Complexity of the major surface protease (msp) gene organization in Leishmania (Viannia) braziliensis: evolutionary and functional implications.

The major surface protease (msp or gp63) of Leishmania plays a major role in the host-parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host-parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

Diagnosis of leishmaniasis in Maltese dogs with the aid of the polymerase chain reaction.

Visceral leishmaniasis due to infection with Leishmania infantum (a member of the L. donovani complex) has been known in Malta since the beginning of the century. In 1946, when human diseases became compulsorily notifiable on the islands, the leishmaniasis figures were 1264 visceral cases, 36 cutaneous cases and 5 unspecified. Five cases of cutaneous infection were reported in 1997 and 23 cases of cutaneous and 3 of visceral infection in January-October 1998. There may be considerable under-reporting of the disease. Figures of between 18% and 47% have been reported for canine leishmaniasis. This large discrepancy between reservoir and human hosts suggests that the canine reservoir could be a serious threat and is worthy of careful examination. This pilot study was carried out to determine the proportion of dogs serologically positive for leishmaniasis in order to assess the necessity for a possible control programme in Malta. Using 60 canine blood samples from the Maltese islands, we tested for deoxyribonucleic acid (DNA) of the L. donovani complex using the polymerase chain reaction (PCR). The samples had all been subjected to the indirect immunofluorescent antibody test (IFAT) and a direct comparison was made. DNA was extracted using the phenol/chloroform method and amplified with primers specific for kinetoplast mini-circle DNA of the L. donovani complex and L. major, Southern blotted and hybridized with a radio-labelled probe specific for the L. donovani complex. Twelve of the samples gave positive results in the IFAT, whilst 37 (62%) were positive by PCR and hybridization. All samples from 36 dogs from a non-endemic area in the UK were negative by PCR. Five of the 12 samples positive by IFAT gave negative PCR results.

Genes coding structural proteins in the Leishmania braziliensis complex.

Acidic ribosomal P1 and P2b proteins, referred to as P proteins, and histone H3 are reported for first time in the Leishmania braziliensis complex. Deoxyribonucleic acid analysis and multiple sequence alignment suggest that both P proteins may maintain their structural function in the ribosomal stalk, in spite of the high rate of mutations detected. The deduced amino acid sequence of protein P1 showed 51% identity with Trypanosoma cruzi protein P1 and protein P2b showed 61% identity with T. cruzi protein P2b. Another conserved protein, L. (Viannia) braziliensis histone H3, showed 82% and 70% identity with histone H3 of L. (Leishmania) infantum and T. cruzi, respectively. The N-terminal end of this histone is divergent in comparison with the consensus eukaryotic sequence. Their predicted tridimensional structure was designed.

Analysis of minicircle classes in Leishmania (Viannia) species.

This paper reports the analysis of minicircle sequence classes from 4 Leishmania species, all belonging to the 'New World' species of the subgenus Viannia: Leishmania braziliensis, L. guyanensis, L. panamensis and L. peruviana. A minicircle library was constructed for each species, and clones were analysed by restriction enzyme digest and sequence analysis. 319 minicircles from the 4 species were examined and 96 of these were wholly or partially sequenced. The sequences of 41 whole minicircles--21 from L. panamensis, 8 from L. guyanensis and 6 each from L. braziliensis and L. peruviana are presented. Sequence classes were identified within which sequences were highly conserved, with only a small number of single base pair changes between them. In contrast, minicircles from different classes differed significantly, displaying sequence homology only over the minicircle conserved region. Some minicircle classes were identified which were shared between species. These minicircles displayed sequence variation which was potentially species-specific, and were analysed phylogenetically. These results question the hypothesis that minicircle sequence is rapidly evolving and also suggest that an as yet unknown selective pressure maintains sequence class conservation over the entire minicircle molecule even in different species, not only over the conserved region and the guide ribonucleic acid gene. A novel hypothesis is proposed to explain these results.

A kinetoplast DNA minicircle probe (B18) specific for the Leishmania subgenus Viannia.

