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PubMed2001

Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.

Adolescent↗
PubMed2002

Detection of measles virus-specific immunoglobulin M in dried venous blood samples by using a commercial enzyme immunoassay.

The optical densities (ODs) of 216 dried venous blood (DVB) samples submitted to the Victorian Infectious Diseases Reference Laboratory as part of enhanced measles surveillance were compared to the ODs of the corresponding serum samples collected at the same time. DVB samples, stored for up to 24 months at 4 degrees C, were tested by the Dade Behring Enzygnost Anti-Measles-Virus/IgM immunoassay. Elution and testing conditions were optimized with the use of spiked DVB samples. The assay showed an overall sensitivity of 90.2% and a specificity of 98.8% for DVB samples compared to the results for serum. When the results were analyzed according to the length of time that the DVB sample had been stored, the assay was 100% sensitive and 97% specific according to the ODs for those samples stored for less than 6 months compared to the results for the corresponding serum samples, with 97.7% agreement between the results for the two sample types. These results demonstrate the potential for the use of DVB samples for the diagnosis of measles in routine diagnostic laboratories.

Adolescent↗
PubMed2003

Comparison between indirect immunofluorescence assay and shell vial culture for detection of mumps virus from clinical samples.

We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy.

Fluorescent Antibody Technique, Indirect↗
PubMed2005

Genetic typing of the porin protein of Neisseria gonorrhoeae from clinical noncultured samples for strain characterization and identification of mixed gonococcal infections.

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Bacterial Typing Techniques↗
PubMed1997

PCR for detection of Chlamydia trachomatis in endocervical, urethral, rectal, and pharyngeal swab samples obtained from patients attending an STD clinic.

OBJECTIVE: To investigate, by use of the Amplicor PCR in a routine setting, the recovery rate of Chlamydia trachomatis in ano-rectal and pharyngeal swab samples obtained from males and females attending an STD clinic in relation to sexual practices, symptoms, and signs. DESIGN: Data regarding sexual practices, and symptoms and signs related to the rectum and pharynx, were obtained from 196 females and 208 males, including 31 homosexuals and eight bisexuals. Swab samples were obtained from the urethra, rectum, and pharynx from all the patients. An additional endocervical swab sample was obtained from the females. METHODS: All samples were analysed by the Amplicor PCR (Roche). SETTING: Rudolph Bergh's Hospital, a clinic for sexually transmitted diseases situated in the centre of Copenhagen, Denmark. RESULTS: The overall prevalence of urogenital C trachomatis infection was 9.2% (37/404). The specificity of the Amplicor PCR was 100% for both ano-rectal and pharyngeal swab samples. In females three (13%) of the 23 infections were detected only by testing an ano-rectal or throat swab sample. In homosexual males two (67%) of three infections were detected only by the anorectal swab sample. Ano-rectal intercourse without use of condom was reported by 44% of females and by 52% of homosexual males. Fellatio without condom use was reported by 91% of females, and 80% of heterosexual males practised cunnilingus. Pharyngeal infection, however, occurred only in females, and the presence of pharyngeal symptoms or signs seemed predictive for pharyngeal C trachomatis infection, for which the time of incubation or colonisation exceeded 3 months. The presence of ano-rectal signs or symptoms was not predictive for an ano-rectal C trachomatis infection. CONCLUSION: The Amplicor PCR can be used on ano-rectal and pharyngeal swab samples. Ano-rectal swab samples should be obtained in females and homosexual males at high risk of being infected. Pharyngeal samples should be taken in females at high risk of being infected, especially when pharyngeal signs or symptoms are present.

Adolescent↗
PubMed1990

Effect of sampling on variability and plateau in oxygen uptake.

To evaluate the effect of the gas exchange sampling interval on variability and plateau in O2 uptake (VO2), 10 subjects underwent steady-state treadmill exercise at 50% maximal VO2 and 6 subjects underwent maximal testing using a ramp protocol. During steady-state exercise, gas exchange data were acquired by using 10 different sampling intervals. The variability in VO2 was greater as the sampling interval shortened (SD = 4.5 ml.kg-1.min-1 for breath-by-breath vs. 0.8 ml.kg-1.min-1 for 60-s samples). The breath-by-breath data suggested a Gaussian distribution, and most of the variability was attributable to tidal volume (51%). During ramp testing, the slope of the change in VO2 (for each sample) was regressed with time. Considerable variability in the slopes was observed throughout exercise, and in each subject the slopes varied about zero, demonstrating both positive and negative values throughout submaximal effort. These observations were made despite the use of large sampling intervals. Shortening the sample resulted in even greater variability. We conclude that 1) the sampling interval can have a major impact on gas exchange data during exercise and 2) considerable variability exists in the slope of the change in VO2 with a consistent change in external work regardless of the sample used, suggesting that a plateau (defined as the slope of a VO2 sample at peak exercise that does not differ significantly from a slope of zero) in VO2 is not a reliable physiological marker for maximal effort.

