Effects of saliva collection using cotton swab on cortisol enzyme immunoassay

Cotton swabs are among the most commonly used devices for collecting saliva, but various studies have reported that their use impacts the results of salivary cortisol assays. These studies, however, estimated this impact by comparing the average of the concentration and/or scatter plots. In the present study, we estimated the impact of cotton swabs on the results of salivary cortisol enzyme immunoassay (EIA) by Bland–Altman plot. Eight healthy males (aged 20–23 years) provided four saliva samples on different days to yield a total of 32 samples. Saliva samples were collected directly in plastic tubes using plastic straws and then pipetted onto cotton swabs (cotton saliva collection) and into clear sterile tubes (passive saliva collection). There was a lower correlation between cotton and passive saliva collection. Individually, four subjects showed a negative correlation between passive and cotton saliva collection. A Bland–Altman plot indicated that cotton swabs causes a proportional bias on the EIA assay result. Our findings indicate a considerable effect of using cotton swabs for saliva collection, and subject-specific variability in the impact. A Bland–Altman plot further suggests possible reasons for this effect.     

It's not that bad: error introduced by oral stimulants in salivary cortisol research

Evidence that oral stimulants can produce interference effects in salivary cortisol assays has led to advice to avoid their use. However, in studies with young children, the use of these saliva-producing substances increases compliance with collection procedures. Four experiments are described to examine the effects of two commonly used stimulants, SweetTarts and Koolaid. Across these experiments, interference effects produced by different quantities of these stimulants (0.025, 0.1, and 0.2 g) and those produced in two commonly used assays, DELFIA and EIA, were explored. The impact of using cotton rolls soaked with an oral stimulant prior to saliva collection was also examined. In general, oral stimulants did not affect the rank ordering of cortisol values, as the results for stimulant-treated samples were highly correlated with those of untreated samples (approximately .90 for serially collected samples and .95 for aliquots of the same saliva pool). Depending on which assay was used, however, oral stimulants increased or decreased the cortisol levels reported, with effects sizes in the small to medium range. Thus, oral stimulants should not be used with only a portion of the subjects in a study, nor should researchers assay stimulant-treated samples from the same study using different assays. When used sparingly, oral stimulants can be employed without compromising the quality of salivary cortisol data.     

Marshmallows used as saliva stimulant do not affect cortisol concentrations: finally a palatable alternative for toddler saliva collection

Two studies were conducted to validate marshmallows as a saliva stimulant for use with toddlers. First, cortisol concentrations from 14 subjects (ages 6-46 years) were compared using three saliva collection methods: (1) plain cotton dental roll, (2) dental roll with one mini-marshmallow, and (3) expectorating into a collection tube using no cotton or stimulant. EIA was used for analyses. There were no significant differences among cortisol concentrations. Second, saliva collection compliance rate was compared for 21-month-olds (n = 51) using either flavored drink crystal- (compliance rate = 16.7%) or marshmallow-flavored (compliance rate = 60%) dental rolls for saliva collection (chi(2) (1) = 4.02, p = .045). These studies indicate that marshmallow is a viable option for saliva stimulation to determine toddler cortisol concentrations using EIA.     

Measuring salivary cortisol in studies of child development: watch out--what goes in may not come out of saliva collection devices

Technical advances that enable the noninvasive measurement of biomarkers in saliva have spawned a generation of investigations that integrate biological variables into behavioral and developmental research. This study examines whether the collection of saliva, using common absorbent devices compromises the measurement of cortisol when saliva specimens have low sample volume. Within subjects (n = 20), saliva samples were prepared to experimentally represent a gradient of lower to higher sample volumes. One aliquot was immediately frozen (no treatment control) and the remaining aliquots were absorbed ("collected") using one of three collection techniques employed in studies of child development (e.g., braided cotton dental rope, Salivette cotton pledget, or hydrocellulose microsponge). The sample volume recovered from each device relative to the initial volume available to be absorbed, and cortisol level recovered from each device relative to the untreated-control condition were measured. Results reveal that for certain collection devices (1) the percent volume recovered is related to the initial volume available to be absorbed, (2) a substantial percentage of cortisol in saliva specimens can remain in absorbent material, and (3) the percent of cortisol recovered can be associated with the initial sample volume available to be absorbed. When research participants, such as young children, produce low volume saliva specimens, some absorbent devices may have the potential to introduce error variance in the measurement of salivary cortisol.     

