On-site testing of saliva and sweat with Drugwipe and determination of concentrations of drugs of abuse in saliva, plasma and urine of suspected users.

Potential drug users participated voluntarily in a Belgian study on the usefulness of the non-instrumental immunoassay Drugwipe (Securetec, Germany) for the screening of cocaine, opiates, amphetamine and cannabinoids in saliva and sweat. If one of the screening assays (urine, oral fluid, sweat) showed a positive result, blood and saliva were collected. The on-site Drugwipe results were correlated with the Drugwipe results for saliva in the laboratory and with the GC/MS results of the corresponding saliva, plasma and urine samples and pharmacological effects at the time of sampling. The Drugwipe assay proved to be sufficiently sensitive for the detection of recent cocaine (n = 6) and amphetamine (n = 15) abuse, whether the device was wiped on the tongue or on the surface of the body, or when a saliva sample was applied to the wiping part. In five of the six potential cocaine users, the saliva concentrations of cocaine exceeded 1,000 ng/ml. In the amphetamine group, the saliva concentrations of amphetamine, MDMA or both were high (> 1,000 ng/ml) in 13 subjects. For cocaine and amphetamine, the positive scores for Drugwipe matched the GC/MS results for the three body fluids. Recent heroin abuse (n = 5) could be demonstrated to some extent with Drugwipe on samples from the tongue but only the two subjects with the highest saliva concentrations of MAM (> 500 ng/ml) and morphine (> 500 ng/ml) were positive. If the legal cut-off value for driving under the influence of opiates in Belgium (20 ng/ml of free morphine in plasma) was taken into account, only three subjects would have been legally positive. For cannabinoids (n = 15), false negatives and even some false positives were observed. Saliva can be considered as a useful analytical matrix for the detection of drugs of abuse after recent abuse when analysed with GC/MS.

Analysis of Drugs in Saliva.

Saliva is presented as an alternative matrix in the establishment of drug abuse. The ultimate salivary concentration is determined by the route of administration, the salivary pH, the degree of plasma protein binding, and the physico-chemical properties of the abused drug. Since the saliva/plasma ratio can exceed 1, saliva might be a better analytical tool than blood during roadside testing of potentially intoxicated drivers. Moreover, saliva can be obtained non-invasively and under supervision. Although drugs of abuse have been determined in saliva for more than a decade, the use of saliva drug testing for forensic purposes is still limited. Several problems have been demonstrated: (a) differences in the collection protocol produce variable results and often, e.g., during roadside testing, only very small volumes of saliva are obtained; (b) the salivary concentrations are much lower than in urine; (c) saliva principally contains the parent drug and until now, no suitable immunoassays have been commercialized. Although salivary drug concentrations are well correlated with pharmacological effects for some drugs, e.g., cocaine, further studies have to prove whether saliva is a suitable matrix to demonstrate "driving under the influence" of psychoactive drugs. Furthermore, an on-site screening assay for drugs of abuse in saliva and the establishment of appropriate cutoff levels should facilitate the use of saliva during roadside testing.

Hymenolepis murissylvatici: humoral response in intestinal lavages of the mouse.

Hymenolepis murissylvatici elicits a humoral response in serum and in the intestine of the mouse from which it is immunologically rejected. In serum, significant differences were recorded 3 days after reinfection, while in intestinal lavages it takes place from day 9 after reinfection. In serum the response is largely the result of IgG and IgM antibodies whereas in the intestine, IgA is the most abundant antibody. Although specific IgE could not be demonstrated in serum, it was present in intestinal lavages, although rather late (i.e. day 14 after reinfection). Treatment of young worms in vitro both with immune serum or intestinal lavages had no apparent effect on their viability. Immune serum produced a complement independent precipitation on the surface of the worms in vitro. This reaction did not affect viability or infectivity of the parasite, as demonstrated by the successful implantation of treated worms in recipient mice. The above-mentioned results, together with the knowledge that the Hymenolepis model has no tissue stages and causes no tissue damage, make it probable that further study of this model will reveal some specific intestinal immunological reactions.

Hymenolepis muris-sylvaticae and H. microstoma: immunological cross-reaction in mice.

A primary infection with Hymenolepis microstoma strongly protects against cross-infection with H. muris-sylvaticae and also against secondary infection with H. microstoma in NMRI mice, resulting in an accelerated loss of worms and a weight reduction of the remaining worms. A primary infection with H. muris-sylvaticae causes an accelerated rejection of secondary infection with H. muris-sylvaticae but it has no effect on cross-infection with H. microstoma, neither with regard to worm recovery nor with regard to worm biomass. Determinations by enzyme-linked immunosorbent assay of antibody concentrations in the mouse sera revealed that: (1) the antibody response evoked by H. microstoma infection is much greater than by H. muris-sylvaticae infection; (2) a cross-infection with H. muris-sylvaticae boosts the antibody response evoked by H. microstoma infection; (3) H. microstoma antigen can be used to measure antibody concentration against both H. microstoma and H. muris-sylvaticae; and (4) although H. muris-sylvaticae is rejected faster in a cross-infection (i.e., after a primary H. microstoma infection) than in a secondary infection (i.e., after a primary H. muris-sylvaticae infection), antibodies evoked by the primary H. microstoma infection show little cross-reaction with H. muris-sylvaticae antigen. This suggests that it is doubtful whether serum antibodies are the direct effectors in worm rejection.

Immunological aspects in the rejection process of Hymenolepis muris-sylvaticae from CFLP and NMRI mice.

When mice were treated with 1.25 mg cortisone acetate thrice weekly, recovery of Hymenolepis muris-sylvaticae was significantly higher than in untreated controls, both in oral infections with six cysticercoids and surgical transplantations of one 7-day or 8-day-old worm. Cortisone treatment also resulted in the worms being located more anteriorly in the small intestine. Evidence of an immunological response against the tapeworm in the intestine is given by: an accelerated rejection of a secondary oral cysticercoid infection and a significant difference of the dry weights of the worms recovered on day 10 in CFLP mice; an accelerated rejection of a secondary surgical infection on days 4 and 6 in CFLP mice and on days 3 and 4 in NMRI mice; an accelerated rejection of a secondary surgical infection given 3 and 6 months after the primary immunizing infection in SWISS-albino mice.

Hymenolepis muris-sylvaticae in laboratory rodents.

The establishment, growth, survival and distribution of Hymenolepis muris-sylvaticae in different laboratory animals (Wistar rats, golden hamsters, CFLP and NMRI mice) after oral infection and surgical transplantation were determined. Worms that establish in CFLP and NMRI mice grow exponentially but are rejected at different times p.i. depending on the strain of mice and mode of infection. Only in golden hamsters will H. muris-sylvaticae develop to a mature worm; in rats there is no growth at all. Worms of all sizes are found in the posterior half of the small intestine in rats and both strains of mice; in hamsters the worms are recovered more anteriorly.