This paper reports the development, sequence and specificity of probe B18, which hybridizes only to the kinetoplast deoxyribonucleic acid (kDNA) minicircle of Leishmania species of the subgenus Viannia. This probe was developed in 1985 and has been used extensively since, on whole kDNA, Leishmania dot-blots and kDNA minicircles amplified by the polymerase chain reaction.

Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico.

An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence.

Members of the Leishmania donovani complex are parasites of the reticulo-endothelial system that are often associated with serious epidemics of a life threatening disease known as visceral leishmaniasis or kala-azar. Twenty-two Leishmania isolates representative of the geographical range of the parasite were analysed for sequence variations in their cytochrome oxidase II gene. In performing phylogenetic analysis, the maximum parsimonious, neighbour joining and maximum likelihood trees were congruent and produced a tree that differentiated between two clades conforming to the current classification of the species complex into two species: Leishmania donovani and Leishmania infantum. Furthermore, the molecular haplotypes were concordant, in general, with the isoenzyme data of the complex. The donovani isolates from the Sudan that possessed the most ancestral sequence were of a single haplotype that significantly resembled the sequence of Leishmania major. Our sequence data tallied with a general neutral model of sequence evolution with manifestations of weak selection. The data allowed an approximate dating of the origin of the complex to a period contemporary to or predating the spread of modern humans out of Africa.

A survey of managed care strategies for pregnant smokers.

OBJECTIVE: The purpose of this study was to measure the content and comprehensiveness of pregnancy specific smoking cessation strategies within managed care organisations (MCOs) responding affirmatively to the national 1997-98 Addressing Tobacco in Managed Care (ATMC) survey. DESIGN: This cross sectional follow up study consisted of a fax survey sent to medical directors and a 37 question telephone survey of program overseers about the smoking cessation strategy. SUBJECTS: 147 MCOs identifying a pregnancy specific smoking cessation strategy on the 1997-98 ATMC survey served as the initial sample; 88 MCOs of 128 eligible plans completed both components, with a response rate of 69%. RESULTS: Pregnancy specific smoking cessation strategies varied. 40% of respondents used the Agency for Health Care Policy and Research guidelines for clinical smoking cessation to design their strategy. Strategies included self help materials, quit classes, telephone support and brief counselling by providers, linkages to quality improvement efforts, and use of patient databases for outreach. Only 42% offered a postpartum relapse prevention element. Lack of patient interest, competing clinic priorities, and the lack of a smoker identification system were the most problematic barriers to implementing strategies, common to at least a quarter of respondents. A majority ranked best practice manuals and web site linkages as the most useful form of technical assistance, followed by peer-to-peer counselling, regional workshops, newsletters, on-site assistance, and national conferences. CONCLUSIONS: The survey provides the first profile of prenatal tobacco treatment strategies in managed care. While design limitations prevent generalisation of these results to all MCOs, such information can help guide technical assistance to plans interested in reducing smoking among pregnant women.

Effect of restrictions on smoking at home, at school, and in public places on teenage smoking: cross sectional study.

OBJECTIVE: To determine the relation between extent of restrictions on smoking at home, at school, and in public places and smoking uptake and smoking prevalence among school students. DESIGN: Cross sectional survey with merged records of extent of restrictions on smoking in public places. SETTING: United States. PARTICIPANTS: 17 287 high school students. MAIN OUTCOME MEASURES: Five point scale of smoking uptake; 30 day smoking prevalence. RESULTS: More restrictive arrangements on smoking at home were associated with a greater likelihood of being in an earlier stage of smoking uptake (P<0.05) and a lower 30 day prevalence (odds ratio 0.79 (95% confidence interval 0.67 to 0.91), P<0.001). These findings applied even when parents were smokers. More pervasive restrictions on smoking in public places were associated with a higher probability of being in a earlier stage of smoking uptake (P<0.05) and lower 30 day prevalence (0.91 (0.83 to 0.99), P=0.03). School smoking bans were related to a greater likelihood of being in an earlier stage of smoking uptake (0.89 (0.85 to 0.99), P<0.05) and lower prevalence (0. 86 (0.77 to 0.94), P<0.001) only when the ban was strongly enforced, as measured by instances when teenagers perceived that most or all students obeyed the rule. CONCLUSIONS: These findings suggest that restrictions on smoking at home, more extensive bans on smoking in public places, and enforced bans on smoking at school may reduce teenage smoking.