Children's noncompliance during saliva collection predicts measures of salivary cortisol

Salivary cortisol has been useful for evaluating children's physiological responses to stress and for identifying factors that predict their magnitude and duration. However, results have been somewhat equivocal across studies, and this has motivated researchers to identify sources of variance and error. Here, we examined the prevalence of preschoolers' noncompliance during saliva collection and aimed to learn about noncompliant children in terms of their hypothalamus-pituitary-adrenal function, behavior in other situations, and symptoms of behavioral problems. Results were based on measures of cortisol, children's behavior during saliva collection and a mother-child teaching interaction, and ratings of problem behavior by teachers and parents. Results show that 12% (21/174) of the sample was noncompliant on at least one of the collection trials. Children, who were noncompliant but did not outright refuse saliva collection, had higher cortisol than did compliant children. Children who were noncompliant during saliva collection were likely to be noncompliant during the teaching episode, and they were perceived as having more internalizing symptoms than compliant children. These results suggest that children's noncompliance during saliva collection can be a source of nonrandom missing data or extreme cortisol values, which should be considered in future studies.     

Methodological considerations in the use of salivary α‐amylase as a stress marker in field research

Salivary α‐amylase recently has been identified as a stress‐related biomarker for autonomic nervous system activity. This study addresses sample collection and handling considerations for field researchers. Saliva was collected by unstimulated passive drool from 14 adults and pooled. Incubation of pooled saliva at 22 or 37°C for 21 days did not diminish amylase activity. However, sodium azide added at concentrations ≤1.12 mg/ml to pooled saliva artificially inflated activity. After dosing cotton rolls within Salivette saliva collection devices with 0.25 to 1.5 ml of unpooled passive drool saliva from six additional adults, recovery of amylase activity was significantly below 100% at all volumes, with increased variance in recovery when the cotton was incompletely saturated (≤1.0 ml). Hence, collection by passive drool instead of cotton‐containing devices for amylase determinations is recommended, particularly whenever it is impossible to ensure full, uniform cotton saturation, and azide should be avoided as a preservative. Am. J. Hum. Biol., 2008. © 2008 Wiley‐Liss, Inc.     

ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998. ESHRE PGD Consortium Steering Committee

The first clinical application of preimplantation genetic diagnosis (PGD) was reported almost a decade ago. Since then, the range of genetic defects that can be detected at single cell level has increased dramatically. At the 13th Annual Meeting of ESHRE in Edinburgh in 1997, a PGD Consortium was formed to undertake the first systematic and long-term study of the efficacy and clinical outcome of PGD. We report here the first data collection covering the period of January 1997 to September 1998. Referral data on 323 couples have been collected for a variety of monogenic and chromosomal disorders, providing information about which patients, at risk for which genetic diseases, are interested in PGD. Data were collected on 392 PGD cycles, resulting in 302 embryo transfers and 66 clinical pregnancies. Because of the importance of follow-up of the children born after PGD, participating centres were asked to contribute data on the pregnancies achieved and the children born after PGD since the start of their PGD programme. Data on 82 pregnancies and 110 fetal sacs were collected, and information was available on 79 children. Finally, biopsy, fluorescence in-situ hybridization and polymerase chain reaction protocols were collected, clearly showing that no consensus exists on technical aspects such as which culture medium to use, and emphasizing the role the PGD Consortium could play in setting up guidelines for good laboratory practice. In conclusion, it is clear that the effort of gathering data on PGD cycles is worthwhile and will be continued in the future, preferably using electronic data collection.     

Brief report: Assessment of children's gastrointestinal symptoms for clinical trials

OBJECTIVE: To conduct a pilot study evaluating a procedure for assessment of daily symptoms and functioning in pediatric patients. METHOD: Participants included 11 parent-child dyads referred to a tertiary care center for evaluation of constipation and abdominal pain. Each family was provided a hand-held computer and modem. For 7 consecutive days, parents and children (ages 6-10 years) responded as a team to questions regarding the level of children's gastrointestinal symptoms and the extent to which symptoms interfered with the day's activities. Parents responded to a telephone interview evaluating the procedure. RESULTS: Parents reported that children understood most questions and that responses entered into the computer were accurate. Parents and children were enthusiastic about the data collection method. Some technical problems arose in use of the computers. CONCLUSIONS: Within the limitations of a small sample, this data collection procedure appears to have promise for evaluating pediatric symptom outcomes.     

