Leishmania donovani complex (Kinetoplastida, Trypanosomatidae): comparison of deoxyribonucleic acid based techniques for typing of isolates from Ethiopia.

In Ethiopia, visceral leishmaniasis (VL) is an increasing public health concern. Recently, a new outbreak of VL claimed the lives of hundreds of Ethiopians. Mapping its distribution and the identification of the causative Leishmania species is important for proper use of resources and for control planning. The choice of appropriate typing technique is the key for determining the infecting species. Here we compared three deoxyribonucleic acid (DNA) based markers. We used, for the first time, cpbE and cpbF (cpbE/F) PCR-RFLP and demonstrated that it clearly differentiates Leishmania donovani from Leishmania infantum. The cpbE/F PCR-RFLP gave identical banding pattern for all L. donovani strains irrespective of their geographic origin. With the K26 (primers) PCR-RFLP, the L. donovani strains gave a banding pattern different from L. infantum and showed variation with geographic origin. The Ethiopian isolates typed as L. donovani by the PCR-RFLP of the cpbE/F (gene) and K26 (primers) showed two types of patterns with the T2/B4 (primers) PCR-RFLP; one group with L. infantum-like and the other L. donovani-like pattern. Phylogenetic analysis using cpbE/F sequences showed variation with geographic origin of strains and the African strains of L. donovani are more distantly related to L. infantum. Moreover, the Ethiopian isolates were seen to be closely related to the Sudanese, Kenyan and Indian strains. Thus, we recommend the use of more than one marker to study the population genetics of L. donovani complex.

Validation of bleach-treated smears for the diagnosis of pulmonary tuberculosis.

SETTING: Health centres in Awassa, southern Ethiopia. DESIGN: Consecutive patients visiting health centre laboratories for the evaluation of suspected pulmonary tuberculosis (TB) between June and September 2006 were investigated. On-the-spot, morning and second on-the-spot sputum samples were pooled for each patient. Direct smears were stained with hot Ziehl-Neelsen (ZN) technique and aliquots cultured for mycobacteria on Löwenstein-Jensen media. The remaining sputum was treated with household bleach, aliquoted and processed with short-term digestion, centrifugation and sedimentation techniques, and stained with ZN. RESULTS: Acid-fast bacilli were detected in respectively 126 (25%), 141 (28%), 169 (34%) and 198 (40%) of the 497 pooled sputum samples processed by the direct, short-term, sedimentation and centrifugation techniques (P < 0.001). The sensitivity of the direct, short-term, sedimentation and centrifugation techniques was respectively 51.1%, 53.2%, 57.6% and 63.6%. The difference between the direct smear and centrifugation (P < 0.001) or sedimentation (P < 0.005) methods was significant. The specificity of the direct, short-term digestion, sedimentation and centrifugation techniques was respectively 97%, 93%, 86.5% and 80.8%. CONCLUSIONS: Bleach treatment of sputum and centrifugation significantly improves the sensitivity of smear microscopy for the diagnosis of TB in a health centre in a high TB burden area. It is more sensitive, but possibly less specific, than other bleach methods.

Identification and Genotyping of the Etiological Agent of Tuberculous Lymphadenitis in Ethiopia

Background: In Ethiopia; little has been done to assess how Mycobacterium bovis has contributed to human tuberculosis; though the population routinely consumes unpasteurized milk and raw meat. The aim of this study was to determine the proportion of M. tuberculosis and M. bovis as etiological agents of tuberculous lymphadenitis (TBLN). Methods: Patients with lymphadenopathy (n = 171) were included in a cross-sectional study at Butajira Hospital; Southern Ethiopia. Lymph node biopsies were cultured. Patients' HIV status was identified. DNA from positive cultures was tested by PCR to identify M. bovis and M. tuberculosis. Isolates were genotyped by multiplex ligation-dependent probe amplification (MLPA) assay. Results: Among 171 patients; 156 had culture results. Of these; 107 (69) were positive for M. tuberculosis complex (MTC). Six of the 10 HIV-positive patients were culture positive. M. tuberculosis specific sequences were identified in the DNA of each of 100 samples as assessed by RD10 targeted PCR; and each of the 95 isolates exhibited the M. tuberculosis specific TbD1 deletion by MLPA analysis. No M. bovis was identified. These results indicate that all the isolates were modern M. tuberculosis strains. Furthermore; MLPA studies confirmed that 42of the isolates showed the Haarlem genotype and 12displayed sequences compatible with INH resistance. No mutations conferring resistance to ethambutol or rifampicin were detected. Conclusions: Our data showed that M. tuberculosis strains had common characteristics with strains causing pulmonary TB; which appears to be the main etiological agent of TBLN

Diagnosis of tuberculous lymphadenitis in Ethiopia: correlation with culture, histology and HIV status.

