Isoenzyme variability of five principal triatomine vector species of Chagas disease in Mexico.

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysed by multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domestic and peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the least polymorphic species (P(0.95)=0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member of the protracta complex, clustered apart from the other studied species by Nei's genetic distance with >1.36, and at least eight loci were found to be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex) than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances between the three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiating among them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied.

Field application of polymerase chain reaction diagnosis and strain typing of Trypanosoma cruzi in Bolivian triatomines.

A new approach for direct identification and characterization of Trypanosoma cruzi stocks in biological samples was tested for field applicability on an extensive sample of feces collected from triatomine vectors from four different species found in Bolivia. The first step of the technique is polymerase chain reaction (PCR) amplification of the hypervariable region of kinetoplast DNA minicircles of T. cruzi parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoma infestans, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, Rhodnius pictipes, Eratyrus mucronatus, and Triatoma sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. cruzi-infected insects in each species. The second step of the procedure is the characterization of the T. cruzi clones by means of hybridization of the PCR products with clone-specific probes generated by the PCR. We used two probes corresponding to major clones circulating in high frequency in Bolivia (as shown by previous population genetic studies using isoenzyme characterization). We obtained four primary results: 1) we confirm the importance of two major clones in Bolivia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoma infestans, up to 22% and 35% in Cochabamba and La Paz departments, respectively; 3) the results favor the absence of interaction between different clones; and 4) we find, for the first time, evidence of the major clones circulating in three species of triatomines that are known as mainly sylvatic species.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct identification of Trypanosoma cruzi natural clones in vectors and mammalian hosts by polymerase chain reaction amplification.

The polymerase chain reaction was used to amplify the highly variable region of the kinetoplast minicircle of Trypanosoma cruzi directly in biological samples (feces of infected Triatomine bugs, blood samples of experimentally infected mice, and artificially infected human blood samples). Hybridization of the amplified DNAs with reference stocks representing different genotypes (natural clones) enabled us to characterize the stocks infecting the biological samples under study. The main interest of this new approach is the diagnosis of T. cruzi infection and simultaneous direct identification of the different natural clones circulating in vectors and mammalian blood without isolation of the stocks. The suitability of this technique for epidemiologic studies is also discussed.

Identification of Trypanosoma brucei gambiense group I by a specific kinetoplast DNA probe.

A set of 26 Trypanosoma brucei stocks from various African countries, previously characterized by multilocus enzyme electrophoresis (MLEE) for 18 polymorphic loci, have been selected to be representative of the three T. brucei classic subspecies. The kinetoplast DNA minicircle variable regions from these stocks have been amplified using the polymerase chain reaction (PCR) technique, and hybridized with the amplified variable regions of three T. brucei reference stocks, previously identified as T. brucei brucei, T. brucei gambiense, and T. brucei rhodesiense, respectively. Both T. b. brucei and T. b. rhodesiense probes hybridized only with their own stocks, but the T. b. gambiense probe specifically hybridized with a group of 12 stocks that represented most of the human stocks from West and Central Africa in our sample. These stocks, which appeared as a clearly separable cluster based on previous MLEE analysis, probably correspond to T. brucei gambiense group I. No other stock hybridized with this amplified fragment. Since the T. b. gambiense probe obtained is specific for many isolates that are pathogenic for humans in Central and West Africa, it appears to be a promising tool for epidemiologic and medical surveys.

High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area.

The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.

Malaria, cause of ahaptoglobinaemia in Africans.

The lack of serum haptoglobin in Africans has been investigated in the Congo, Central Africa, where HpO prevalence is about 30%. This study shows that it is possible to suppress ahaptoglobinaemia within a few weeks by antimalarial chemoprophylaxis, that it does not occur in protected individuals, that ahaptoglobinaemia reappears at its original incidence levels after interruption of chemoprophylaxis, and that some individuals are more susceptible in relation to Hp2 gene. Malaria is the only significant cause of ahaptoglobinaemia in subjects both with and without detectable parasitaemia. The possible mechanisms involved are discussed.

Low probability of transmission of Trypanosoma cruzi to humans by domiciliary Triatoma sordida in Bolivia.

The role of Triatoma sordida in the domestic transmission of Trypanosoma cruzi was assessed in 7 rural localities in Velasco Province, Department of Santa Cruz, Bolivia. Tri. sordida, the only triatomine species identified in these localities, was found inside 58.0% of houses but not in large numbers (3.1 bugs per infested house on average). A total of 220 faecal samples from domiciliary bugs was examined microscopically and by the polymerase chain reaction for the presence of trypanosomes: 21.4% were infected. Analysis of blood meals of domiciliary Tri. sordida showed that humans were the commonest host (70.4%), followed by chickens and dogs. Four of 418 persons tested were seropositive for Tryp. cruzi. Only 2 of a second group of 62 persons living in dwellings infested by Tri. sordida were seropositive. Tryp. cruzi infection was demonstrated in dogs and domestic rats. Three other species of small mammals were found to be infected with trypanosomes. In our study area, domestic Tri. sordida are mainly incriminated in the transmission of Tryp. cruzi to synanthropic animals, whereas transmission to humans is very rare. The presence in houses of small populations of Tri. sordida infected with Tryp. cruzi is therefore currently insufficient for this insect to constitute a major epidemiological risk factor.