Leishmania (Viannia) guyanensis: a new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes.

A new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes. Experimental Parasitology 94, 143-149. In this paper we describe a new minicircle class exclusive to Leishmania (Viannia) guyanensis. The minicircle class was obtained with the aid of a total DNA cosmid library. The library was screened with an EcoRI fragment isolated from L. (V.) guyanensis (M4147). After screening seven clones were selected which showed strong hybridisation. Clones were digested and hybridised with the same probe. After hybridisation only one clone containing the desired fragment was positive. The fragment sized around 1000 bp was subcloned into pBluescript for sequencing. Sequence analysis using the GCG programme showed no substantial homology with any sequences previously reported, apart from the expected homology with the conserved region of Leishmania kDNA sequences. The probe hybridised strongly only to L. (V.) guyanensis kDNA after medium stringency washing.

Aetiology of visceral leishmaniasis in Mexico.

Two children with visceral leishmaniasis (VL), were studied by DNA analysis. DNA from liver biopsy samples from both patients, was amplified by PCR with broad primers specific for the Leishmania subgenus. DNA from the patient from Chiapas was also amplified with primers specific for the Leismania donovani complex and hybridised with a probe specific for L. donovani complex. The second patient, who is the first reported case of visceral leishmaniasis in the Mexican state of Tabasco, where localised cutaneous leishmaniasis and DCL predominate, had a co-infection with Toxoplasma gondii. The DNA from this patient was not amplified with primers specific for the L. donovani complex, did not hybridise with a probe specific for the L. donovani complex, but did hybridise with kDNA from a Mexican Leishmania mexicana strain used as a probe. We therefore, suggest that members of the L. donovani or L. mexicana complexes cause VL in Mexico.

Use of PCR to detect Leishmania (Viannia) spp. in dog blood and bone marrow.

A PCR-based protocol for the detection of Leishmania (Viannia) parasites in canine blood, buffy coat, and bone marrow was developed and was then tested with field samples taken from a random sample of 545 dogs from villages in Peru where Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana are endemic. Comparative tests with cultured parasites mixed with dog blood showed that the PCR assay's sensitivity was significantly dependent on the DNA extraction protocol and the PCR primers used. Mass screening of field samples by the preferred PCR protocol detected American cutaneous leishmaniasis (ACL) in 44 of 545 (8.1%) dogs; 31 of 402 (7.7%), 20 of 223 (9.0%), and 8 of 46 (17.4%) were PCR positive when whole blood, buffy coat, and bone marrow aspirates, respectively, were tested. The high prevalence of Leishmania in both asymptomatic (7.6%) and symptomatic (18.0%) dogs provides further circumstantial evidence for their suspected role as reservoir hosts of ACL and indicates that hematogenous dissemination of parasites may be a more common pathological phenomenon than has previously been acknowledged. However, unlike for zoonotic visceral leishmaniasis, the comparatively low prevalence of Leishmania (Viannia) in the blood of symptomatic dogs indicates that PCR with blood cannot be the "gold standard" for the (mass) screening of samples in epidemiological studies.

Leishmania donovani: development and characterisation of a kinetoplast DNA probe and its use in the detection of parasites.

The polymerase chain reaction is used increasingly widely for the diagnosis of both cutaneous and visceral leishmaniasis and for the identification of asymptomatic carriers in the population in endemic disease areas. The use of complex-specific hybridisation probes in conjunction with the polymerase chain reaction increases the specificity as well as the sensitivity of the diagnostic procedure as it discriminates between different infecting Leishmania species. A minicircle kinetoplast DNA probe, B4 Rsa, which hybridizes to all members of the Leishmania (L.) donovani complex has been identified and characterised. It is a segment of a minicircle highly conserved in Bangladeshi and Indian L. (L.) donovani isolates.

The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy.

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.