Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

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.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Domestic and peridomestic transmission of American cutaneous leishmaniasis: changing epidemiological patterns present new control opportunities

Predictions that deforestation would reduce American cutaneous leishmaniasis incidence have proved incorrect. Presentations at a recent international workshop, instead, demonstrated frequent domestication of transmission throughout Latin America. While posing new threats, this process also increases the effectiveness of vector control in and around houses. New approaches for sand fly control and effective targeting of resources are reviewed.

Clinical picture of cutaneous leishmaniases due to Leishmania (Leishmania) mexicana in the Yucatan peninsula, Mexico

Localized cutaneous leishmaniasis (LCL), known as "chiclero's ulcer" in southeast Mexico, was described by Seidelin in 1912. Since then, the sylvatic region of the Yucatan peninsula has been identified as an endemic focus of LCL. The purpose of the present work was to describe the clinical picture of LCL caused by Leishmania (Leishmania) mexicana in the Yucatan peninsula. A total of 136 cases of LCL, based on isolation and characterization of L. (L.) mexicana by isoenzymes and/or monoclonal antibodies, were selected. Some variability of clinical features regarding number, type, size, form, location and time of evolution of the lesions was observed. The most frequently observed presentation was a single, ulcerated, rounded small lesion, located on the ear, with an evolution time of less than three months, with neither cutaneous metastases nor lymphatic nor mucosal involvement. This picture corresponds to previous studies carried out in the same endemic area where an organism of the L. mexicana complex has been incriminated as a major aetiological agent of classical "chiclero's ulcer", confirming that in the Yucatan peninsula LCL due to L. (L.) mexicana when located on the pinna of the ear is a remarkable characteristic.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Distribution of sand flies (Diptera: Psychodidae) at different altitudes in an endemic region of American cutaneous leishmaniasis in the State of Espírito Santo, Brazil

The involvement of different sand fly species in the transmission of American cutaneous leishmaniasis (ACL) at different altitudes was evaluated in the municipality of Afonso Cláudio in the State of Espírito Santo, Brazil, from November 1995 to February 1997. CDC light traps and Shannon traps baited with human volunteers were hung simultaneously at three altitudes of a river valley: (a) 650-750 m in an area of active ACL transmission; (b) 750-850 m in a transitional area; and (c) 850-950 m in an area where no ACL transmission occurred. A total of 13,363 specimens belonging to 28 species was collected. The five most abundant man-biting species were Lutzomyia intermedia, which constituted 24.3% of the total, Lu. migonei (22.3%), Lu. whitmani (15.4%), Lu. fischeri (14.9%) and Lu. monticola (5.8%). Analysis of the distribution of these species at the three altitudes provided evidence that Lu. fischeri and Lu. monticola were not involved in ACL transmission, whereas Lu. migonei and Lu. whitmani might act as secondary vectors of Leishmania (Viannia) braziliensis and Lu. intermedia was probably the principal vector of the parasite. These results reinforce those published in the existing literature, which indicate that Lu. intermedia is the main vector of Le. (V.) braziliensis in Southeast Brazil, while Lu. migonei and Lu. whitmani are of secondary importance.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Comparison of the Proportion Method With Mycobacteria Growth Indicator Tube and E-test for Susceptibility Testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method.

Presence of American cutaneous leishmaniasis vectors surrounding the city of Medellín, Colombia

The presence of Lutzomyia (Helcocyrtomyia) hartmanni, as a vector of Leishmania colombiensis and L. columbiana (Verrucarum group), recently incriminated in the transmission of leishmaniasis, and L. pia (Verrucarum group) are reported for the first time in a periurban area of Medellín city. There is thus a risk of leishmaniasis transmission in this town.

Concurrent cutaneous, visceral and ocular leishmaniasis caused by Leishmania (Viannia) braziliensis in a kidney transplant patient

Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia) braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V.) braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.

Seasonality of Lutzomyia intermedia and Lutzomyia whitmani (Diptera: Psychodidae: Phlebotominae), occurring sympatrically in area of cutaneous leishmaniasis in the State of Rio de Janeiro, Brazil

Lutzomyia intermedia and Lutzomyia whitmani occur sympatrically in the district of Posse, municipality of Petrópolis, in the State of Rio de Janeiro (114 km from the city of Rio de Janeiro), where sand flies were captured weekly for three consecutive years in both the peridomicile and forest, in two first years from 18:00 to 22:00 h, using a Shannon trap and CDC light traps. The anthropophilic habit was assessed by recording the specimens that bit the human captors. Considering the different capture types and sites, the following species were identified: Brumptomyia brumpti, B. cardosoi, B. guimaraesi, Lutzomyia amarali, L. longipalpis, L. microps, L. costalimai, L. edwardsi, L. firmatoi, L. migonei, L. termitophila, L. tupynambai, L. fischeri, L. pessoai, L. lutziana, L. shannoni, L. barrettoi, L. intermedia, L. whitmani, L. lloydi, and L. minasensis. L. intermedia (38.3%) and L. whitmani (38.6%) predominated. L. intermedia predominated in the peridomicile, where it displayed more intense anthropophily, while L. whitmani displayed greater density in the banana plantations and forest, preferential sites for biting humans. Studies on seasonality showed that while L. intermedia displayed greater density in the hottest months of the year (December, January, and February), L. whitmani was abundant in the coolest months (June, July, and August), although both occurred throughout the year.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 ± 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.