Trypanosoma cruzi genotypes associated with domestic Triatoma sordida in Bolivia.

Triatoma sordida is the second species of Triatominae considered of epidemiological significance in Bolivia. Associated with Triatoma infestans in various regions, it is as yet the only triatomine species established in human dwellings in localities of Velasco province, Department of Santa Cruz. This domestication is considered as primary. Flagellate parasites were detected in 16.2% of domiciliary T. sordida and the kDNA-PCR confirmed the presence of Trypanosoma cruzi. Frequencies of T. cruzi clonets 20 and 39, common clonets in Bolivian domestic cycle (T. infestans), were established by their direct detection in feces using PCR and hybridization. These clonets present low frequencies in T. sordida and synanthropic mammals. Forty-six stocks were isolated and analysed by multilocus enzyme electrophoresis (MLEE). The MLEE showed a higher clonal diversity than in T. infestans domestic cycle and the genotypes were clustered in the two principal lineages of T. cruzi. Within each lineage, a broad variability was observed. Mixture of genotypes was mostly observed in mammals. The large diversity of T. cruzi in this cycle should be related to its sylvatic origin. Moreover, the current limited sample of stocks suggests a lineage association with specific hosts.

Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes.

Here we define a new approach for the detection and characterisation of Leishmania complexes by polymerase chain reaction (PCR) and specific hybridisation. The first step consists of PCR amplification of kDNA minicircles using general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second step is the identification of the Leishmania species complexes by hybridisation of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by electrophoresis and the banding patterns compared with multi-locus enzyme electrophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely related strains and for fingerprinting individual strains. The degree of kDNA-PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three complex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania braziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combination of kDNA-PCR fingerprinting and hybridisation with kDNA probes was found to be useful for both sensitive detection and direct identification of Leishmania species complexes.

Sylvatic triatominae of the phyllosoma complex (Hemiptera: Reduviidae) around the community of Carrillo Puerto, Nayarit, Mexico.

Research on domestic and sylvatic triatomines within the community of Carrillo Puerto and neighboring areas of Nayarit, Mexico, documented that Triatoma longipennis (Usinger) and Triatoma picturata (Usinger) were infected with Trypanosoma cruzi (L.) in both habitats. T. picturata was the predominant species in both habitats. Mouse baited-traps increased the effectiveness of collecting sylvatic triatomines, which were difficult to sample by inspecting habitats such as burrows, caves, and cliffs. The colonization of sylvatic and peridomestic habitats by Triatoma, the occurrence of high rates of infection with T. cruzi and the possibility that bugs move between habitats may require modification of current control strategies in Mexico.

Smallness of the panmictic unit of Triatoma infestans (Hemiptera: Reduviidae).

The population genetic structure of Triatoma infestans (Klug), the principal vector of the causative agent of Chagas disease in Bolivia, was investigated by enzyme electrophoresis at 15 loci, of which 3 were polymorphic. A total of 1,286 adults and nymphs was collected from 19 localities of the Cochabamba (high endemicity) and La Paz (low endemicity) departments. Previous results were confirmed, including a low level of polymorphism (0.20), low genetic distance between geographic areas, and a population structure compatible with an isolation by distance model. However, a high proportion (26.3%) of the surveyed localities showed a significant excess of homozygotes, disputing previous conclusions that considered the village as the probable panmictic unit. The excess of homozygotes was reduced when smaller subunits, such as individual houses or chicken coops, were considered, indicating a Wahlund effect.

Sylvatic triatomines (Hemiptera: Reduviidae) in Bolivia: trends toward domesticity and possible infection with Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae).

