Chronic meningitis in immunocompromised adult Ethiopians visiting Tikur Anbessa Teaching Hospital and Ye'huleshet Clinic from 2003-2004.

BACKGROUND: Chronic meningitis is inflammation of the meninges where signs and symptoms develop and last for at least four weeks without alleviation. Little is known about the current etiology and incidence of the disease in adults living in developing countries. OBJECTIVE: The objective of this study was to elucidate the most common etiologies of chronic meningitis in adult Ethiopian patients and give an aid in the empiric therapy. METHODOLOGY: A total of 53 adult patients (median age 32 years) having chronic meningitis and who were admitted at Tikur Anbessa Teaching Hospital and Ye'huleshet Clinic, Addis Ababa, Ethiopia were recruited between 2003 and 2004. Of the 53 patients, bacteriological, molecular and immunological investigations were done for 52 of the study participants to detect Cryptococcus neoformans, Mycobacterium tuberculosis, Toxoplasma gondii, Brucella and Neisseria meningitides infections. RESULTS: Forty eight of the participants were HIV positive and 15% (8/52) of the CSF were positive with Cryptococcal latex antigen detection test; in addition, M. tuberculosis DNA was detected using PCR from CSF ofpatients infew of the patients. Multiple infections were observed in studyparticipants with < 0.1 to 1 CD4 to CD8 ratio. CONCLUSION: Chronic meningitis mostly occurred in HIV infected patients, where most of the infections were attributed to Cryptococcus neoformans whereas M. tuberculosis appeared secondary.

A pilot study on detection and genotyping of humanpapilloma virus isolated from clinically diagnosed Ethiopian women having cervical intraepithelial neoplasia.

BACKGROUND: Human Papilloma virus associated cervical cancers are more prevalent in developing countries compared to developed countries. Cervical cancer is reported as the most frequent malignancy among women visiting hospitals in Ethiopia. This study is a pilot study designed to examine the prevalence and genotypes of HPV in twenty Ethiopian women, clinically diagnosed to have cervical neoplasia, while visiting gynecology unit of a tertiary level referral hospital in Addis Ababa. The objective of this study was to detect the presence of HPV L1 gene and respective genotypes among women clinically diagnosed with different grades of cervical neoplasia. METHODOLOGY: A total of 20 fresh biopsy samples were collected from clinically diagnosed cases, DNA extracted and further amplified using PCR for HPV L1 and beta globin genes. The PCR amplicons were denatured and allowed for hybridization onto a nitrocellulose strip containing the type-specific probes for 27 HPV genotypes representing both high and low risk groups as well as beta globin genes. Socio-demographic characteristics and clinical findings of the participants were recorded on structured questionnaires. RESULTS: Amplification of HPV L1 gene by PCR detected 17 cases out of 20. Based on reverse line blot hybridization assay, the most frequent genotype identified was HPV16 (13/20). Mixed infection of HPV 16 with HPV 33, HPV 35, HPV 45 and HPV 58 was detected from other four study participants. CONCLUSION: Human papilloma virus type 16 was the most prevalent genotype identified from the subjects screened. Further investigation with statistically sound sample size would help to clearly visualize the existing trend in Ethiopia regarding factors for high risk HPV positivity and multiple gravidity, young age at first coitus and cervical neoplasia.

Genotyping of human papillomavirus in paraffin embedded cervical tissue samples from women in Ethiopia and the Sudan.

Cervical cancer is the most frequent female malignancy in most developing countries. Previous studies have demonstrated a strong association of human papillomavirus (HPV) infection with dysplasia and carcinoma of the uterine cervix. The objective of this study was to identify the prevailing HPV genotypes responsible for the development of cervical cancer among women in Ethiopia and the Sudan. A molecular characterization of HPV was done on 245 paraffin embedded cervical biopsy samples collected from the two countries. Amplification of HPV and subsequent genotyping was done using SPF10 primers and Line probe assay. Of samples collected from Ethiopian patients, 93% (149/160) and 13% (21/160) had high risk and low risk HPV genotypes, respectively. Among samples collected from the Sudan, 94% (80/85) harbored high risk and 11.7% (10/85) low risk HPV genotypes. Human papillomavirus 16 was the most frequent genotype identified in samples from Ethiopia (91%, 136/149) and the Sudan (82.5%, 66/80). HPV 52, 58, and 18 were the second, third and fourth common genotypes identified in Ethiopia, whereas HPV 18, 45, and 52 were the second, third, and fourth genotypes identified in samples collected from the Sudan. Thus, individuals living in different geographical localities should receive vaccines based on the specific genotypes circulating in the area and a vaccine targeting HPV 16, 18, 45, 52, and 58 may be optimal for the control of cervical cancer in the two countries.