SETTING: Butajira, Southern Ethiopia. OBJECTIVE: To compare the diagnostic capacity of the clinical criteria for tuberculous lymphadenitis (TBLN) with histological and/or culture results and to assess the association of human immunodeficiency virus (HIV) with tuberculosis (TB) lymphadenitis. DESIGN: Patients (n=171) were included in the study from October 2005 until July 2006 at Butajira Hospital. Laboratory tests were performed to confirm TBLN. HIV status was identified in TBLN patients and retrospectively in 1608 healthy individuals. RESULT: A total of 136/161 (84.5%) patients were diagnosed with TBLN by histology. TBLN was culture-confirmed in 107/156 (68.6%) patients. The sensitivity, specificity, positive and negative predictive values of histology were respectively 92.5%, 49%, 79.8% and 75% when compared to culture as gold standard. Patients positive for TBLN by cytology and Ziehl-Neelsen (ZN) were also positive by histology and culture. Among the 143 confirmed TBLN patients, nine (6.3%) were HIV-positive. Of the 1608 healthy individuals, 77 (4.8%) were HIV-positive. Younger age (P=0.0001), female sex (P=0.016), not being married (P=0.0001) and illiteracy (P=0.016) showed a strong association with HIV in healthy individuals. CONCLUSION: Clinical criteria alone over-diagnosed TBLN by 15.4% compared to histological and/or bacteriological results. The HIV prevalence in TBLN patients and healthy individuals was the same.

Effect of skin testing and segregation on the prevalence of bovine tuberculosis, and molecular typing of Mycobacterium bovis, in Ethiopia.

In 2002, the prevalence of bovine tuberculosis (tb) among 500 cattle on Holeta Farm, near Addis Ababa, Ethiopia, was 48 per cent, and the farm was divided into positive and negative herds. After three consecutive rounds of skin testing and segregation of skin test-positive and -negative animals, the prevalence of bovine tb was reduced from 14 per cent to 1 per cent in the negative herd in a year. Spoligotyping of 41 isolates from 17 cows gave an identical and unique spoligotype pattern, which can be represented as the binary number 1100000101111110111111100010000000000100000, where 1 indicates the presence of a spacer and 0 represents a loss. This spoligotype pattern had not previously been reported on the Mycobacterium bovis spoligotype database, and it was therefore designated SB1176, Ethiopian M bovis strain 1 (EMbs1). The variable number tandem repeat (VNTR) profile of the strain was 5254(*)33.1, which differed from the VNTR profile of strains reported in Great Britain.

Knowledge and practice of private practitioners in TB control in Addis Ababa.

SETTING: Private clinics and hospitals in Addis Ababa, Ethiopia. OBJECTIVES: To assess the knowledge of private practitioners (PPs) with regard to tuberculosis (TB) control and their practice of TB diagnosis, treatment and monitoring. DESIGN: A descriptive survey was conducted among PPs. A total of 120 responded to a self-administered questionnaire. RESULTS: According to 81.5% of the PPs, at least two to five TB cases were diagnosed in their clinic per week. The correct anti-tuberculosis treatment regimens recommended by the National Tuberculosis Programme (NTP) were mentioned by only 9.7% of the doctors, while 63% listed 68 regimens. The majority (41.0%) monitored treatment using chest X-ray (CXR) alone, while 21.0% use CXR in combination with another diagnostic tool. Eighty per cent of the PPs did not keep a TB register, and case holding was non-existent. CONCLUSIONS: PPs in Addis Ababa diagnose a high number of TB cases. However, there is a huge lack of information on anti-tuberculosis treatment. This shows the likely irrational use of the few available anti-tuberculosis drugs, which may favour the emergence and spread of drug resistance.

Very high DDT-resistant population of Anopheles pharoensis Theobald (Diptera: Culicidae) from Gorgora, northern Ethiopia.

