Amalia Monroy-Ostria

Identification of Mexican Leishmania species by analysis of PCR amplified DNA

Leishmania parasites isolated into culture from patients with LCL or DCL from four different Mexican states were characterised using polymerase chain reaction (PCR), hybridisation with specific probes, and isoenzymes. PCR of the parasites showed that 10 of 11 of those isolates were members of the mexicana complex. This was confirmed in seven cases by isoenzymes. Restriction enzyme digests of PCR products of Mexican isolates showed the isolates to be different from the L.(L.) mexicana reference strain BEL21. Two (C2 and AM) of the isolates were shown to be a possible mixed infection between mexicana and braziliensis complex members. With a second set of samples from different patients from Campeche state, PCR of 14 biopsies indicated the presence of braziliensis complex members in six of the samples. The results showed that most of our isolates of Leishmania which come from the states of Tabasco and Veracruz are members of the Leishmania mexicana complex, but they seem to be different from the L.(L.) mexicana BEL21 reference strain. By hybridisation most of the biopsies (seven out of 14) from Campeche belong to the L. braziliensis complex and two out of 14 to L. mexicana complex and three out of 14 hybridised with both complexes, and two biopsies were negative. In Campeche, which is very close to Tabasco state and has border with Guatemala, we found members of the L. mexicana and L. braziliensis complexes.

Visceral Leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection

A 36 year old male was admitted in December 1997 to hospital with afternoon fever, malaise and hepatosplenomegaly. He also had a dry cough, dyspnoea and anaemia. Pneumonia caused by Pneumocystis carinii and human immunodeficiency virus (HIV) infection were documented. The HIV infection was confirmed in 1997 with 290,000 virus copies. The patient had been in the Mexican State of Chiapas which is known to be endemic for visceral leishmaniosis (VL) and localized cutaneous leishmaniosis (LCL). The visceral symptoms were diagnosed as VL and the causal agent was identified as Leishmania (L. ) mexicana. Identification of Leishmania was carried out by the analysis of amplified DNA with specific primers belonging to the Leishmania subgenus and by dot blot positive hybridisation of these polymerase chain reaction derived products with kDNA from the L. (L. ) mexicana MC strain used as probe. This is the first case in Mexico of VL caused by a species of Leishmania that typically produces a cutaneous disease form.

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.

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.

Genetic similarity between cysticerci of Taenia solium isolated from human brain and from pigs

Mitochondrial (mt) cox1 and ribosomal ITS1 DNA sequences from Taenia solium cysticercus isolates from pigs and cysticerci (racemose and cellulose types) from patients with neurocysticercosis were amplified by the polymerase chain reaction (PCR). The amplicons were sequenced in order to determine the genetic relationship between these types of cysticerci. Phylogenetic trees were constructed and evolutionary distances were calculated. ITS1 and mt cox1 cysticerci sequence data were compared with previously published Taenia spp. sequences. The variation in the ITS1 and cox1 sequences of samples collected from Mexico was minimal, regardless of geographical origin, size or colour of cysticerci from either pigs or human brain. These results suggest that the racemose and cellulose types represent genetically identical metacestodes of T. solium. Alignment of the mt cox1 sequences of the Mexican samples with sequences of other Taenia taxa showed that most were very similar to T. solium from Mexico and T. solium from Colombia; one T. solium Mexican isolate and Taenia hydatigena were placed in the same group close to Taenia crassiceps. The ITS1 sequences for the Mexican T. solium samples indicated the majority were in the same group as the Latin American T. solium. Two Mexican T. solium samples and T. solium from Philippines were placed together in a different group.

Lactobacillus casei ssp. rhamnosus enhances non specific protection against Plasmodium chabaudi AS in mice

OBJECTIVE To evaluate the capacity of Lactobacillus casei ssp. rhamnosus to enhance resistance against Plasmodium chabaudi chabaudi AS. MATERIAL AND METHODS NIH mice were IP injected with viable lactobacillus casei seven days (LC1 group) or 7 and 14 days (LC2 group) before the challenge (day 0) with Plasmodium chabaudi parasitized red blood cells (pRBC). Control mice were inoculated with pRBC only. When parasitaemia was resolved, naive mice were injected with spleen cells from each group. The parasitaemia was measured. Nitric oxide (NO*) in serum was determined. RESULTS Mice from the LC1 group presented a reduction in parasitaemia, with a prepatent period of five days, parasitaemia lasted 11 days, and the peak was (36.3 % pRBC) on the 12th day post-infection. Mice from the LC2 group showed a prepatent period of five days, parasitaemia lasted eight days, and the peak (30 % pRBC) was of on the 11th day. In the control, the prepatent period was three days, the parasitaemia lasted 15 days, and the peak (51% pRBC) was on day nine. Mice inoculated with spleen cells from the LC2 group showed a prepatent period of 21 days, parasitaemia lasted seven days, and the peak (13.5% pRBC) was on the 26th day. CONCLUSION L. casei enhanced nonspecific resistance to P. chabaudi, as indicated by longer prepatent periods, reduced parasitaemia, and reduction in the viability of the parasites recovered from the spleen of infected mice, along with high concentrations of NO* in serum.