Expression of apoptosis-related genes in an Ethiopian cohort study correlates with tuberculosis clinical status.

Mycobacterium tuberculosis remains one of the world's deadliest pathogens in part because of its ability to persist in the face of an active immune response. It has been suggested that apoptosis of infected macrophages is one way in which the host deals with intracellular pathogens and that M. tuberculosis can inhibit this process. To assess the relevance of this process for human disease, we compared the expression of multiple genes involved in the activation of the extrinsic ("death receptor initiated") pathway of apoptosis in 29 tuberculosis patients, 70 tuberculosis contacts and 27 community controls from Ethiopia. We found that there is a strong upregulation of genes for factors that promote apoptosis in PBMC from individuals with active disease, including TNF-alpha and its receptors, Fas and FasL and pro-Caspase 8. The anti-apoptotic factor FLIP, however, was also upregulated. A possible explanation for this dichotomy was given by fractionation of PBMC using CD14, which suggests that macrophage/monocytes may regulate several key molecules differently from non-monocytic cells (especially TNF-alpha and its receptors, a finding confirmed by protein ELISA) potentially reducing the sensitivity to apoptotic death of monocyte/macrophages--the primary host cell for M. tuberculosis. This may represent an important survival strategy for the pathogen.

Modulation of cell death by M. tuberculosis as a strategy for pathogen survival.

It has been clearly demonstrated that in vitro, virulent M. tuberculosis can favor necrosis over apoptosis in infected macrophages, and this has been suggested as a mechanism for evading the host immune response. We recently reported that an effect consistent with this hypothesis could be observed in cells from the blood of TB patients, and in this paper, we review what is known about evasion strategies employed by M. tuberculosis and in particular consider the possible interaction of the apoptosis-inhibiting effects of M. tuberculosis infection with another factor (IL-4) whose expression is thought to play a role in the failure to control M. tuberculosis infection. It has been noted that IL-4 may exacerbate TNF-α-induced pathology, though the mechanism remains unexplained. Since pathology in TB typically involves inflammatory aggregates around infected cells, where TNF-α plays an important role, we predicted that IL-4 would inhibit the ability of cells to remove M. tuberculosis by apoptosis of infected cells, through the extrinsic pathway, which is activated by TNF-α. Infection of human monocytic cells with mycobacteria in vitro, in the presence of IL-4, appears to promote necrosis over apoptosis in infected cells-a finding consistent with its suggested role as a factor in pathology during M. tuberculosis infection.

Chloroquine-resistant Plasmodium vivax malaria in Debre Zeit, Ethiopia.

BACKGROUND: Plasmodium vivax accounts for about 40% of all malaria infection in Ethiopia. Chloroquine (CQ) is the first line treatment for confirmed P. vivax malaria in the country. The first report of CQ treatment failure in P. vivax was from Debre Zeit, which suggested the presence of chloroquine resistance. METHODS: An in vivo drug efficacy study was conducted in Debre Zeit from June to August 2006. Eighty-seven patients with microscopically confirmed P. vivax malaria, aged between 8 months and 52 years, were recruited and treated under supervision with CQ (25 mg/kg over three days). Clinical and parasitological parameters were assessed during the 28 day follow-up period. CQ and desethylchloroquine (DCQ) blood and serum concentrations were determined with high performance liquid chromatography (HPLC) in patients who showed recurrent parasitaemia. RESULTS: Of the 87 patients recruited in the study, one was lost to follow-up and three were excluded due to P. falciparum infection during follow-up. A total of 83 (95%) of the study participants completed the follow-up. On enrolment, 39.8% had documented fever and 60.2% had a history of fever. The geometric mean parasite density of the patients was 7045 parasites/microl. Among these, four patients had recurrent parasitaemia on Day 28. The blood CQ plus DCQ concentrations of these four patients were all above the minimal effective concentration (> 100 ng/ml). CONCLUSION: Chloroquine-resistant P. vivax parasites are emerging in Debre Zeit, Ethiopia. A multi-centre national survey is needed to better understand the extent of P. vivax resistance to CQ in Ethiopia.

