFP). At 6-9 weeks after transplantation, the frequency of transduced human cells in the bone marrow (BM) (40.5% +/- 2.4% [mean +/- SE]) and spleen (SPL) (36.4% +/- 3.2%) in recipients of UCB cells was significantly higher (p < 0.001) than that observed in the BM (2.2% +/- 1.8%) and SPL (2.0% +/- 2.6%) in recipients of MPB. In subsequent studies, MPB was cultured for 2-8 days in cytokines prior to transduction to determine if longer prestimulation was required for optimal gene transfer. A significant increase in gene transfer into CD45(+) human cells and clonogenic cells derived from MPB SRCs was observed when cells were prestimulated for 6 days compared to 2 days prior to transduction (p = 0.019). However, even after 6 days of prestimulation, transduction was still significantly less than UCB. A substantial discrepancy exists in the ability to introduce genes effectively via retrovirus vectors into SRCs derived from MPB as compared to UCB.

Multiplex polymerase chain reaction for diagnosis of viral and chlamydial keratoconjunctivitis.

PURPOSE: To develop a multiplex polymerase chain reaction (PCR) for the detection of adenovirus, herpes simplex virus, and Chlamydia trachomatis in conjunctival swabs. METHODS: Oligonucleotide primers for detection of the 3 agents were combined in one reaction and evaluated for optimal performance using control DNAs of adenovirus type 2, herpes simplex virus, and C. trachomatis plasmid. The multiplex PCR was evaluated prospectively against its corresponding uniplex PCRs, virus isolation, Chlamydia Amplicor PCR, and an immunoassay technique (immune dot blot test) in a total of 805 conjunctival swabs from patients with suspected viral and chlamydial keratoconjunctivitis. RESULTS: The multiplex PCR was as sensitive as uniplex PCRs for the detection of the agents in clinical specimens. In the prospective study, 48 of 49 (98%) clinical specimens were positive for adenovirus by the multiplex PCR compared with 26 of 49 (53%) by adenovirus isolation. For herpes simplex virus detection, the multiplex PCR had a sensitivity of 92% (34/37) compared with 94.5% (35/37) by cell culture. The multiplex PCR produced identical results to the Amplicor PCR (21/21; 100%) compared with 71% (15/21) by the immune dot blot test. CONCLUSIONS: With clinical specimens the multiplex PCR was as sensitive as its respective uniplex PCRs but more sensitive than adenovirus isolation and as sensitive as herpes simplex virus isolation or C. trachomatis Amplicor PCR. It has the potential to replace several diagnostic tests with consequent savings in cost. The test also reduces the risk of misdiagnosis by the clinicians.

Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.

PURPOSE: To evaluate newly designed primers in a polymerase chain reaction (PCR) for the detection of adenovirus DNA in conjunctival swabs. METHODS: Oligonucleotides were derived from the adenovirus hexon gene and modified such that a maximum of only two mismatches occurred with adenovirus types 2 through 5, 7, and 16. Specificity was determined against adenovirus types 2 through 4, 7, 8 through 11, 14, 19, 37, 40, and 41 and from non-adenoviral DNA and the sensitivity by PCR amplification of purified adenovirus type 2 DNA. The assay was compared retrospectively with cell culture and a PCR with different primers on 59 stored conjunctival swab samples. The new PCR also was used prospectively in comparison with cell culture on 2743 conjunctival swabs. RESULTS: The 140-bp product was amplified from all the adenovirus serotypes tested except types 40 and 41, which have not been isolated from the eye. There were no amplified products from the non-adenoviral DNA tested. With adenovirus type 2 DNA, despite two deliberate mismatches, 40 copies of the target were detectable after PCR and ethidium bromide-staining. In the retrospective study, 51 of 55 (92.7%) were positive by this new PCR compared with 42 of 55 (76.4%) by the older PCR and 40 of 55 (72.7%) by cell culture. In the prospective study, the new PCR detected 386 of 415 (93%) adenovirus-positive specimens compared with 248 of 415 (59.8%) by cell culture. Of 167 specimens positive for herpes simplex virus by cell culture, none were positive by the adenovirus PCR. CONCLUSIONS: PCR with the newly designed primers shows a much increased sensitivity over cell culture and previous PCRs for the detection of adenoviruses in conjunctival swabs.

Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human beta-globin DNA.

Human cytomegalovirus and human herpesvirus-6 are closely related viruses which cause similar diseases, have similar cellular repositories of latent infection, and may be detected largely in the same types of clinical specimens. DNA amplification appears likely to play an increasing role in the diagnosis of recent and remote infection with these agents. A sensitive multiplex polymerase chain reaction was therefore developed for the two viruses and for human beta-globin DNA. Optimization of parameters such as the primers, primer concentrations, magnesium concentration, and buffer constituents was crucial in achieving a sensitive assay. Preliminary results indicated that the assay could simultaneously monitor DNA extraction from clinical specimens and allow detection of HCMV or HHV-6 in patients with diseases possibly caused by either pathogen.

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.