Adult↗
PubMed1987

The influence of the sampling point on testicular steroid concentrations in spermatic venous blood: a physiological approach to evaluate testicular secretion.

In the present study, we have evaluated the influence of the location of the blood sampling in the spermatic vein on the steroid concentrations observed. Simultaneous blood sampling at two different points of the spermatic vein (iliac level and pampiniform plexus) was perfomed in the same patients during a surgical protocol for varicocelectomy. In order to further evaluate which of the two sampling points is more useful to investigate testicular secretion, we have performed both forms of sampling in 4 volunteers given an HCG stimulation 24 h before the surgical procedure. It was found that levels of testosterone (T) and 17 alpha-hydroxyprogesterone (17-OHP) were higher in the pampiniform plexus (scrotal) than at the iliac sampling point (T scrotal 1,168.343 +/- 142.65 nmol/l, iliac 850.63 +/- 143.411 nmol/l, n = 21, p less than 0.01; 17-OHP scrotal 260.130 +/- 43.14 nmol/l, iliac 164.46 +/- 31.02 nmol/l, n = 17, p less than 0.01). This indicates that spermatic blood collected at the scrotal sampling point has received more blood coming from the testis than the blood collected at the iliac point. We did not observe significant differences in progesterone and delta 4-androstenedione (delta 4) levels between the two samplings. The T/delta 4 ratio was significantly lower in the iliac than in the scrotal sampling (T/delta 4 scrotal 31.420 +/- 6.69; iliac 15.41 +/- 3.84; p less than 0.05). After HCG stimulation, testosterone concentrations were higher in the pampiniform plexus than in the iliac sample. This suggests that the first sampling point is more proper for studying testicular secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

17-alpha-Hydroxyprogesterone↗
PubMedDate not supplied

Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.

OBJECTIVE: To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN: To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month-old samples. Smears of approximately 500 base pairs were observed in the 1-year-old and 10-year-old samples. Successful mRNA amplification was possible from microdissected cell populations. CONCLUSION: Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.

Cervix Uteri↗
PubMed1975

The reliability of intermittent ECG sampling in arrhythmia detection.

Although intermittent ECG sampling is a commonly used method for arrhythmia detection there are no controlled studies of its efficiency. In the present study a continuous ECG was recorded for three hours in 52 patients with ventricular tachyarrhythmias. All ECGs were interpreted minute to minute to get the true arrhythmia content. Intermittent ECG samples were simulated by analyzing the first two and first five minutes of every 15 minutes in the ECG material. Two minute long ECG samples were also used every 30 minutes. The 15 and 30 minute long periods were classified as containing arrhythmias or not according to the arrhythmia content of the samples. These findings were subsequently compared with the true arrhythmia content. The arrhythmia detection rate depends on the sampling time and true arrhythmia occurrence. The distribution of arrhythmias within the periods was also of importance. With a five minute long ECG sample about 80% of the intervals containing any type of ventricular tachyarrhythmia will be detected. This is reduced to about 50% when one minute long ECG strips are used. Infrequent types of VPCs such as R on T or ventricular tachycardia are very poorly detected also when sampling as much as one-third of the total time. The study also included a comparison between the results obtained from the clinical material and the results obtained by the use of computerized arrhythmia models. It is concluded that intermittent ECG sampling besides a low detection rate for infrequent arrhythmias and short ECG samples, also brings a risk of underestimating or overemphasizing the arrhythmia occurrence depending on a periodic type of arrhythmia distribution among the patients. These factors make intermittent ECG sampling an unsuitable method for evaluating antiarrhythmic drugs.

Adult↗
PubMed2003

Sampling requirements for ambulatory blood pressure monitoring in the diagnosis of hypertension in pregnancy.