Outbreak of cutaneous leishmaniasis in Silti woreda, Ethiopia: risk factor assessment and causative agent identification.

An outbreak of skin lesions was reported in June 2005 in the district of Silti woreda, 150 km south of Addis Ababa, by the Christian Children's Fund (CCF) and confirmed to be cutaneous leishmaniasis (CL) by our group from the Armauer Hansen Research Institute in July 2005. A house-to-house survey of 1907 residents in three kebeles of Silti woreda conducted in April 2006 showed a prevalence of 4.8%. RFLP analysis of the internal transcribed spacer RNA (ITS1) showed that Leishmania aethiopica was the causative agent. In the survey, it was found that the age group 11-20 years was the most affected. Environmental factors such as proximity of the house to the gorge where hyraxes reside, presence of the plants Adhatoda schimperiana and Acacia spp. in the compound and sharing the same room with domestic animals were significantly associated with developing CL. The prevalence of active disease was higher in Kibet town (10.4%) compared to the rural kebeles. The identified risk factors of CL in the area need further study. The appearance of leishmaniasis in Silti, which was not known to be endemic for the disease, underlines the need to initiate a leishmaniasis control program in Ethiopia to limit its expansion.

A phase I, open-label trial on the safety and immunogenicity of the adjuvanted tuberculosis subunit vaccine H1/IC31® in people living in a TB-endemic area.

BACKGROUND: H1/IC31® is a tuberculosis (TB) subunit vaccine candidate consisting of the fusion protein of Ag85B and ESAT-6 (H1) formulated with the IC31® adjuvant. Previous trials have reported on the H1/IC31® vaccine in M. tuberculosis (Mtb)-naïve, BCG-vaccinated and previously Mtb-infected individuals. In this trial, conducted between December 2008 and April 2010, the safety and immunogenicity of H1/IC31® was assessed in participants living in Ethiopia - a highly TB-endemic area. METHODS: Healthy male participants aged 18-25 years were recruited into four groups. Participants in group 1 (N = 12) and group 2 (N = 12) were Tuberculin Skin Test (TST) negative and QuantiFERON-TB Gold in-tube test (QFT) negative (Mtb-naïve groups), participants in group 3 (N = 3) were TST positive and QFT negative (BCG group), and participants in group 4 (N = 12) were both TST and QFT positive (Mtb-infected group). H1 vaccine alone (group 1) or H1 formulated with the adjuvant IC31® (groups 2, 3 and 4) was administered intramuscularly on day 0 and day 56. Safety and immunogenicity parameters were evaluated for up to 32 weeks after day 0. RESULTS: The H1/IC31®vaccine was safe and generally well tolerated. There was little difference among the four groups, with a tendency towards a higher incidence of adverse events in Mtb-infected compared to Mtb-naïve participants. Two serious adverse events were reported in the Mtb-infected group where a relationship to the vaccine could not be excluded. In both cases the participants recovered without sequelae within 72 h. Immunogenicity assays, evaluated in the 29 participants who received both vaccinations, showed a stronger response to TB antigens in the Mtb-naïve group vaccinated with the adjuvant. CONCLUSION: The trial confirmed the need for an adjuvant for the vaccine to be immunogenic and highlighted the importance of early phase testing of a novel TB vaccine candidate in TB-endemic areas. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT01049282. Retrospectively registered on 14 January 2010.

Field evaluation of FD-DAT, rK39 dipstick and KATEX (urine latex agglutination) for diagnosis of visceral leishmaniasis in northwest Ethiopia.

