The biological diagnosis of leishmaniasis in HIV co-infected pacients

This publication emphasises the particular difficulties encountered in conforming a suspected case of cutaneous or visceral leishmaniasis when that case is co-infected with HIV and a recommended, standardized procedure for the diagnosis of leishmaniasis in HIV-infected patients is presented. Document in PDF format, required Acrobat Reader.

Enzymatic polymorphism during Leishmania/HIV co-infection: a study of 381 Leishmania strains received between 1986 and 2000 at the international cryobank in Montpellier, France.

Between 1986 and 2000, 381 Leishmania strains isolated from 288 HIV-positive patients were studied at the international cryobank in Montpellier, France. Most (95.1%) of the strains came from cases of visceral leishmaniasis but 4.9% were from HIV-positives with cutaneous leishmaniasis. The majority of the strains came from patients infected in the Mediterranean region, with a few originating in sub-Saharan Africa and South America. Isoenzymatic characterization revealed 28 zymodemes in four different species: L. infantum (which was predominant), L. donovani, L. major and L. guyanensis. The strains belonging to the L. infantum complex included 20 zymodemes, some of which have so far only been found in cases of Leishmania/HIV co-infection.

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.

Crossing of antinuclear antibodies and anti-leishmania antibodies.

Visceral leishmaniasis or Kala-azar is an endemic parasitic infection in Mediterranean countries. We report an interesting case occurring in a 38-year-old woman suffering from systemic lupus erythematosus and secondary antiphospholipid syndrome. In our patient visceral leishmaniasis occurred during high dose-steroids treatment mimicking a flare of lupus. As the lupus resolved under immunosuppressive treatment, a reactivation of visceral leishmaniasis was observed and was confirmed by the successive serological tests which showed crossing of leishmania and antinuclear antibody titers. Our case shows that, faced with fever occurring in lupus patients in an endemic area, visceral leishmaniasis should be searched for before intensifying immunosuppressive treatments.

Flow cytometric assessment of amphotericin B susceptibility in Leishmania infantum isolates from patients with visceral leishmaniasis.

Amphotericin B susceptibility was measured by a flow cytometric membrane potential assay in Leishmania infantum promastigotes isolated from 11 immunocompetent children treated with liposomal amphotericin B and 19 HIV-infected young adults treated with intralipid amphotericin B. Susceptibility levels were measured by the 90% inhibitory concentrations (IC90) representing the concentrations of drug that induced a 90% decrease in membrane potential compared with the control culture. In immunocompetent children, treatment was fully effective whatever the susceptibility of isolates to amphotericin B. In immunocompromised adults, on the contrary, unresponsiveness and relapses could be observed in all cases and IC90 increased in the course of successive treatments: a decrease of amphotericin B susceptibility in both promastigote and amastigote forms could be observed in a patient who had six relapses. These results suggest that the success of amphotericin B treatment depends greatly on patient immunity status, and indicate that successive relapses could enhance emergence of amphotericin B resistant isolates. The results demonstrate that the flow cytometric membrane potential assay can be used as an easy and reliable tool for studying the evolution of interactions between amphotericin B and the parasite membrane during long-term treatments.

[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.

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.

In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis.

Primary and secondary unresponsiveness to meglumine has long been described in human visceral leishmaniasis. However, no studies have been performed to elucidate if these therapeutic failures were due to strain variability in meglumine sensitivity or were related to host factors. We have studied the in vitro sensitivity of 37 strains of Leishmania infantum isolated from 23 patients (11 human immunodeficiency virus-infected and 12 immunocompetent patients) with visceral leishmaniasis. Sensitivity tests were performed by infecting murine macrophages with Leishmania parasites and culturing them in medium containing different concentrations of meglumine. For each test we calculated a 50% effective dose (ED50) corresponding to the meglumine concentration at which 50% of the Leishmania parasites survived. In vitro results were strongly correlated to immediate clinical outcome. All strains requiring an ED50 of >70 microg/ml were related to therapeutic failures, whereas all strains requiring an ED50 of <40 microg/ml corresponded to an initial efficiency of meglumine. Among those patients who were initially improved, relapses occurred in all immunocompromised patients and in most immunocompetent patients who had a short duration of treatment (15 days). Finally, we found that in vitro sensitivity of strains decreased progressively in relapsing patients treated with meglumine. Consequently, the physician may be encouraged to alternate meglumine with other treatments such as amphotericin B or pentamidine, especially in the case of relapsing patients.

[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.

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)

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.

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).

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.

[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.