Use of agricultural insecticides in Benin

The use of insecticides in households and in agriculture has been incriminated in the emergence of insecticide resistance in insect vectors. For farming staff, the emergence of vector resistance is due to indoors spray of insecticides using aerosols and other low quality products in rural and urban settings against mosquitoes. On the other hand, public health specialists believe that the phenomenon of resistance could be due to massive use of insecticides in agriculture for field pests control. In Turkey, the implication of agricultural use of pesticides in the selection of vector resistance is clearly established. This study was framed to identify potential practices favouring the emergence of insecticide resistance in the Republic of Benin. Interviews and focus group discussions were organized with cotton, rice and vegetables farmers. The final aim of these surveys was to point out practices likely to favour the emergence of resistance. The research is conducted in 3 cotton fields, 2 rice fields and 2 vegetable plantations. After filling and signing concerned forms, farmers are subjected to quantitative and qualitative questionnaires to generate data on: insecticides being used, the various doses applied for pests eradication, the frequency of treatments, the cost of treatments (cost/hectare/year) the origin of insecticides, the place of purchase, safety precautions and related health hazards. The results of this study have shown that the use of insecticides in agriculture is a clear fact. During treatments, insecticide residues get in contact with mosquito breeding sites where they diffuse into water and exercise a selection pressure on larvae. This partially explains the high levels of resistance recorded in with strains of Anopheles gambiae collected in agricultural settings under insecticides pressure. Pyrethroids and more specifically deltamethrin and cyfluthrin are the insecticides mainly used in studied localities. Bedrooms of farmers are used as storage place for half-used and un-used insecticides containers. For a proper management of insecticides, cotton and vegetable plantations farmers receive assistance from the ministry of rural development. Because of the importance of cotton production in the Republic of Benin, trainings on management of insecticides in agricultural settings are frequently organised by the ministry of rural development and are opened to farmers and their family members (husbands, wives, children and relatives...). In the long run, the whole family learns and becomes very versant with the use of insecticides, spraying frequencies, spraying devices and spraying techniques.     

Preliminary studies for the development of a second generation granulocytapheresis (G-CAP) column. Part II: in vitro and ex vivo studies

Our previous studies concluded Egyptian cotton was the most appropriate material for making a second generation granulocytapheresis (G-CAP) column as structural dimensions of the cotton fibers are able to attract granulocytes. Unfortunately, it is considered to be blood incompatible as its fibers are of non-synthetic origins. In this study we examined the alteration of the removal rates of blood cells with different surface modifications of Egyptian cotton to enhance its blood compatibility. The surface-modified cotton fibers were compared after three kinds of combination treatments. There were no differences in the removal rates of white blood cells (WBCs) and particularly neutrophils with the use of three kinds of biolized cottons. Next, an ex vivo animal study with a healthy dog was performed with the prototype of the G-CAP column. The dog’s blood pressure (BP) decreased to approximately 80% of the initial values of BP at 20 min after the start of the extracorporeal circulation. The decrease in BP gradually reverted to normal. WBCs and particularly neutrophils decreased significantly at 15 min after the start of the extracorporeal circulation and remained low during the extracorporeal circulation. The ability of this column to remove WBCs was maintained during extracorporeal circulation. Especially, neutrophils at the inlet of the column were thoroughly removed for 1 h. Based upon these results, a second generation G-CAP column could be fabricated with Egyptian cotton and applied for clinical use on the condition that the biocompatibility of the Egyptian cotton needs to be improved by the appropriate biolization method.     

Respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats

Palivizumab (PZ) is the only monoclonal antibody in human use against an infectious disease. PZ is a humanized monoclonal antibody that recognizes the fusion protein of respiratory syncytial virus (RSV). PZ prophylaxis reduces the likelihood of hospitalization for young children at risk for severe RSV infections. The quasispecies nature of RNA viruses allows rapid emergence of viruses with a selective advantage. A PZ resistant virus was selected by passage of RSV in the presence of PZ in cell culture. The cell culture-derived virus was completely resistant to PZ prophylaxis in cotton rats. The increasing use of PZ, and in particular, the use of PZ in immunosuppressed patients, provide opportunities for resistant viruses to emerge. Whether such viruses will appear and be of clinical significance for humans is unknown. Preclinical studies in cotton rats predicted the efficacy of PZ in humans; these results suggest that if PZ resistant viruses arise in humans, PZ prophylaxis may be ineffective.     