Three novel diagnostic tests for visceral leishmaniasis (VL), namely FD-DAT, rK39 dipstick and KATEX, were evaluated under field conditions using 101 clinical cases suspected of having VL enrolled in a trial either by active (63 patients) or passive (38 patients) surveillance. VL was confirmed in 49 patients: 35 by both aspirate smear microscopy and NNN culture, 10 by NNN culture alone and 4 by aspirate smear microscopy alone. Based on tests performed in the field, sensitivity for FD-DAT, rK39 dipstick and KATEX was determined to be 95.3% (95% CI 82.9-99.2%), 71.7% (95% CI 56.3-83.5%) and 57.4% (95% CI 42.3-71.4%), respectively. Similarly, the specificity was determined to be 62.7% (95% CI 48.1-75.5%), 82.4% (95% CI 68.6-91.1%) and 84.3% (95% CI 70.9-92.5%), respectively. A higher sensitivity of KATEX (73.9% vs. 41.7%) and higher specificity of FD-DAT (100.0% vs. 48.6%) were demonstrated under passive case detection compared with active case detection. FD-DAT is recommended for confirmation of VL diagnosis in hospital settings, whereas its use in the field will be limited to exclude VL in clinical suspects. The sensitivity of KATEX and rK39 dipstick tests needs to be improved to promote their use as first-line diagnostic tests in the field setting of northwestern Ethiopia.

Strategic emphases for tropical diseases research: a TDR perspective.

Setting priorities for health research is a difficult task, especially for the neglected diseases of the poor. A new approach to priority setting for tropical diseases research has been adopted by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (known as the TDR). Priorities are defined on the basis of a comprehensive analysis of research needs and research opportunities for each of the ten major tropical diseases in the TDR portfolio. The resulting strategic emphases matrix reflects the priorities for tropical diseases research from the perspective of the TDR. Its purpose is not to impose global research priorities, but we believe the results could be useful to other organizations.

Strategic emphases for tropical diseases research: a TDR perspective.

Setting priorities for health research is a difficult task, especially for the neglected diseases of the poor. A new approach to priority setting for tropical diseases research has been adopted by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (known as the TDR). Priorities are defined on the basis of a comprehensive analysis of research needs and research opportunities for each of the ten major tropical diseases in the TDR portfolio. The resulting strategic emphases matrix reflects the priorities for tropical diseases research from the perspective of the TDR. Its purpose is not to impose global research priorities, but we believe the results could be useful to other organizations.

Treatment response of cutaneous leishmaniasis due to Leishmania aethiopica to cryotherapy and generic sodium stibogluconate from patients in Silti, Ethiopia.

Cutaneous leishmaniasis in Ethiopia is caused mainly by Leishmania aethiopica. In this study, the response of L. aethiopica to sodium stibogluconate (SSG) and liquid nitrogen in Silti has been investigated. Patients were divided into two groups by the treating physician and were treated with either liquid nitrogen or SSG. Punch biopsy samples were collected from 54 patients with mean age of 20.61 (± 9.87 SD) years for histological characterization. The histological spectrum found to be type-1, type-2, type-3 and type-4 were 37.0%, 3.7%, 37.0% and 22.2% respectively. One hundred and three patients with a mean age of 18.4 (± 11.7 SD) years were treated with liquid nitrogen. The mean duration of the lesions before the onset of treatment was 8.5 months (± 6.7 SD). Of the 103 patients 80.6% (83/103) were cured, 13.6% (14/103) were dropouts and 5.8% (6/103) did not respond. Twenty patients with a mean age of 19.55 (+1.64 SD) years were treated with Pentostam on conventional dose. Of the 20 patients 85.0% (17/20) were cured, 10.0% (2/20) were unresponsive and 5.0% (1/20) were dropouts. The per protocol cure rate for cryotherapy and Pentostam was 93.3% and 89.5% respectively. Hence, liquid nitrogen can be used as one of the treatment options especially in resource poor settings.

TB case detection: can we remain passive while the process is active?

TB remains a major public health problem despite all the efforts that have been made since it was declared a global emergency in 1993. Different strategies have been implemented to curb the spread of the epidemic. Early case detection and treatment is one of the pillars of the TB control program. In 1991, WHO set targets for increasing case detection and treatment success rates to 70% and 85% respectively. Although the target of treatment success rate has been achieved, the case detection rate remains far below target at currently less than 50%. It is high time that control programs move from simple passive to a more systematic active case finding in order to accelerate TB control.

