Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction.

To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).

Early postnatal development of EEG and sleep-waking cycle in two inbred mouse strains.

The postnatal maturation of brain electrical activity and sleep-waking cycle were studied in two inbred mouse strains (C57BL and BALBc), previously differentiated in their sleep patterns at adult age. Genetic differences are evident during the first postnatal period (until day 12) in the maturation of electrical activity which is both earlier and slower in C57BL than in BALBc. On the other hand, from day 12 onwards, as soon as the sleep-waking cycle can be defined by using EEG morphology to select quiet sleep (QS) and active sleep (AS) C57BL is characterized by a higher amount of AS and a lower amount of waking (W) than BALBc, as found in juvenile and adult mice. These differences appear a little later when the recordings are performed on animals which are isolated instead of being left with the rest of the litter.

Sleep variations in C57BL and BALBc mice from 3 weeks to 14 weeks of age.

The evolution of sleep patterns during the diurnal period was studied in C57BL and BALBc mice from 3 to 14 weeks of age. Quantitative analysis of the hypnograms reveals that the main sleep changes occur between weeks 3 and 4 in the two strains. This period is characterized by a decrease of the percentage of paradoxical sleep in BALBc strain, due to a simultaneous drop in the mean number and duration of the episodes. During this time C57BL strain exhibits an increase in the percentage of slow wave sleep, resulting from a rise in the mean number of episodes. At the same time the percentage of awake state decreases in C57BL mice. In the adults these changes result in differences between the strains in sleep pattern that are characterized by a higher percentage of paradoxical and slow wave sleep in C57BL than in BALBc mice. As regards the results obtained, the maturation of sleep mechanisms and particularly of the paradoxical sleep seems to be different in C57BL and in BALBc mice.

Visceral leishmaniasis and HIV-1 co-infection in southern France.

Between 1986 and 1993 visceral leishmaniasis (VL) was diagnosed in 50 adult patients with human immunodeficiency virus type 1 (HIV-1) infection (8 females, 42 males: 31 intravenous drug users, 11 homosexual or bisexual men, 6 heterosexual individuals, 2 blood recipients) from 5 hospital centres in southern France. Diagnosis of VL was by demonstration of Leishmania and isolation of promastigotes by culture in Novy-McNeal-Nicolle medium. Leishmania isolates were identified by their isoenzyme profile in 28 patients. All the patients were immunocompromised when VL was diagnosed. Their median CD4 cell count was 25 x 10(6) (0-200). However, only 21 patients (42%) fulfilled the 1987 CDC criteria for the acquired immune deficiency syndrome before VL developed. Fever (84%), splenomegaly (56%), hepatomegaly (34%), and pancytopenia (62%) were the most common presenting features. Clinical signs were lacking in 10% of patients. Anti-leishmanial antibodies were detected by indirect immunofluorescence or enzyme-linked immunosorbent assay in 26/47 cases (55%). Combining these techniques with Western blotting (WB) gave a positivity rate of 95%. Amastigotes were demonstrated in bone marrow aspirates in 47 cases (94%). Unusual sites for parasites were found in 17 patients (34%), mainly in the digestive tract but also skin and lung. Viscerotropic L. infantum zymodeme MON-1 was characterized in 86% of cases. Dermotropic zymodemes MON-24, MON-29, MON-33, and a previously undescribed zymodeme MON-183, were isolated from 4 patients. The response rate to pentavalent antimony was 50% and to amphotericin B 100%, but clinical relapses were noted in both groups. In endemic areas, VL should be considered as a possible opportunistic infection in HIV-infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Resistance of Leishmania infantum to Glucantime: risk factors and therapeutic management]. / Résistance de Leishmania infantum au Glucantime: circonstances de survenue et prise en charge thérapeutique.

BACKGROUND: Resistance to antimonial drugs is rarely observed in immunocompetent patients. CASE REPORT: A 1-year-old girl was admitted suffering from persistent fever. A diagnosis of visceral leishmaniasis was made. The patient was given two courses of meglumine antimoniate (Glucantime) (60 mg/kg/d for 15 days) and one course of 12 injections of pentamidine (4 mg/kg). She relapsed 8 months later and failed to respond to Glucantime. Immunological tests performed during the relapse showed a suppression of the T cell response to Leishmania antigen and no production of interferon gamma. The patient was then successfully given liposomal amphotericin B (3 mg/kg/d for 10 days). She was asymptomatic 9 months later and had acquired specific cellular immunity against Leishmania. CONCLUSION: Deficient cell-mediated immunity and interferon gamma production are some factors responsible for decreased sensitivity to antimonial drugs. The WHO recommendations treating visceral leishmaniasis with prolonged administration of Glucantime may prevent relapses. Liposomal amphotericin B could be an alternative treatment.

[Treatment of infantile visceral leishmaniasis]. / Traitement de la leishmaniose viscérale infantile.