Evaluation and modification of off-host flea collection techniques used in northwest Uganda: laboratory and field studies

Quantifying the abundance of host-seeking fleas is critical for assessing risk of human exposure to flea-borne disease agents, including Yersinia pestis, the etiological agent of plague. Yet, reliable measures of the efficacy of existing host-seeking flea collection methods are lacking. In this study, we compare the efficacy of passive and active methods for the collection of host-seeking fleas in both the laboratory and human habitations in a plague-endemic region of northwest Uganda. In the laboratory, lighted "Kilonzo" flea traps modified with either blinking lights, the creation of shadows or the generation of carbon dioxide were less efficient at collecting Xenopsylla cheopis Rothchild and Ctenocephalides felis Bouché fleas than an active collection method using white cotton socks or cotton flannel. Passive collection using Kilonzo light traps in the laboratory collected significantly more X. cheopis than C. felis and active collection, using white socks and flannel, collected significantly more C. felis than X. cheopis. In field studies conducted in Uganda, Kilonzo traps using a flashlight were similar in their collection efficacy to Kilonzo traps using kerosene lamps. However, in contrast to laboratory studies, Kilonzo flea traps using flashlights collected a greater number of fleas than swabbing. Within human habitations in Uganda, Kilonzo traps were especially useful for collecting C. felis, the dominant species found in human habitations in this area.     

Experimental studies on detoxication of mercury salts by means of ethylenediaminetetra-acetic acid

Summary It was experimentally proved that mercury may be fixated with the aid of ethylendiaminetetraacetic acid. The latter, likewise, promotes excretion of mercury from the body. It was established in experiments on isolated frog's heart that the ratio of the quantity of sublimate and ethylendiaminetetraacetic acid should be 1 : 120 for total fixation of mercury. However, it was impossible to fixate mercury in the intact organism, even in presence of this ratio.Presented by Prof. S. V. Anichkov, Member AMS USSR     

Guidelines for buccal smear collection in breast-fed infants.

Buccal smear analysis is a noninvasive, fast, and relatively inexpensive diagnostic method. It is used commonly where rapid gender identification is necessary or, more recently, for detection of aneusomy, microdeletion syndromes, and a variety of polymerase chain reaction-based molecular genetic tests. Previously we have shown that maternal cells can contaminate buccal smears taken from breast-fed infants, resulting in difficulty with test interpretation. The aim of this study was to determine optimal timing and technique for buccal smear collection in breast-fed infants in order to avoid diagnostic errors. We analyzed prospectively 50 breast-fed male infants for presence of cells with XX signal pattern from buccal mucosa scrapings at different times after breast feeding. The efficiency of mucosal cleaning on elimination of maternal cells was evaluated by comparing the proportion of XX cells before and after wiping of buccal mucosa with a cotton swab. Maternal cells were present in 23 of 48 (47.9%) samples collected within 5 min of feeding. The proportion of XX signal pattern was significantly (P = 0.001) reduced in samples collected at 30 min (8/48, P = 0.001) and > or =60 min (2/29, P = 0.0002) after feeding. Mucosal cleaning prior to smear collection significantly decreased the number of XX positive samples from 23 of 48 to 10 of 48 (P = 0.002). Buccal smears should not be obtained in nursing neonates until at least 60 min after breast feeding. In addition, prior to sample collection, buccal mucosa should be cleaned thoroughly with a cotton swab applicator. The same guidelines are applicable to older nursing infants.     

Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants

In community-based studies of stress and immunity, saliva samples offer a non-intrusive way of gathering biological data. Cotton-based devices are widely used in cortisol research, but some may affect assay results. We compared assay reliability and perceived acceptability of three saliva collection methods: passive, cotton 'salivettes' and cellulose-cotton tip 'eyespears'. Compared to passive collection, salivettes reduced the concentration of cortisol (p = .001) and sIgA (p = .002). Eyespears did not reduce cortisol or sIgA concentration, and showed less interference in the rank ordering of cortisol (r(eyespear with passive) = .90) and sIgA scores (r(eyespear with passive) = .96) compared to salivettes (r cortisol(salivette with passive) = .79; r sIgA(salivette with passive) = .66). The comfort and acceptability of both cotton-based devices were rated positively. Cotton-cellulose eyespears could offer methodological advantages for collecting saliva to measure cortisol and sIgA levels, and, because they can be held during sampling, may be useful for research with children and the frail elderly.     

ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001)

The ESHRE PGD Consortium was formed in 1997 to undertake a long-termstudy of the efficacy and clinical outcome of preimplantationgenetic diagnosis (PGD). Here, the third report of the ESHREPGD Consortium is presented, collating data received from 25centres on referrals, cycles, pregnancies and babies born afterPGD. The second report, published in December 2000, reportedon 886 referrals, a total of 1318 started cycles (of which 465for aneuploidy screening, 386 for fluorescence in-situ hybridization(FISH) and 385 for PCR going beyond oocyte retrieval), 163 pregnanciesand 162 children born. This year, 675 referrals from 12 centreswere added giving a total of 1561 referrals, 370 regular PGDcycles, 334 PGD-aneuploidy screening (PGD-AS) cycles and 78cycles for social sexing from 24 centres and 215 pregnanciesand 117 babies from 12 centres. Because more in-depth informationwas asked for the cycles, this year's data will be shown separatelyas well as cumulatively. One striking feature of this year'sdata collection is the appearance of the first data for genderscreening on preimplantation embryos for social reasons. Theethical concerns regarding social sexing will be discussed,as well as the forthcoming changes in timing of data collection.When the data collection was discussed at the last meeting ofthe members of the ESHRE PGD Consortium in Lausanne, Switzerland(June, 2001), it appeared that the current system of data collection,although yielding results very quickly, showed fundamental flaws.The ESHRE PGD Consortium Steering Committee intends to remedyto these problems, on the one hand by introducing a new typeand timing of data collection, and on the other hand by re-analysingand correcting the data which have already been sent in duringthe past 4 years.     