Previous studies on ambulatory blood pressure monitoring as a potential screening test for hypertension in pregnancy have not carefully considered sampling requirements. We have examined the impact of duration and frequency of blood pressure sampling in the reproducibility of mean values in pregnancy. We analyzed 2430 blood pressure series sampled every 20 minutes during the day and every 30 minutes at night for 48 hours every 4 weeks from the first obstetric visit until delivery in 235 normotensive and 168 hypertensive pregnant women. Blood pressure series were decimated to generate shorter series with data sampled every 1, 2, 3, or 4 hours for 48 hours, as well as at the original rate for the first day. Reproducibility of mean blood pressure as well as sensitivity and specificity in the diagnosis of hypertension were compared between the original and the decimated series. Sensitivity and specificity of the 24-hour blood pressure mean are similar for the values calculated from the original series and for those obtained from shorter profiles up to data sampled every 3 hours but reduced by 5% to 12% when diagnosis is based on data sampled at 20- to 30-minute intervals for the first 24 hours. Results also indicate that the 24-hour blood pressure mean is better reproduced with data sampled at 3-hour intervals for 48 hours than by data sampled at 20- to 30-minute intervals for 1 day only. This study demonstrates that reproducibility of mean blood pressure values is more dependent on duration of sampling than on sampling rate.

Adolescent↗
PubMed2006

Acceptability of unsupervised HPV self-sampling using written instructions.

OBJECTIVES: The study measured the acceptability of self-sampling for human papillomavirus (HPV) testing in the context of cervical cancer screening. Women carried out self-sampling unsupervised, using a written instruction sheet. SETTING: Participants were women attending either a family planning clinic or a primary care trust for routine cervical screening. METHODS: Women (n = 902) carried out self-sampling for HPV testing and then a clinician did a routine cervical smear and HPV test. Immediately after having the two tests, participants completed a measure of acceptability for both tests, and answered questions about ease of using the instruction sheet and willingness to use self-sampling in the future. RESULTS: The majority of women found self-sampling more acceptable than the clinician-administered test, but there was a lack of confidence that the test had been done correctly. Significant demographic differences in attitudes were found, with married women having more favourable attitudes towards self-sampling than single women, and Asian women having more negative attitudes than women in other ethnic groups. Intention to use self-sampling in the future was very high across all demographic groups. CONCLUSION: Self-sampling for HPV testing was highly acceptable in this large and demographically diverse sample, and women were able to carry out the test alone, using simple written instructions. Consistent with previous studies, women were concerned about doing the test properly and this issue will need to be addressed if self-sampling is introduced. More work is needed to see whether the demographic differences we found are robust and to identify reasons for lower acceptability among single women and those from Asian background.

Adult↗
PubMed2004

A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.

Animals↗
PubMed2004

A comparison of routine culture with polymerase chain reaction technology for the detection of Mycoplasma species in feline nasal samples.

Nasal flush samples were collected from 20 cats and submitted for Mycoplasma culture and polymerase chain reaction (PCR). Nasal biopsy samples were also obtained from each cat and simultaneously evaluated for Mycoplasma by standard culture and PCR. Concordance of the test results was determined through calculation of the kappa statistic. In 6 cats, nasal flush samples were culture positive for Mycoplasma. PCR was positive in each culture-positive cat and also positive in 1 flush sample that was culture negative. DNA sequencing of the PCR product from the culture negative flush sample identified the organism as Mycoplasma arginini. All other flush samples that were culture negative were also PCR negative (kappa = 0.89). Nasal biopsy samples from 7 cats were culture positive for Mycoplasma, and all were PCR positive. Biopsy samples that were culture negative for Mycoplasma were also PCR negative (kappa = 1.0). Results of culture and PCR for both nasal flush and biopsy were concordant in 19 of 20 cats, and PCR was able to identify an unusual Mycoplasma species that did not grow in culture. In most cats, organisms could be detected in either nasal flush or biopsy samples. In this study, PCR provided rapid and sensitive detection of Mycoplasma species in nasal samples from cats and detected 1 organism that did not grow in culture.

Animals↗
PubMed2004

Collecting, archiving and processing DNA from wildlife samples using FTA databasing paper.

BACKGROUND: Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. RESULTS: FTA databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. CONCLUSIONS: FTA paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler.

Animals↗
PubMedDate not supplied

Field evaluation and comparison of five methods of sampling lead dust on carpets.

