Pooled Urine Research Articles

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Neutralising antibodies (VNT titres > or = 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Development of a Selective ESI-MS Derivatization Reagent: Synthesis and Optimization for the Analysis of Aldehydes in Biological Mixtures

In LC-MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC-MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/ MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 degrees C). As a result, an in-vial derivatization can be performed before analysis with an LC-MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3-33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.

Use of acupuncture in equine reproduction

The goal of this review is to provide evidence supporting the physiologic basis for the use of acupuncture as a treatment in equine reproduction, and to discuss current uses of acupuncture for the treatment of anestrus, urine pooling, uterine infection and/or fluid, infertility, maintenance of pregnancy, cryptorchidism, musculoskeletal pain, and poor libido in stallions. The benefits achieved through acupuncture treatment are thought to be a result of hormonal regulation, altered smooth muscle motility, and general stress and/or pain relief from musculoskeletal or environmental conditions.

First Prize:Novel Uropathogen-Resistant Coatings Inspired by Marine Mussels

Success in the prevention of urinary device infections has been elusive, largely due to multiple bacterial attachment strategies and the development of urinary conditioning films. We investigated a novel anti-fouling coating consisting of mussel adhesive protein mimics conjugated to polyethylene glycol (mPEG-DOPA(3)) for its potential to resist conditioning film formation and uropathogen attachment in human urine. Model TiO(2) -coated silicon disks ( approximately 75 mm(2)) were either coated with mPEG-DOPA(3) or left uncoated and sterilized using ethylene oxide gas. For bacterial attachment experiments, coated and uncoated surfaces were separately challenged with bacterial strains comprising six major uropathogenic species for 24 hours at 37 degrees C in human pooled urine. Starting inoculum for each strain was 10(5) CFU/mL and 0.5 mL was used per disk. Following incubation, the disks were thoroughly rinsed in phosphate buffered saline to remove non-adherent and weakly-adherent organisms and cell scrapers were employed to dislodge those that were firmly attached. Adherent bacteria were quantitated using dilution plating. Representative disks were also examined using scanning electron microscopy, energy dispersive x-ray analysis, and live/dead viability staining. The mPEG-DOPA(3) coating significantly resisted the attachment of all uropathogens tested, with a maximum >231-fold reduction in adherence for Escherichia coli GR-12, Enterococcus faecalis 23241, and Proteus mirabilis 296 compared to uncoated TiO(2) disks. Scanning electron microscopy and viability staining analyses also reflected these results and demonstrated the ability of the coating to resist urinary constituent adherence as well. Model surfaces coated with mPEG-DOPA(3) strongly resisted both urinary film formation and bacterial attachment in vitro. Future in vitro and in vivo studies will be conducted to assess whether similar findings can be demonstrated when these polymer coatings are applied to urologic devices.

Determination of urinary phytoestrogens by HPLC–MS/MS: A comparison of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)

A comparison of the analytical performance of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) for the quantitative determination of six urinary phytoestrogens (daidzein, O-desmethylangolensin, equol, enterodiol, enterolactone and genistein) by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is presented here. Both APCI and ESI were suitable for the analysis of these compounds; however, ESI did improve measurement imprecision and sensitivity in certain cases. Method imprecision (between-run coefficients of variation [CVs] from duplicate analysis of three quality control [QC] urine pools across 20 runs) was 5.6–12% for ESI, as opposed to 5.3–30% for APCI. At low concentrations (3–60 ng/mL, analyte dependent) imprecision was lower with ESI, whereas both techniques were generally commensurate at high concentrations (200–1000 ng/mL, analyte dependent). Method accuracy (spiked analyte recovery from the QC pools) was comparable between techniques: 86–114% for ESI; 95–105% for APCI. Limits of detection (LODs) were equivalent or better with ESI compared to APCI, with the most significant LOD improvement observed for equol (ESI: 0.3 ng/mL; APCI: 2.7 ng/mL). This translated into a substantial increase in equol detection frequency (% of sample results above LOD) within a random patient sample subset (98% for ESI, compared to 81% for APCI, n = 378). Correlation (Pearson) and agreement (Deming regression, Bland-Altman bias) between ESI and APCI results in the patient subset was better in cases where imprecision and sensitivity was similar for both techniques (daidzein, enterolactone, genistein: r = 0.993–0.998; slope = 0.98–1.03; bias = −4.2 to −0.8%); correlation and/or agreement was poorer for analytes, where APCI imprecision and sensitivity were inferior (equol, O-desmethylangolensin, enterodiol). Baring significant factors arising from differences in ionization source design, these observations suggest that ESI is more appropriate for urinary biomonitoring of these compounds by LC–MS/MS.

