Treatment failure in Trichomonas vaginalis infections in females. II. In-vitro estimation of the sensitivity of the organism to metronidazole.

We have developed a standard procedure to measure the susceptibility of Trichomonas vaginalis to metronidazole in vitro, by controlling the state of oxygenation of the test cultures, the size of the inocula in terms of infectivity, and providing multiple tests at each drug concentration. Estimates were made of the in-vitro sensitivity to metronidazole of T. vaginalis isolated from 11 randomly selected patients, all, except for three defaulters, known to have been treated successfully, and from six 'treatment failure' patients. Stocks isolated from 'successfully treated' patients, were all highly sensitive to metronidazole. Eighteen stocks isolated from the 'treatment failure' patients fell into two groups: (a) those clearly separable from sensitive stocks, with ED50 values (the concentration of drug at which growth is prevented in 50% of test wells) in N2 of up to 0.51 mg/l and 3.5 to 11 mg/l in N2/1%O2; (b) those giving intermediate ED50 values of up to 0.20 mg/l in N2 and 0.7 to 1.8 mg/l in N2/1%O2. The current procedure demonstrates clearly that repeated treatment failure in patients without other complication is associated with enhanced resistance to metronidazole of T. vaginalis, and it may discriminate between T. vaginalis infections that are likely to respond to higher dosage and those that are not.

Treatment failure in Trichomonas vaginalis infections in females. I. Concentrations of metronidazole in plasma and vaginal content during normal and high dosage.

Concurrent estimation by high pressure liquid chromatography (HPLC) of the concentrations of metronidazole in plasma and vaginal content in 12 patients with infections due to Trichomonas vaginalis who did not respond to normal and high-dose treatment has shown that the two levels are closely related to each other and to the dose. Individual idiosyncrasies in absorption of drug from the gut lumen or in its transfer into the vaginal content are unlikely to be the cause of treatment failure.

Miniature anion-exchange/centrifugation technique for the diagnosis of microfilaraemia in the field.

The miniature anion-exchange/centrifugation (AEC) technique, developed originally for the detection of low parasitaemias in laboratory rodents, was adapted to field use for the diagnosis of trypanosomiasis in man in Africa and was tested in The Gambia. During this field study it was found that microfilariae of Dipetalonema perstans could also pass through the anion exchange column and appear in the centrifugate as 'medusa heads'. One locality-group (Mansafa Bolon) showed a generally higher prevalence and prevalences in women over 40 years old were higher than in the corresponding male groups in every locality. The potential usefulness of this technique in epidemiological studies of filariasis and ways of improving the accuracy of numerical estimates are briefly discussed.

Trypanosoma brucei: Miniature anion-exchange centrifugation technique for detection of low parasitaemias: Adaptation for field use.

The miniature anion-exchange/centrifugation (AEC) method, originally developed for the detection of submicroscopic trypanosomaemias in laboratory rodents, has been adapted for the diagnosis of trypanosomiasis in man in the field using blood samples obtained by finger-prick. It has been tested in a survey in The Gambia. The method is shown to be highly sensitive and to fulfil the first essential criteria for exploitation in the field, namely, that it can be operated in the open air under tropical conditions, and that an adequate number of subjects can be examined in a normal working day at an acceptable cost. The method also offers two advantages over the other highly sensitive method applicable to small blood samples, the microhaematocrit buffy-coat microscopy (MBCM) method, namely, that it minimized the requirements for highly critical microscopy and provides, in the same operation, samples of diluted plasma which can be used for serological study.

Absence of detectable local antibody in genitourinary tract secretions of male contacts of women infected with Trichomonas vaginalis.

Samples of semen and urine were obtained from 37 male contacts of women with proved Trichomonas vaginalis infection; on culture, eight (22%) of the men were shown to harbour the parasite. However, significant amounts of antitrichomonal antibody were found in only two of these samples, and the amounts present were very small. A further 10 samples were tested but none was found to contain antibody. The asymptomatic nature and low parasite numbers commonly described in infections in men is thus unlikely to be due to a vigorous local immune response.