Five methods of sampling lead-contaminated dust on carpets were evaluated and compared in 33 New Jersey homes of children with elevated blood lead levels. The five sampling methods were (1) wipe, (2) adhesive label, (3) C18 sheet, (4) vacuum, and (5) hand rinse. Samples were collected side by side on the same carpets within the homes. Among the five methods the wipe and vacuum methods showed high percentages of detectable samples, good reproducibility, and significant correlations with other methods. C18 sheets and adhesive labels collected the least quantity of lead dust, with high percentages of undetectable samples. Because of the limited ability of sampling lead on carpets and the relatively high cost for laboratory analysis, C18 sheets or adhesive labels are not considered feasible sampling techniques. The hand rinse method also was not feasible for carpet sampling, because it was difficult to conduct in the field and laboratory, and it was subject to inconsistency and cross contamination. Wipes, which collected lead dust from carpet surfaces, were believed to be the most appropriate method for measuring lead from carpets accessible to children. However, because of the low pickup from carpets, wipes may not be an appropriate measuring tool to assess the levels of total lead contamination in carpets. The authors recommend using surface wipe sampling to measure accessible lead from carpets for exposure assessment, and vacuum sampling to obtain the information on total lead accumulation.

Child↗
PubMed1983

The effect of processing and storage on key enzymes, B vitamins, and lipids of mature human milk. I. Evaluation of fresh samples and effects of freezing and frozen storage.

A study was initiated to evaluate the effects of several methods of processing and storage on key enzymes, B vitamins, and lipid components of mature human milk. In order to establish standard values for the nutrient components with which to compare processed samples, a total of 30 individual raw samples of mature human milk were analyzed. There was considerable sample to sample variation as indicated by the large range of values for each component. Freezing and frozen storage had little effect on the enzymes of pooled samples of milk. Lactoperoxidase activity decreased from 36 in raw pooled samples to 17 in pooled samples slow frozen and stored for 3 months at -25 degrees C (P less than 0.05). Similarly, quick freezing and storage for 3 months significantly decreased the lactoperoxidase activity of pooled samples from 93 to 14 (P less than 0.05). Quick freezing and frozen storage tended to increase lipase activity although the changes were not significant. Freezing and frozen storage did not significantly affect the levels of biotin, niacin, and folic acid. Similarly, the total lipid fatty acid level and relative % of each fatty acid were not significantly different in the frozen samples as compared to the raw samples.

Enzymes↗
PubMed2004

Pneumatic tube system induced haemolysis: assessing sample type susceptibility to haemolysis.

BACKGROUND: The pneumatic tube system (PTS) has been implicated in inducing haemolysis. It is not known whether certain sample types are more susceptible to haemolysis than others. We assessed the level of haemolysis in commonly used sample types in the clinical biochemistry department when transported through the PTS. METHOD: Blood was collected in pairs for different sample types and sent to the laboratory via the pneumatic tube or delivered by a porter. Haemolysis indices were measured spectrophotometrically and compared for each pair of sample type. RESULTS: Our results suggest that plain serum samples are more susceptible to haemolysis than the other sample types when sent through our PTS (P <0.0001). Compared with serum with gel samples, plain serum samples are more prone to haemolysis (P <0.001). This suggests that gel may confer some protection against haemolysis. CONCLUSION: Different hospitals will have varying system configurations and use different sample types. We recommend that each hospital investigate their own system to assess whether haemolysis is a recurring problem in any of the sample types transported.

Blood Specimen Collection↗
PubMed2003

Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination.

We correlated results of microbiologic culture and histopathologic examination for 2,891 consecutive samples from autopsy tissue, surgical or biopsy tissue, and bronchoalveolar lavage (BAL) or bronchial washing (BW) specimens. For 23 autopsy cases with suspected invasive septate mold infections by histopathologic examination, culture yielded a mold in 12 cases (52%). For 1,683 surgical or biopsy samples, histopathologic evidence of invasive septate mold infection was present in 30 samples, 9 of which also grew mold by culture (30%); 20 additional samples grew mold in culture alone, possibly representing culture contamination. Of 1,185 BAL and BW samples, mold was evident in 28 by cytologic examination and culture, 20 by cytologic examination alone, and 68 by culture alone. These results suggest a positive concordance for culture and histologic-cytologic examination of 23%, although both methods were negative in 96% of surgical and biopsy tissue and BAL and BW samples. The septate molds cultured from these samples were Aspergillus fumigatus (19), Aspergillus flavus (15), Aspergillus terreus (13), Aspergillus niger (7), Fusarium species (3), and Scedosporium apiospermum (2). A flavus was isolated significantly more frequently from tissue than from BAL and BW samples.

Aspergillosis↗