Evaluation of a Modified Surgical Technique to Correct Urine Pooling in Cows

Urovagina of cows and horses often results in infertility by inducing necrotizing endometritis. This condition is seen most commonly in old, pluriparous cows and mares with poor vaginal and vestibular conformation. Adverse changes result from numerous pregnancies or from dysfunction of the constrictor vestibuli muscle caused by damage to this muscle during parturition. Cranioventral tipping of the pelvis, the result of numerous pregnancies, or damage to the constrictor vestibuli muscle causes the cranial portion of the vagina to be positioned ventral to the external urethral orifice, leading to collection of urine in the vaginal fornix. Various surgical techniques have been used to create a mucosal extension from the urethral orifice to the labia to correct urovagina in cows, but a fistula often forms in the extension, causing the surgical remedy to fail. The objective of the trial reported here was to determine if the incidence of fistula formation could be decreased by covering submucosal tissue on the floor of the vestibule, exposed by transposing mucosal flaps to create the extension, with a mucosal graft.

Exercise and Calcium Supplementation

Exercise-induced sweat calcium losses have been reported as substantial in male athletes. The first aim of the study was to quantify the increase in 24-h total dermal calcium losses and the net changes in calcium retention in active sportswomen after a 1-h strenuous exercise session. A second aim was to determine the effectiveness of calcium supplementation to offset any calcium loss. Twenty-six premenopausal sportswomen completed three 8-d intervention phases in a randomized-order, crossover design. The three phases were placebo+no exercise (control), placebo+exercise, and 400 mg of calcium as calcium carbonate (TUMS Ultra) twice daily+exercise. The supervised exercise was 1 h.d(-1) cycling at 65-70% of heart rate reserve. A controlled diet of approximately 450 mg.d(-1) of calcium and 24-h pooled urine and fecal collections allowed determination of calcium balance on days 5-8 of each phase. Twenty-four-hour dermal collections were made at the end of each phase using a whole-body washdown procedure. Exercise increased (P<0.05) dermal calcium losses (means+/-SD, 92+/-49 vs 79+/-31 mg.d(-1) in the nonexercise intervention period), which was no longer significant (P=0.14) when calcium supplementation was provided (83+/-49 mg.d(-1)). Higher (P<0.01) urinary calcium excretion during calcium supplementation is suggestive of higher net calcium absorption. Exercise did not affect urinary calcium excretion indicating lack of compensation for dermal losses. Net calcium retention was positive only during the exercise+calcium supplementation intervention period. Calcium supplementation can correct for negative calcium balance attributable to low calcium dietary intake and additional dermal losses from exercise.

Effect of urinations on the ammonia emission from group-housing systems for sows with straw bedding: Model assessment