Trypanosoma brucei: detection of low parasitaemias in mice by a miniature anion-exchanger/centrifugation technique.

A method is described for the detection of trypanosomaemia in mice using the samples of about 55 microliter of blood which can be readily obtained from mice by puncture of the retro-orbital plexus with heparinized glass microhaematocrit tubes. The samples, mixed with an appropriate diluent, are passed through small (2 ml) anion-exchanger columns by means of peristatic pumps and the eluates collected in tubes which can be centrifuged so as to concentrate any organisms in the terminal part of the tube, of calibre about 200 micrometer, for microscopical examination. Evidence is presented to show that the method is more sensitive than other methods in use. It is also quick, requiring only some five minutes per mouse examined.

Numerical immunotaxonomy of Leishmania. I. Differentiation of four strains of Leishmania by serological tests.

This paper describes the application of numerical methods to the arrangement of four leishmanial strains according to their reactivity and cross-reactivity in tests of parasite agglutination, indirect immunofluorescence and passive cutaneous anaphylaxis with antisera prepared by immunization or infection of rabbits, guinea-pigs and mice. Using corresponding pools of animal sera as test 'reagents' the antigenic reactivity of the four leishmanial strains (L. enriettii, L. tropica major, L. aethiopica and L. mexicana amazonensis) was scaled by reference to end-point serum titres; and antigenic relationships between individual strain pairs were expressed as mean similarity coefficients, giving equal weight to the results of the different serological tests. Overall analysis of the results revealed that L. mexicana amazonensis and L. tropica major were the two most closely related strains, clustering with an overall similarity coefficient of 89%, whereas coefficients of similarity between other strain combinations fell between 75 and 80%. Although different sera had different discriminatory capacity for the leishmanial strains, two combinations of serum reagent and test system yielded relationships between the four strains that most closely approximated to the overall values. These were: (a) immunofluorescence tests with mouse antisera; and (b) agglutination tests with selected rabbit antisera. The results illustrate the use of a number of immunological parameters in relating micro-organisms of a given genus, and reveal a serological classification of the four leishmanial strains at variance with their geographical origin.

In vitro sensitivity of Entamoeba histolytica to furazolidone and iodochlorhydroxyquin, separate and combined.

Entamoeba histolytica trophozoites isolated in Robinson's medium from faeces containing cysts, transported from India, have been tested in vitro against the drugs furazolidone, iodochlorhydroxyquin, and both drugs combined as Dependal. Results show the levels at which drug action is significant and that interaction between the drugs occurs at two concentrations.

Filaments of Trypanosoma brucei: some notes on differences in origin and structure in two strains of Trypanosoma (Trypanozoon) brucei rhodesiense.

Filaments attached to trypanosomes of two strains of T. (T.) brucei were studied by electron microscopy and two distinct types identified: short-thick and long-thin. The former are associated with stumpy trypanosomes and are secretions, via the flagellar pocket, which originate in the area of the Golgi complex, during the infection of the host. They are referred to as 'secretory filaments'. Their diameter is 0.09 to 0.14 mum. The long-thin filaments are associated with slender forms of trypanosome in various artificial situations; those shown by negative staining are believed to be cytoplasmic extrusions from the anatomically weak extremities of the parasite and are referred to as 'plasmanemes'. Their diameter is 0.06 mum. Both types appear to maintain their structure without the aid of the normal type of unit membrane as myelin formations.

Antitrichomonal antibody in the vaginal secretions of women infected with T. vaginalis.

Vaginal secretions from 508 women were examined for evidence of infection by Trichomonas vaginalis, and for antibodies directed against this organism; 42 women (8-3 per cent.) were found to be infected. Secretions from 29 of these women were assayed and antibody apparently directed against T. vaginalis was found in 22 (76 per cent.) of them. Eight out of nineteen secretions (42 per cent.) from apparently uninfected women also contained antibody. Amongst the infected women, no correlation could be found between the presence or absence of antibody and the degree of inflammation, duration of symptoms, use of oral contraceptives, or additional sexually-transmitted disease; there was, however, a slight suggestion that low parasite counts in the vaginal secretions were associated with the presence of antitrichomonal antibody.