Prevalence of naturally occurring viral infections, Mycoplasma pulmonis and Clostridium piliforme in laboratory rodents in Western Europe screened from 2000 to 2003.

In this report prevalence rates of rodent viruses in laboratory animals are presented based on routine serological screening of mouse and rat colonies from European institutes. The prevalences found during the period 2000-2003 are compared with those reported for 1981-1984 and 1990-1993. It is shown that some infections were eliminated from laboratory animal colonies (e.g. K-virus and polyomavirus) by taking preventative measures whereas other infections such as mouse hepatitis virus and parvoviruses remained at a high rate. Further decreases in prevalence rates in the last 10 years were found for infections such as pneumonia virus of mice, reovirus type 3, Sendai virus, sialodacryoadenitis/rat coronavirus and Mycoplasma pulmonis. The introduction of new detection methods showed that mouse parvovirus and rat parvovirus, both members of the Parvoviridae family, remain a major threat to laboratory mice and rats. Guinea pig cytomegalovirus and para-influenza virus appeared to be the most prevalent agents among laboratory guinea pigs. The importance of a standardized, up-to-date screening programme is discussed.

Prenatal diagnosis and treatment of congenital Toxoplasma gondii infections: an experimental study in rhesus monkeys.

The efficacy of treatment in fetuses in whom congenital Toxoplasma gondii infection has ben established has been investigated using rhesus monkeys as a model for humans. A polymerase chain reaction has been developed for the detection of Toxoplasma gondii. Using this polymerase chain reaction congenital infection can be established within 2 days of receiving an amniotic fluid sample. The polymerase chain reaction has subsequently been used to monitor the effect of treatment on fetal infection. The results show that early treatment with the combination of pyrimethamine and sulfadiazine was clearly effective in reducing the number of parasites in the infected fetus. The parasite was no longer detectable in the amniotic fluid 10 to 13 days after treatment was started. Spiramycin, on the other hand, has to be administered for at least 3 weeks to achieve the same effect. Moreover, pharmacokinetic studies revealed that spiramycin does not reach the brain. Pyrimethamine and sulfadiazine are able to pass the blood-brain barrier. Pyrimethamine appears to accumulate in the brain tissue and reaches concentrations which are also effective in vitro.

Anti-toxoplasma effect of pyrimethamine, trimethoprim and sulphonamides alone and in combination: implications for therapy.

The aim of the present study was to determine the in-vitro susceptibility of Toxoplasma gondii to dihydrofolate reductase inhibitors and sulphonamides alone, and in combination. It was found that pyrimethamine had the most potent anti-Toxoplasma activity, while sulphadiazine, sulphamethoxazole and sulphametrole were approximately equally effective, but only at a high concentration. The 50% inhibitory concentration of sulphamethoxazole was determined using different fixed concentrations of pyrimethamine or trimethoprim. It was found that a ten-fold increment of the concentration of pyrimethamine, reduced the IC50 of sulphamethoxazole 1000 times. The influence of trimethoprim on the IC50 of sulphamethoxazole was less concentration dependent. Addition of dihydrofolate or tetrahydrofolate did not influence the IC50 of pyrimethamine. In conclusion, the present dose recommendations for sulphonamides, when combined with pyrimethamine, may be unnecessarily high. There may be a justification for dose reduction of sulphonamides in order to prevent side effects.

Hepatitis C virus: biological and clinical consequences of genetic heterogeneity.

Hepatitis C Virus infection accounts for the majority of post-transfusion and sporadic hepatitis. In Western Europe, anti-HCV is detected in 0.4-1.5% of healthy blood donors. There is a high frequency of progressive chronic hepatitis, ranging from 50 to 80%, which leads to cirrhosis in 20-50% of patients after 10-20 years. Viremic patients with minimal biochemical abnormalities may have chronic liver disease histologically. There is growing evidence that virological features of HCV are associated with different clinical manifestations and response to therapy. The RNA genome consists of a 5' and 3' Untranslated Region, a structural domain encoding the core and envelope proteins, and a non-structural domain. Different HCV isolates show a high sequence heterogeneity, which has led to the classification of currently six genotypes and several subtypes. There is a marked difference in the geographic distribution of HCV genotypes, with types 1, 2 and 3a being most frequently found in western countries. In The Netherlands, subtype 1b accounts for approximately 60% of all cases of chronic HCV. Serologic diagnosis based on recombinant C-100 antigens (first generation immunoassays) only reliably detected type 1, due to the heterogeneity of the NS4 region; inclusion of more conserved proteins c22 and c33 (second generation assays) has largely improved sensitivity of anti-HCV testing. Genotype 1b is associated with more severe liver disease and with lower response rates for antiviral therapy, compared with types 2 and 3. Quasispecies nature and escape mutants may enable viral persistence and the development of chronic liver disease. As cross-reactivity between genotypes is unlikely, prevention of HCV disease may be dependent on the development of multivalent vaccines.