A model was developed as a tool for designing straw-bedded sow group-housing systems with low ammonia emission. Using mechanistic and empirical relationships it calculates the total ammonia emission by integrating ammonia volatilisations from all the urine pools in the house. The reference data were from a house with a floor comprising 60% straw bedding, 14% drinking area (slatted floor with pit), 3% waiting area (slatted floor with pit) and 23% alley (solid floor). Simulations were performed to elucidate the effect of the distribution of the urinations over the different surfaces, relating this to the size and distribution of the urine pools, and the area of the surfaces urinated upon. The results were compared with emission data from an entire sow house. When the default settings were a urine production of 7 l/d per sow, a urination frequency of 5 times a day per sow, and urinations distributed evenly over the four emitting surfaces the model estimated the ammonia emission from the entire house as 11.7 g/d per sow, and the relative contributions of the straw bed, the drinking area, the waiting area and the alley as, respectively, 27%, 22%, 9% and 42%. By comparison, the actual emission from the house was 8.7 g/d per sow. The 90% confidence interval was 6.5–10.9 g/d per sow. Increasing the size of the urine pool from 0.14 m2 to 1.40 m2 in the model simulations caused ammonia emission initially to increase from 9.7 to 12.1 g/d per sow when the pool volume was 0.47 m2. If the pool was bigger, emission fell to 10.6 g/d per sow because, though the larger emitting area increases ammonia emission, the increase is outweighed by the reduction in emission caused by successive, superseding urinations on the same spot. If the entire emitting area was assumed to be straw bedding, the calculated emission from the house was 5.8 g/d per sow. Assuming slatted and concrete floors without straw bedding increased the emission to 16.5 g/d per sow. It is concluded that measures to reduce the ammonia emission from the bedded sow house should be aimed at decreasing the emission from the solid floor and/or allowing more urinations on the straw bed. The model is a useful design tool for achieving emission reduction from group-housing systems for sows with straw bedding. Its predictive power would be improved by inputting data on the actual size of the urine pool and urinating behaviour of sows.

Evaluation of a modified surgical technique to correct urine pooling in cows

Various surgical techniques to correct urovagina in cows describe creating a mucosal extension from the urethral orifice to the labia; however, a fistula often forms in the mucosal extension. The objective of the present study was to determine if the incidence of fistula formation could be decreased by covering transposed submucosal tissue on the dorsal aspect of the urethral extension with a mucosal graft. Cows in both the control group (19) and the experimental group (19) received a modified McKinnon technique of urethral extension; cows in the experimental group also had a sheet of mucosa, obtained from the dorsal aspect of the vestibule, grafted to submucosa exposed during creation of the urethral extension. During histological examination of the biopsy of the graft and its recipient site (harvested 1 week after surgery), neither inosculation nor revascularization of the graft was evident in any sample. Fourteen of 19 (74%) cows in the control group and 10 of 19 (53%) cows in the experimental group developed a fistula in the extension ( P = 0.18). We concluded that application of a mucosal graft to the subcutaneous tissue exposed to the vestibule using the McKinnon technique of creating a urethral extension was of little or no benefit in preventing the formation of a fistula in the extension. Furthermore, during evaluation of the extensions, digital palpation alone was often insufficient for detection of a fistula.

Stable isotope dilution analysis of N‐acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry

N-acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a 'dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d(3)-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C(8) minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d(3)-NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 micromol/L with limit of quantification at 1 micromol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9-102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases.

Urinary excretion of equol by postmenopausal women consuming soymilk fermented by probiotic bifidobacteria

To study the effects of consuming isoflavone aglycone-enriched soymilk fermented by bifidobacteria on urinary excretion of equol with respect to fermentation, daidzein dose, supplementation duration and background diet. Double-blind crossover pilot study comprising three 14-day supplementation periods separated by a washout. Victoria University, Melbourne, Australia. Sixteen postmenopausal women. SUBJECTS randomized into two groups consuming either fermented (FS) or non-fermented soymilk (NFS), ingested three daily dosages of daidzein via soymilk and collected pooled urine specimens. Daidzein and equol were quantified using high-performance liquid chromatography. After 14-days supplementation six women (38%) excreted equol (>1 micromol equol/day), including four from the FS group, two of whom were classified as non-producers at day 4. Bifidobacteria ingestion, composition of daidzein and its glucosides, and carbohydrate intake appeared to influence equol formation among equol producers. Pilot-study group mean urinary equol excretion results provided insufficient evidence (P>0.05) that FS consumption instigates equol production in women predetermined as non-producers.