Research hurdles complicating the analysis of infertility treatment and child health

Research aimed at the empirical evaluation of infertility treatment including assisted reproductive technologies (ART) on child health and development is hampered by investigators' inability to methodologically separate possible treatment effects from underlying fecundity impairments. While the literature continues to identify ART as a risk factor for many child health outcomes, less attention has been paid to the methodologic rigor needed to answer this question. We identify aspects of fecundity and the nuances of medical practice that need to be considered and captured when designing epidemiologic investigations aimed at assessing ART and child health. These include: (i) the use of prospective study designs in which the unit of analysis (cycle versus individual versus couple) is defined; (ii) data collection on relevant time-varying covariates at, before and during treatment; and (iii) the use of statistical techniques appropriate for hierarchical data and correlated exposures. While none of these issues in and by itself is unique to ART research, attention to these issues has been lacking in much of the published research limiting our ability to evaluate health consequences for children. Longitudinal studies of children conceived with ART will benefit from attention to these issues and, hopefully, produce answers to lingering questions about safety.     

The fine morphology of the basal cell in the frog's taste organ

We investigated the fine morphology of basal cell in the frog's taste organ by means of transmission electron microscopy. Results show that basal cells are placed at the base of the disc and are highly polarized; the cell body is peripherally located and a long cell process reaches the central region of the taste disc without branching. The cell body contains the nucleus, the Golgi apparatus and large lysosomes; junctions between more peripherally located 'marginal' cells prevent passage of macromolecules from oral ambient to basal cells as shown by horse-radish peroxidase experiments. The cell process, running just over the basement membrane in the taste disc epithelium, is rich in microtubules, filaments and clustered secretory granules arranged near the plasmalemma. Nerves interrupting the basement membrane make synaptic-like junctions with basal cells. The cell process ends in the central region of the taste disc; here, the basal cell is expanded to contain filaments, secretory granules and mitochondria in characteristic arrangement and contacts intraepithelial nerve endings as well as basal processes of sensory and supporting cells. Marginated granules are found where basal cell contacts nerve ending and also where nerve and sensory cells contact basal cell. Our findings are consistent with the hypothesis that basal cells are under nerve control and that they may have a diffuse (paracrine) influence on neighbouring cells in the frog's taste organ.     

Influence of cotton dust inhalation on free lung cells in rats and guinea pigs.

The influence of cotton dust inhalation on pulmonary leukocyte recruitment has been examined in rats and guinea pigs in an attempt to develop an animal model which can be used to assess the relative pulmonary toxicity of dusts from various varieties of cotton. Statistically significant increases in leukocyte populations in the lungs occur as the result of dust inhalation. Although the response is not influenced by the sex or age of the test animal, species differences do occur; guinea pigs are more sensitive to the effects of exposure than rats. Also, the exposure regimen tends to influence the degree of the response, with intermittent exposure producing fewer cells than continuous exposure for the same period of time. Future investigations will use the information gathered here to employ the most appropriate animal species as a tool to screen varieties of cotton, and thereby to rank them according to their potential to aggravate or to initiate certain pulmonary pathologies.     

Stretch receptors stimulation in frog's lungs

Summary The response of the stretch receptors of the frog's lung to inflation together with pulmonary volume and pressure have been quantitatively evaluated by counting the action potentials led off from the vagus nerve in isolated lung-vagus preparations. It is seen that sustained and repeated inflations deeply change the mechanical properties of the lung and the receptors response to inflation. The response of the receptors, however, appears to be constantly and linearly related with the tension of the pulmonary wall considered as a stretched elastic lamina. It is concluded that tension of the pulmonary wall is the natural stimulus for the receptors of the frog's lung. Adaptation of the receptors to the stimulus appeared to be negligible over the inflation period.This study was supported by grants from the Consiglio Nazionale delle Ricerche (Impresa Elettrofisiologia), Roma (Italy).