Visceral leishmaniasis is an endemic disease in the Mediterranean Basin. Children are one of the targets of the infection. Treatment usually requires parenteral injections of pentavalent antimony (Glucantime or Pentostam), but the high frequency of adverse events and the occurrence of primary or secondary resistance cases limit the use of these medications. Diamidines (Pentacarinat) or amphotericin B derivatives are alternatives to antimony. Unfortunately, pharmacokinetics and optimal dosage of diamidines are not well-known, and numerous adverse events are described. Liposomal preparations of amphotericin B enhance its efficiency and tolerance, and the duration of treatment may be reduced to 5 days. Moreover, primary resistance to amphotericin B is not described in immunocompetent children. Allopurinol associated with antimony seems no more efficient than antimony alone. Aminosidine is not evaluated.

[A simple method for cloning Leishmania spp]. / Méthode simple de clonage de Leishmania spp.

A rapid and simple technique for isolation of clones of Leishmania spp. was developed. This method consists to adapt promastigote forms of Leishmania infantum to an easily realized medium: the Panmede solid medium. We obtained cloned-population of this parasite for epidemiologic and toxonomic studies.

Modulation of the vestibulo-ocular reflex by serotonin in the rat.

The effects of intraventricular injection of serotonin (5-HT) and its agonists and antagonists on the amplitude of the vestibulo-ocular reflex were studied in chronic implanted rats. 5-HT (10(-5) M) triggers an increase of the amplitude of the reflex which lasts 30 min. Similar results are obtained when N,N-dimethyl-5-methoxytryptamine (10(-3) M) is introduced into the ventricular cannula. The increasing effects observed both with 5-HT and N,N-dimethyl-5-methoxy-tryptamine are abolished by methiothepin, a potent antagonist of 5-HT receptors. Injection of indirect agonists like pargyline, a monoamine oxidase inhibitor, or fluoxetine, a potent inhibitor of 5-HT reuptake, is followed by an increase of the amplitude of the vestibulo-ocular reflex. These results indicate that 5-HT can modulate the activity of the vestibulo-ocular pathway and muscular tone of extraocular muscles. Location and involvement of various modulating 5-HT sites are discussed.

Electrophysiological and morphological properties of type C vagal neurons in the nodose ganglion of the cat.

Some passive and active electrical properties of type C neurons were studied intracellularly, in situ, in the nodose ganglia of adult cats. From the neuronal responses to hyperpolarizing and depolarizing rectangular current pulses it was possible to determine the input resistance (34.4 M omega) and specific membrane resistance (2373 omega.cm2). Significant changes in magnitude and duration of the action potential evoked by vagal stimulation result from changes in the resting potential caused by the passage of steady polarizing currents across the cell membrane. The action potentials evoked by infranodose vagal stimulation had a long duration, a long latency and comprised several components. The fast main spike was followed by a long post-hyperpolarization. The double shock technique showed that the fast main potential was composed of an initial segment spike ('A spike') and a somatic spike ('S spike'), and made it possible to determine the somatic refractory periods. After electrical identification of the cells, horseradish peroxidase was injected ionophoretically into the soma, and it was shown that the central processes were about four times smaller in diameter than the peripheral processes.

Comparison of brain serotoninergic systems in C57BL and BALBc mice during development.

Adult C57BL and BALBc mice will exhibit differences in behaviour and in their sleep patterns which may be correlated with monoamine content. In this study, we measured the endogenous levels of serotonin (5-HT), tryptophan (TRP), 5-hydroxyindole acetic acid (5-HIAA), noradrenaline (NA) and dopamine (DA) in the forebrain and hindbrain regions of these strains of mice from day 1 to 12 weeks of age. Using the micropunch technique and a radioenzymatic method, we also determined the endogenous 5-HT levels in the nuclei raphe dorsalis, centralis, magnus-pallidus and obscurus. During development, NA levels in the brain stem are higher in the C57BL than in the BALBc mice. From 5 weeks of age onwards, the levels of 5-HT, DA and NA in the forebrain are also higher in the C57BL mice. However, no differences in 5-Ht and DA content was found for the brain stems of these two strains. When the 5-HT levels in the different raphe nuclei were analyzed, the nucleus raphe dorsalis of the BALBc showed a higher 5-HT content.

Rapid identification of causative species in patients with Old World leishmaniasis.

Conventional methods for the identification of species of Leishmania parasite causing infections have limitations. By using a DNA-based alternative, the present study tries to develop a new tool for this purpose. Thirty-three patients living in Marseilles (in the south of France) were suffering from visceral or cutaneous leishmaniasis. DNA of the parasite in clinical samples (bone marrow, peripheral blood, or skin) from these patients were amplified by PCR and were directly sequenced. The sequences observed were compared to these of 30 strains of the genus causing Old World leishmaniasis collected in Europe, Africa, or Asia. In the analysis of the sequences of the strains, two different sequence patterns for Leishmania infantum, one sequence for Leishmania donovani, one sequence for Leishmania major, two sequences for Leishmania tropica, and one sequence for Leishmania aethiopica were obtained. Four sequences were observed among the strains from the patients: one was similar to the sequence for the L. major strains, two were identical to the sequences for the L. infantum strains, and the last sequence was not observed within the strains but had a high degree of homology with the sequences of the L. infantum and L. donovani strains. The L. infantum strains from all immunocompetent patients had the same sequence. The L. infantum strains from immunodeficient patients suffering from visceral leishmaniasis had three different sequences. This fact might signify that some variants of L. infantum acquire pathogenicity exclusively in immunocompromised patients. To dispense with the sequencing step, a restriction assay with HaeIII was used. Some restriction patterns might support genetic exchanges in members of the genus Leishmania.