The risk of domestic transmission of Trypanosoma cruzi (Chagas) by sylvatic triatomines was assessed in an isolated area of the subandean region of Bolivia. None of the 390 residents examined had serological evidence of infection. Two sylvatic triatomine species, Eratyrus mucronatus (Stål) and Triatoma sordida (Stål), were found in houses and in peridomestic structures. The collection of nymphal instars of both species from some houses indicated possible domesticity. Microscopic examination of feces from 92 insects showed no parasites, and cultures from the guts of 30 insects were negative. Nevertheless, a polymerase chain reaction (PCR) test performed on the same fecal samples showed the presence of T. cruzi DNA in 19.1 and 12.5% of E. mucronatus and T. sordida, respectively. These 16 PCR-positive samples were hybridized with 2 T. cruzi-specific probes known from the domestic cycle in Bolivia (clones 20 and 39). At least 1 of these clones was identified in 7 bugs (5 E. mucronatus and 2 T. sordida). Moreover, no hybridization was observed with these probes in S E. mucronatus and 1 T. sordida samples that showed an amplified band by PCR. These data indicated that T. cruzi clones, genetically unrelated to clones 20 and 39, also were circulating in this area. Based on these results, the 2 sylvatic triatomine species encountered in Apolo should not be overlooked as possible local vectors of T. cruzi.

Trypanosoma cruzi: data supporting clonality in Mexican stocks.

To further study genetic heterogeneity of Mexican stocks of Trypanosoma cruzi, genomic Southern analyses from 54 Mexican isolates and 5 South American reference stocks were carried out. The membranes were hybridized with a homologous cDNA clone from the ribosomal protein S4 that identifies allelic bands from a single gene type locus. These allelic bands were sequentially numbered depending on their relative size. Mexican T. cruzi stocks were quite homogeneous: 31 cases (57%) showed a homozygous genotype 3/3, and 21 isolates (38%) exhibited the heterozygote genotype 2/3. Just 2 Mexican stocks (3%) showed a different genotype 2/5, but the potential parental homozygous 2/2 was never observed. Being that T. cruzi is a diploid organism, the apparent absence of the presumptive parental homozygous genotype 2/2 argues against sexual reproduction within the population, at least as a common event. Therefore, these data support a clonal population structure of T. cruzi in Mexico.

[Plasmodium falciparum and drepanocytic gene in Popular Republic of Congo. I. Prevalence of malaria and drepanocytic trait among school children in Brazzaville area (author's transl)]. / Le paludisme à Plasmodium falciparum et le gène de la drépanocytose en République Populaire du Congo. I. -- Prevalence du paludisme et du trait drepanocytaire, en milieu scolaire dans la région Brazzavilloise.

From 1978 to 1979, 5 surveys, among schoolchildren, were carried out during the rainy season in the neighbourhood of Brazzaville (R.P. Congo): 3 in PK 45 village (northern part of the capital), 2 in Djoumouna village (southern part), and 1 in "Talangai" (a suburb of the capital). 868 exams (plasmodic and splenic index fitted with hemoglobin composition [Hb AA or Hb AS]) were done. It appeared that 19,6% of schoolchildren examined were heterozygous sicklers (AS). This percentage confirmed the previous results from other authors in different countries of Central Africa. On the other hand, in spite of an intense transmission, both plasmodic and splenic index were, on the average, relatively low (24,5 and 24,8% respectively). Plasmodium falciparum was largely predominant (95,3% of infections) but P. ovale and P. malariae were also found (1,9% for each species). From our study no obvious "protecting effect" can be attributed to sickle cell trait because plasmodic index of children AA and AS were similar (23,8 and 27,6% respectively). A slight decrease of splenic index was noticed in AS in regard to AA (19,4 and 26.1% respectively). It is difficult to consider this no significative regression as a definitive proof of a premunition stronger in AS than in AA. Effectively some splenic infarctus are well known to be a regular physiopathological process occurring in homozygous SS but often in heterozygous AS too. In such highly endemic and stable malaria area the problem of a suitable antimalaria strategy remains to be solved.

[A case of a combination heterozygote with hemoglobin K Woolwich and hemoglobin C (Hb Kw/HbC) discovered in Bobo-Dioulasso (Burkina Faso)]. / Remarques à propos d'un hétérozygote composite présentant une hémoglobine K Woolwich et une hémoglobine C (Hb Kw/HbC) dépisté à Bobo-Dioulasso (Burkina Faso).

During a study on malaria in pregnant women in Burkina Faso, the authors gave a particular attention to the hemoglobin of a mother and her new-born child (blood of cord) and they noticed an hemoglobin migrating before the HbA which was identified by isoelectric focusing (IEF). The child is a composite HbK/HbC heterozygote. A survey was carried out to check the transmission of such a K Woolwich hemoglobin within the family of the mother. Out of 40 people, 17 got HbKw. A noticeable anemia was found in HbKw/HbC heterozygote. The authors tried to identified a possible thalassemia. There was little probability for an association of a minor alpha-thalassemia in the absence of Bart's hemoglobin in the blood of the cord (IEF test) and there was no associated beta-thalassemia.

Differential infectivity and immunopathology in murine experimental infections by two natural clones belonging to the Trypanosoma cruzi I lineage.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1 > SO34 c14) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium.

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain reaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization.

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.