Standard WHO insecticide bioassay tests were carried out in Gorgora, northern Ethiopia to evaluate the susceptibility status of Anopheles pharoensis Theobald for the insecticides DDT, malathion, permethrin and deltamethrin. The mortality and when appropriate knockdown effect of the insecticides were observed. The results indicated that this species was resistant to DDT. A high mortality was obtained after exposure to permethrin and deltamethrin but below 97 % which is the limit for susceptibility according to WHO. A prolonged knockdown time was noted for DDT and the two pyrethroids. An. phoaroensis was found to be susceptible to malathion.

Malaria vaccine development: current status.

The development of an effective malaria vaccine represents one of the most important approaches that would provide a cost-effective intervention for addition to currently available malaria control strategies. Here, Howard Engers and Tore Godal review recent advances. Over the past decade there has been considerable progress in the understanding of immune mechanisms involved in conferring protection to malaria and in the identification of vaccine candidate antigens and their genes. Several new vaccines have entered Phase I/II trials recently, new adjuvants have been developed for human use and new approaches, such as DNA vaccines and structural modification of antigens to circumvent some of the strategies the parasite uses to avoid the immune response, are being applied. Thus, from the TDR perspective, global malaria vaccine development is entering a crucial period with unprecedented opportunities.

Progress on vaccines against parasites.

There has been significant progress in attempts to develop effective vaccines against parasitic diseases, including malaria, leishmaniasis and schistosomiasis. In malaria, in addition to field trials with SPf66, the Colombian malaria vaccine, several Plasmodium falciparum candidate vaccines are under Phase I testing, including NYVAC-7, a multi-antigen, attenuated recombinant vaccinia virus. Additional candidate antigens are at an advanced stage of pre-clinical development. In leishmaniasis, Phase III clinical trials on first generation vaccines (killed Leishmania, with or without BCG) are proceeding in several countries. Use of IL-12 as an adjuvant for use with killed Leishmania vaccine is being studied in non-human primates. A genetically constructed (gene knock-out) live avirulent Leishmania is being developed in preclinical studies as a potential live vaccine. Research is also underway to evaluate several recombinant proteins. The genes coding for such leading candidate antigens are also being incorporated into various live vectors to yield recombinant organisms with vaccination potential. In schistosomiasis, a strategy for the development of a vaccine against Schistosoma mansoni has been established, focussing on six priority recombinant antigens. An Asian S. japonicum vaccine development network has also been established, initially to develop a vaccine to block transmission in cattle and oxen, important reservoirs of the disease in Asia, and ultimately a human vaccine. As all of the above-mentioned vaccines will be used in the field in disease-endemic tropical countries, optimal stability will be of paramount importance.

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice.

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.

Antibodies to the repetitive epitope of Plasmodium falciparum circumsporozoite protein in a rural Tanzanian community: a longitudinal study of 132 children.

An ELISA employing a novel synthetic peptide consisting of 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was used to detect antibodies against P. falciparum circumsporozoite protein in 132 children, 1 month to 15 years old, from a rural community (Kikwawila village) of Tanzania, a region where malaria is hyperendemic. The children were surveyed comprehensively over 3 consecutive years for clinical, parasitological, and serological parameters. Entomological data were also gathered for selected households in this village. The following results were obtained: anti-(NANP)40 antibodies increased as a function of age; the majority of children over 10 years showed a stable positivity for such antibodies during the longitudinal study; a negative correlation was observed between the levels of anti-sporozoite antibodies and both spleen enlargement and the presence of parasites in thick smears; no relationship was found between anti-(NANP)40 antibodies and asexual blood stage antibodies; children living in two representative households with comparable indoor resting mosquito densities showed markedly different frequencies of anti-(NANP)40 antibodies, in spite of comparable clinical, parasitological, and serological parameters. Thus, in addition to the exposure to infectious mosquito bites, other (e.g., genetic) factors, may play a role in the ability of certain individuals to mount an antibody response against this immunodominant repetitive epitope. The results presented in this paper confirm that the (NANP)40-ELISA represents a simple, reliable means for the detection of anti-(NANP)40 circumsporozoite protein antibodies and suggest that such antibodies may contribute to the immune protection against malaria in humans.

Functional activity in vivo of effector T cell populations. III. Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone.

The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v. injection of virus-specific T lymphocytes into T cell-deficient "B mice". Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v. into syngeneic "B mice" injected simultaneously at a distant site with the virus. These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection. To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement. The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors. To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system. The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity. However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used. To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis. The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v. 25 days earlier. On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals. The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v. into M-MuLV tolerant mice.