[Persistence of visceral leishmaniasis in the southeast of France and recorded frequency of the disease in adults. Apropos of recent cases]. / Persistance de la leishmaniose viscérale dans le sud-est de la France et fréquence marquée de l'affection a l'age adulte. A propos d'observations récentes.

The authors report on 9 cases of mediterranean visceral leishmaniasis (Kala-Azar) collected in the south-eastern part of France in 1978. They emphasize the more frequent occurrence of this disease in adults than children. Out of the 40 cases detected in this region between January 1975 and December 1978, 22 affected adults (55 percent) moreover, Kala-Azar appears to occur particularly in adults with decreased general resistance and diagnosis may be difficult because classical clinical features are no complete.

[Excessive 7-14-sec positive spikes during REM sleep in monozygotic non-epileptic twins with speech retardation (author's transl)]. / Quantité excessive de 7-14 positive spikes pendant la P.M.O. chez deux jumeaux monozygotes avec retard du langage.

6-14/sec positive spikes (PS) (in our cases 7-14) were observed during 6 all-night sleep, recordings in one pair of monozygotic twins (aged 7 years), who had severe speech retardation, no epilepsy and were otherwise normal (CAT were normal). The EEG during wakefulness and sleep showed multifocal independent spikes over the left mid-temporal and right parieto-occipital area. The 7-14 PS, which were similar in both twins, occurred slightly during light sleep, were absent during slow sleep and were most prominent during REM sleep (mean=6.3 sec of PS bursts/min of REM). During REM sleep, the 7-14 PS bursts were negatively related to bursts of eye movements; PS were 7 times more frequent in the intervals between than during bursts of eye movements. In addition, long bursts of PS (up to 6 sec) might interupt the bursts of eye movements suggesting a functional antagonism between mechanisms (still unclear) responsible for PS and for REM. The predominance of PS during REM sleep and the inverse relationship with eye movements are not peculiar to our case, since similar findings have been reported in other cases (TSUZUKI 1967; OKUMA et al. 1968). During the sleep stages when Ps occurred spontaneously, PS could also be evoked by a click or a tone, with a latency of 1, 5-2 sec.

The biological diagnosis of leishmaniasis in HIV-infected patients.

This review emphasises the particular difficulties encountered in confirming a suspected case of cutaneous or visceral leishmaniasis when that case is co-infected with HIV. HIV infection appears to have a more profound impact on the development of visceral leishmaniasis than on the evolution of the purely cutaneous disease. The various techniques available for immunological, parasitological and molecular diagnosis are presented and evaluated. The value of serodiagnosis for the detection of antileishmanial antibodies is in part dependent on the antigens used. Western blots may have a use not only in diagnosis but also in predicting the cases of HIV infection that are at most risk of developing symptomatic leishmaniasis. The presence of leishmanial parasites may still only be demonstrated incontrovertibly by the microscopical examination of smears or the culture of blood or biopsy samples. The use of cultures not only permits diagnosis but also detailed study of the parasites. The potential use of PCR in diagnosis is explored and related to other possible tests. A recommended, standardized procedure for the diagnosis of leishmaniasis in HIV-infected patients is presented.

Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral Leishmaniasis in immunocompromised patients.

A PCR assay amplifying a repeated sequence from the Leishmania infantum genome was compared with direct examination of bone marrow aspirate, myeloculture, and serology for the diagnosis of visceral leishmaniasis in immunocompromised patients. Of 73 patients living in an area endemic for leishmaniasis and where visceral leishmaniasis was suspected by physicians, only 10 had an indisputable diagnosis of visceral leishmaniasis. None of the diagnostic tests performed in the study achieved 100% sensitivity for diagnosing visceral leishmaniasis. PCR exhibited superior sensitivity (82%) in comparison with bone marrow aspirate examination (55%) and myeloculture (55%). Our PCR assay also showed good specificity (97%), negative predictive value (97%), and positive predictive value (82%) even when all unconfirmed PCR results were scored as false positives. Serology exhibited good sensitivity (80%) and excellent specificity (100%), negative predictive value (98%), and positive predictive value (100%) in diagnosing new cases of visceral leishmaniasis but failed to diagnose relapses. We also observed consistent negative serological results using several different immunological detection methods for 2 of the 10 patients with confirmed cases of visceral leishmaniasis. This lack of serological reactivity persisted throughout the course of their infections. These results demonstrate the importance of using PCR as an aid in the diagnosis of visceral leishmaniasis in immunocompromised patients.