SEROEPIDEMIOLOGICAL STUDIES OF CUTANEOUS LEISHMANIASIS IN THE CAMPECHE STATE OF MEXICO

Seroepidemiological studies of cutaneous leishmaniasis were carried out in 169 individuals in a rural area of the Campeche state of México. Fifty showed cutaneous lesions suggestive of leishmaniasis, 70% were parasite positive and 96% skin test positive. An overall 40% positivity to skin test with Montenegros antigen was found. Most of the affected individuals were males from 11 to 30 years-old. Antibodies were determined by immunofluorescent antibody test (IFA) and by Western blot. Two antigen preparations were used, one from a Leishmania mexicana strain which produced localized cutaneous leishmaniasis (LCL) and the other from a diffuse cutaneous leishmaniasis (DCL). In the general population from the area of study 19% gave positive IFA tests with DCL antigen and 20% with LCL antigen while for the patients 67% gave positive IFA tests with DCL and 71% with LCL. By Western blot analysis most of the patients recognized more antigens in the DCL than in the LCL strain. In the DCL strain 78% of patients recognized a 105 kDa, 34% a 139 kDa, 28% a 117 kDa and 26% a 205 kDa MW antigen. In the LCL strain 40% of patients recognized a 205 kDa and 22% a 175 kDa antigens.

Molecular probes and the polymerase chain reaction for detection and typing of Leishmania species in Mexico

Leishmaniasis in Mexico is a public health problem because all the clinical forms have been recorded in most Mexican states. We studied patients showing clinical symptoms of any form of leishmaniasis, from several endemic areas. Bone marrow samples, aspirates or skin biopsies were taken and deoxyribonucleic acid (DNA) was extracted and amplified by the polymerase chain reaction (PCR) with universal primers AJS1 and DeB8, specific for the Leishmania subgenus Leishmania. The PCR products were then hybridized by dot- or Southern blotting and probed with probe 9.2, specific for the L. mexicana complex. If hybridization did not occur, the DNA was amplified with primers D1 and D2, specific for members of the L. donovani complex, and PCR products were hybridized with probe B4Rsa, also specific for the L. donovani complex. DNA was also amplified with primers B1 and B2, specific for the subgenus Viannia, and the PCR products were hybridized with probe B18, specific for the L. braziliensis complex. It was found that in Tabasco and Veracruz, Mexico, localized cutaneous leishmaniasis (LCL) is caused by infection with members of the L. mexicana complex, whereas in the states of Nayarit and Campeche it was due to infection with the L. mexicana and/or L. braziliensis complexes. Visceral leishmaniasis was caused by L. (L.) chagasi, mainly in the states of Chiapas and Guerrero, and by L. (L.) mexicana in one immunocompromised patient from Tabasco.

ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast

American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L.) mexicana, 5% showed a mixed pattern compatible with L. (L.) mexicana and L. (V.) braziliensis, eight with L. (L.) amazonensis and L. (L.) mexicana, and one to L. (V.) braziliensis. Most of the clinical samples, 109/116 (94%), gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L.) mexicana and L. (V.) braziliensis or L. (L.) mexicana and L. (L.) amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

NRAMP1 Polymorphisms like Susceptibility Marker in Mexican Focus of Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is endemic in Campeche state, Mexico. Host and parasite factors are involved in the establishment and development of CL. Host factors include immune response and genetic background. NRAMP1 (Natural Resistance Associated Macrophage Protein 1) is important in innate immunity. Polymorphisms in NRAMP1 have been associated with susceptibility or resistance to infectious and autoimmune diseases. To study the association of NRAMP1 mutations with CL in patients from Calakmul, Campeche, samples from 115 CL patients and 69 samples of healthy people from the same area were evaluated. Five regions in NRAMP1 were amplified and digested, looking for mutations in the promoter region (−524G/C), exon 3 (274C/T), exon 8 (823 C7T), and exon 15 (G/A) and deletion of 4 bp in the 3′UTR region. We found a statistical association between polymorphisms in 3′UTR region and exon 8 and CL [χ 2 = 13.26; p < 0.05; OR = 17.00; IC of 95% (2.24–128.99)]. Some patients who needed more than 40 doses of Glucantime® to heal injuries presented mutations in exons 3, 8, and 15. Multiple or ear lesions were not associated with NRAMP1 polymorphism.