Metabolism of two analgesic agents, tramadol-n-oxide and tramadol, in specific pathogen-free and axenic mice

The in vivo metabolism of both tramadol-N-oxide (TNO) and tramadol was investigated in urine pools obtained from 0–24 h after a single 300 mg kg−1 oral dose administration of each compound to specific pathogen-free and axenic mice. Unchanged TNO (≤42% of the initial drug sample), tramadol, and 23 metabolites from TNO-treated mice and unchanged tramadol (≤15% of the sample) plus 20 metabolites from tramadol-treated mice were profiled, quantified and tentatively identified on the basis of atmospheric pressure ionization mass spectrometry (API-MS) and tandem mass spectrometry (MS/MS) data. Of the tramadol metabolites, five (M1–5) have been previously identified in mice. Of the tramadol and TNO metabolites, six (M18–23) are new metabolites. The tramadol and TNO metabolites were formed via the following seven metabolic pathways: N-oxide reduction (TNO), O/N-demethylation, cyclohexyloxidation, oxidative N-dealkylation, dehydration (TNO), N-oxidation (tramadol), and glucuronidation. Pathways 1–3 appear to be predominant steps forming four major O/N-desmethyl and hydroxycyclohexyl metabolites, and in conjunction with pathway 7, formed six minor glucuronides. Both tramadol-N-oxide and tramadol are extensively metabolized in mice, and no significant qualitative or quantitative differences in metabolism were observed between specific pathogen-free and axenic mice with the exception of a greater amount of unchanged TNO in axenic mice than in specific pathogen-free mice, more M2 in specific pathogen-free mice than in axenic mice in the TNO-dosed mice, and visa versa for M2 of tramadol-dosed mice.

Assessment of urinary N-telopeptide: Point-of-care testing, sample types, and relationship to urinary helical peptide excretion

Background A point-of-care (POC) device would be useful in the space program for measuring N-telopeptide (NTX), a marker of bone resorption. This study was done to establish whether NTX measurements from a POC device compare favorably with standard (ELISA) techniques. We also compared results from fresh and frozen urine samples, samples collected on consecutive days, and second voids (of the day) and 24-h urine pools. Helical peptide (HP), another crosslink, was compared in second voids and 24-h urine pools. Methods Ten subjects collected urine for 96 h. NTX was measured with the POC device and by ELISA, and HP measured by ELISA. Seven subjects collected 24-h urine pools, and samples were analyzed fresh and after 27 days of freezing. Results Excretion of NTX was greater ( P < 0.05) when measured by the POC device than when measured by ELISA, but was not different between second voids and 24-h urine pools, or among days. HP was similar in second voids and 24-h pools. Less NTX ( P < 0.05) was found in fresh 24-h pools [mean (S.D.) values, 38.4 (11.6) and 33.6 (9.2) nmol/mmol creatinine for the POC device and ELISA] than in previously frozen 24-h pools [42.7 (17.4) and 41.5 (12.5) nmol/mmol creatinine for the POC device and ELISA]. Conclusions Comparisons between NTX measurements from frozen and fresh samples and those analyzed by POC and ELISA techniques should be made with caution.

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.

Validation of an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and S-phenylmercapturic acid

The aim of this study is to validate isotope-dilution electrospray ionization tandem mass spectrometry (ESI–MS–MS) method with a dual-loop cleanup device for simultaneous quantitation of two benzene metabolites, trans, trans-muconic acid ( ttMA) and S-phenylmercapturic acid (SPMA), in human urine. In this study, a pooled blank urine matrix from rural residents was adopted for validation of the analytical method. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of ttMA and SPMA standards spiked into two kinds of urine matrixes over a wide concentration range, 1/32–8-fold biological exposure indices (BEIs) values, showed good linearity ( R > 0.9992). The detection limits in pooled urine matrix for ttMA and SPMA were 1.27 and 0.042 μg g −1 creatinine, respectively. For both of ttMA and SPMA, the intra- and inter-day precision values were considered acceptable well below 25% at the various spiked concentrations. The intra- and inter-day apparent recovery values were also considered acceptable (apparent recovery >90%). The ttMA accuracy was estimated by urinary standard reference material (SRM). The accuracy reported in terms of relative error (RE) was 5.0 ± 2.0% ( n = 3). The stability of sample storage at 4 or −20 °C were assessed. Urinary ttMA and SPMA were found to be stable for at least 8 weeks when stored at 4 or −20 °C. In addition, urine samples from different benzene exposure groups were collected and measured in this system. Without tedious manual sample preparation procedure, the analytical system was able to quantify simultaneously ttMA and SPMA in less than 20 min.