Isolation, identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and rhesus monkey by high-performance liquid chromatography.

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by beta-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N4-acetylsulfadiazine (N4), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 microgram/ml (S 0.31, N4 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 microgram/ml). In urine it varies between 0.6 and 1.1 micrograms/ml (S 0.75, N4 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 micrograms/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.

In vitro effects of sulfadiazine and its metabolites alone and in combination with pyrimethamine on Toxoplasma gondii.

Sulfadiazine and the metabolites N4-acetyl-sulfadiazine, 4-OH-sulfadiazine, 5-OH-sulfadiazine, 5-OH-glucuronide-sulfadiazine, and 5-OH-sulfate-sulfadiazine were tested separately and in combination with pyrimethamine for the inhibitory activity on Toxoplasma gondii growth in vitro. Except for N4-acetyl-sulfadiazine, all sulfa compounds possessed anti-Toxoplasma activity. The addition of 0.05 micrograms of pyrimethamine per ml, a concentration which in itself is not inhibitory, potentiated the microbial activity of sulfadiazine and its metabolites 100 fold.

Study of treatment of congenital Toxoplasma gondii infection in rhesus monkeys with pyrimethamine and sulfadiazine.

The efficacy of the combination of pyrimethamine and sulfadiazine for the treatment of congenital Toxoplasma gondii infection in rhesus monkeys was studied. The dosage regimen for pyrimethamine and sulfadiazine was established by pharmacokinetic studies in two monkeys. Those studies showed that the distributions of both drugs followed a one-compartment model. The serum elimination half-lives were found to be 5.2 h for sulfadiazine and 44.4 h for pyrimethamine. Sulfadiazine reached a maximum concentration in serum of 58.7 micrograms/ml, whereas a maximum concentration in serum of 0.22 micrograms/ml was found for pyrimethamine. Ten monkeys were infected intravenously with T. gondii at day 90 of pregnancy, which is comparable to the second trimester of organogenetic development in humans. Treatment was administered to six monkeys, in whose fetuses infection was diagnosed antenatally. From the moment that fetal infection was proven, the monkeys were treated throughout pregnancy with 1 mg of pyrimethamine per kg of body weight per day and 50 mg of sulfadiazine per kg of body weight per day orally. The therapy was supplemented with 3.5 mg of folinic acid once a week. No toxic side effects were found with this drug regimen. The parasite was no longer detectable in the next consecutive amniotic fluid sample, taken 10 to 13 days after treatment was started. Furthermore, T. gondii was also not found in the neonate at birth. The parasite was still present at birth in three of four untreated fetuses that served as controls. Both drugs crossed the placenta very well. Concentrations in fetal serum varied from 0.05 to 0.14 micrograms/ml for pyrimethamine and from 1.0 to 5.4 micrograms/ml for sulfadiazine. In addition, pyrimethamine was found to accumulate in the brain tissue, with concentrations being three to four times higher than the corresponding concentrations in serum. Thirty percent of the sulfadiazine was found to reach the brain tissue when compared with the corresponding serum concentration. when administered early after the onset of infection, the combination of pyrimethamine and sulfadiazine was clearly effective in reducing the number of parasites in the fetus to undetectable levels.

Pharmacokinetics of spiramycin in the rhesus monkey: transplacental passage and distribution in tissue in the fetus.

Transplacental transfer of spiramycin was investigated in a rhesus monkey model to study whether the antibiotic reaches therapeutic levels in the fetus. Spiramycin concentrations were measured by bioassay and high-performance liquid chromatography. Pharmacokinetic parameters were determined for bioactive spiramycin as measured by the bioassay. Pharmacokinetic pilot studies showed that spiramycin distribution follows a two-compartment model in rhesus monkeys. Following a single intravenous dose of 50 or 250 mg, dose-dependent kinetics were observed. At a dose of 50 mg, 10% of the dose was excreted unchanged in the urine. At the higher dose of 250 mg, an oliguric effect was observed. Spiramycin concentrations in fetal serum were measured over time while the maternal concentration was maintained at a constant level. During a 5-h experiment, a maximum fetal-maternal serum ratio of 0.27 was found. In three fetuses, concentrations in serum and tissue were measured following intravenous administration of 50 mg of spiramycin twice daily to the mother for at least 7 days. The fetal-maternal serum ratios were found to be 0.4 to 0.58 after intravenous administration of the final dose of 50 mg to the mother. It appeared that spiramycin accumulated in the soft tissues, especially in the liver and spleen, of both the mother and the fetus. The concentration in placental tissue appeared to be 10 to 20 times that of the concentration in fetal serum. The concentration of spiramycin in amniotic fluid was about five times higher than the concentration in fetal serum. Another important observation was that absolutely no spiramycin was found in the brain.

Effectiveness of spiramycin for treatment of congenital Toxoplasma gondii infection in rhesus monkeys.

The effectiveness of spiramycin for the treatment of rhesus monkey fetuses congenitally infected with Toxoplasma gondii was studied. Eight monkeys were infected at day 90 of pregnancy. This is comparable to the second trimester of organogenetic development in humans. Transmission of infection was found prenatally in five of the eight monkeys by detection of the parasite in the amniotic fluid. Treatment with spiramycin (20 mg/kg/day in two intermittent doses given intravenously) was started as soon as fetal infection was proven and was continued until birth. Nine to 14 days after initiation of treatment, the parasite was still detectable in amniotic fluid samples from four of these five cases. However, the parasite was detected only by PCR and not by mouse inoculation. T. gondii was also detected only by PCR in the placenta of one monkey that delivered prematurely. This monkey received spiramycin treatment for only 2 weeks. In the four monkeys that received treatment for about 7 weeks, the parasite was not present at birth in the placenta nor in amniotic fluid or neonatal organs. Spiramycin accumulates mainly in maternal tissues. Although concentrations in neonatal tissue were found to be 5 to 28 times higher than the corresponding concentrations in neonatal serum, the concentrations in neonatal tissue were still 11 to 16 times lower than those found in the mothers. However, no spiramycin was found in the fetal brains. Early treatment with spiramycin may prevent transmission of infection to the fetus but most probably cannot interrupt an existing brain infection, which is the most severe outcome of congenital toxoplasmosis in humans.

Congenital toxoplasmosis: an experimental study in rhesus monkeys for transmission and prenatal diagnosis.

This paper describes a rhesus monkey model to study congenital transmission and the prenatal diagnosis of Toxoplasma gondii infection. Transmission to the fetus was investigated after maternal infection in the second (Day 90) or third trimester (Day 130) of pregnancy. A parasitemia was induced and lasted for about 10 days as proven by mouse inoculation and nested PCR on whole blood samples and peripheral blood mononuclear cells. Transmission of T. gondii was proven in 4 of 9 (44%) fetuses in the second trimester and in 3 of 9 fetuses in the third trimester of gestation. Six of the 7 proven fetal infections could be diagnosed antenatally. Postnatally, 4 additional fetuses that most likely were congenitally infected were found, although this could not formally be proven. The mothers of these 4 fetuses were infected in the third trimester. If we accept this group as being antenatally infected, an overall transmission rate is found of 11 of 18 cases (61%). This rate is similar to that found in humans. This is the first description of a monkey model for congenital toxoplasmosis, that is suitable to study the approach of antenatal diagnosis and the effectivity of treatment, with obvious relevance for Toxoplasma infections in humans.

Value of the polymerase chain reaction for the detection of Toxoplasma gondii in cerebrospinal fluid from patients with AIDS.

To investigate whether the polymerase chain reaction (PCR) on the BI gene of Toxoplasma gondii could contribute to the diagnosis of cerebral toxoplasmosis in patients with AIDS, we retrospectively tested CSF samples from 20 patients with AIDS suspected of having cerebral toxoplasmosis for the presence of T. gondii. Suspicion of cerebral toxoplasmosis was based on accepted criteria. Nine patients with AIDS with IgG antibodies to T. gondii but who were not suspected of having cerebral toxoplasmosis and four patients with AIDS seronegative for T. gondii served as negative control patients. T. gondii was demonstrated by PCR in the CSF from 13 of the 20 patients with AIDS suspected of having cerebral toxoplasmosis but was not demonstrated in the CSF samples from the nine control patients seropositive for T. gondii and the four control patients seronegative for T. gondii. The data were statistically evaluated. This study shows the value of PCR for the detection of T. gondii in CSF for the diagnosis of cerebral toxoplasmosis in patients with AIDS.