Calcium oxalates grown in human urine under different batch conditions

Calcium oxalate (CaOx) crystallisation in solution is often studied as it is the major crystalline phase in kidney stones, and as such contributes to a large proportion of pathological mineralisation. A variety of different methods are used to produce CaOx in urine, particularly with the goal of investigating mineral/organic interactions. Interpretation of growth phenomena in these studies often neglects the methodological differences, which are not always trivial. CaOx was grown simultaneously from a single pool of urine using six different protocols comparable to those found in the literature. The variations between the CaOx populations generated was great, with CaOx trihydrate detected as the most common hydromorph in two of the methods used, even though it is usually discounted in stone research. Crystal morphologies, density, particle size and surface area varied in all the methods tested, and a common technique used to remove organic matter from the crystal surface resulted in total phase transformation in one of the crystal populations. In conclusion, inter-assay comparisons of CaOx product are likely to be meaningless unless strict protocols are adhered to. Results indicate that certain crystalline properties considered relevant to stone formation may actually be a symptom of the experimental conditions.

Automated On-Line Column-Switching HPLC-MS/MS Method with Peak Focusing for the Determination of Nine Environmental Phenols in Urine

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using 1 autosampler, 2 HPLC pumps, and a 10-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in 100 microL of urine were retained and concentrated on a C18 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.1-0.4 ng/mL for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.

Practice corner: will it happen again doctor? Prognosis after a first seizure

Susan attends with her mother for a consultation at the end of Monday morning surgery. On the previous Friday evening, 22 year old Susan was alone in the kitchen when...

Diagnosis of congenital cytomegalovirus infection by detection of viral DNA in urine pools

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Diagnosis of this infection is important because 5–17% of asymptomatic infected babies will develop late sequelae and should be followed closely. Most of these children will remain undetected, since screening of all newborns by viral culture is too expensive. The aim of this study was to demonstrate that pool testing could be used to detect HCMV congenital infection in newborns. For this purpose, a nested-PCR technique was tested in urine pools. In phase 1, urine specimens were tested alone by nested-PCR and compared with viral culture, followed by cross experiments to test the reliability of detecting one positive specimen in a 20 samples in a urine pool. In phase 2, this pool method was applied to all urine specimens from children received in the virology laboratory of the Centro Hospitalar Cova da Beira for diagnosis of HCMV infection, between January 2002 and March 2003. In phase 1, 74 urine specimens were tested simultaneously by shell-vial culture and nested-PCR; 17 were positive and the remaining 57 negative by both methods. The negative specimens were divided into three pools and each pool was tested alone and crossed with each of the positive specimens by nested-PCR. Although the three pools were negative when tested alone, all 51 crossed results were positive. In phase 2, 15 out of the 180 urine samples tested positive by shell-vial culture and were detected by this pool method. These results suggest that urine pools can be used to detect HCMV positive urines in children, with similar sensitivity and specificity when compared with the standard method, but with a substantial labour reduction. This significant reduction in labour and consequently in cost per test, opens the possibility of applying PCR to urine pools for screening the HCMV congenital infection in newborns.

A modified surgical technique for repairing third-degree perineal lacerations in mares

A modified surgical technique has been developed for repairing third-degree perineal lacerations in mares. Complications of the currently used methods include rectovaginal fistula formation, urine pooling, complete dehiscence of the repair, constipation, tenesmus and difficulty of performance in the practice. The modified method is simpler and more practical. This method was performed on eight Thoroughbred mares with third-degree perineal lacerations after delivery. The rectovestibular septum was reconstructed by three lines of sutures in a transverse direction in relation to the longitudinal axis of the rectum. In one of the eight cases pneumorectum was observed after using the new method. The conception rate obtained after using the new surgical technique was 62.5%. Pregnant mares delivered normally without any new lacerations at the subsequent parturition. It can be concluded that this new surgical technique can be used successfully for repairing third-degree perineal lacerations in mares.