Usefulness of the rK39-immunochromatographic test, direct agglutination test, and leishmanin skin test for detecting asymptomatic Leishmania infection in children in a new visceral leishmaniasis focus in Amhara State, Ethiopia.

In areas where visceral leishmaniasis is anthroponotic, asymptomatically infected patients may play a role in transmission. Additionally, the number of asymptomatic patients in a disease-endemic area will also provide information on transmission dynamics. Libo Kemkem and Fogera districts (Amhara State, Ethiopia) are now considered newly established areas to which visceral leishmaniasis is endemic. In selected villages in these districts, we conducted a study to assess the usefulness of different approaches to estimate the asymptomatic infection rate. Of 605 participants, the rK39 immunochromatographic test was able to detect asymptomatic infection in 1.5% (9 of 605), direct agglutination test in 5.3% (32 of 605), and leishmanin skin test in 5.6% (33 of 589); the combined use of serologic methods and leishmanin skin test enabled detecting asymptomatic infection in 10.1% (61 of 605). We conclude that the best option to detect asymptomatic infection in this new visceral leishmaniasis-endemic focus is the combined use of the direct agglutination test and the leishmanin skin test.

Multidrug resistant Salmonella Concord is a major cause of salmonellosis in children in Ethiopia.

INTRODUCTION: S. Concord in Ethiopia. The objective of this study was to determine the aetiology of febrile and diarrhoeic illness in Ethiopian children focussing on Salmonella. METHODOLOGY: Paediatric patients (n = 1,225) presenting with diarrhoea or fever from the paediatric outpatient department of Tikur Anbessa University Hospital, Addis Ababa (n = 825), and Jimma University Hospital, South West Ethiopia (n = 400), were investigated for pathogens from January to August 2006. RESULTS: Parasites were detected in 337 cases, Salmonella in 65, and Shigella in 61. Serotyping of Salmonella (including 48 stored isolates) demonstrated the dominance of S. Concord:  S. Concord (85), S. Typhimurium (7), S. Paratyphi B (2), S. Haifa (1), S. Typhi (2),  S. Enteritidis (4), S. Butantan (2), S. Infantis (1), S. Pomona (1), Salmonella group M (28:y:-)  (1), and S. Oskarshamn (1). Six isolates in serogroups B and D were untypeable. Of 81 S. Concord isolates, 30% were invasive, most (86.5%) were positive for ESBL production by E-test and 70% were multiply resistant to trimethoprim-sulphamethaxole, ceftriaxone, chloramphenicol and gentamicin, of which over one quarter (27%) also showed reduced susceptibility to ciprofloxacin. CONCLUSION: Multi-drug resistant S. Concord was the major cause of salmonellosis in two regions of Ethiopia. The strain isolated was highly invasive, highly antibiotic-resistant, and represents a threat to heath care globally.

Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation.

BACKGROUND: Anopheles arabiensis is the major vector of malaria in Ethiopia. Malaria vector control in Ethiopia is based on selective indoor residual spraying using DDT, distribution of long lasting insecticide treated nets and environmental management of larval breeding habitats. DDT and pyrethroid insecticides are neurotoxins and have a similar mode of action on the sodium ion channel of insects. It was therefore necessary to verify the insecticide susceptibility status of An. arabiensis, to better understand the status of cross-resistance between DDT and the pyrethroids in this species as well as to detect a resistant gene. METHODS: Standard WHO insecticide susceptibility tests were conducted on adults reared from larval and pupal collections from breeding sites at three villages namely: Sodere in the Rift Valley, Gorgora in the north and Ghibe River Valley in the south west of Ethiopia. The occurrence of cross-resistance between pyrethroids and DDT was determined using a DDT selected laboratory colony originally collected from Gorgora. Phenotypically characterized mosquitoes were tested for the presence of knockdown resistance (kdr) alleles using the standard polymerase chain reaction assay. RESULTS: All An. gambiae s.l. specimens assayed by PCR were identified as An. arabiensis. The knockdown and mortality results showed An. arabiensis resistance to DDT in all villages, resistance to deltamethrin and permethrin in the Ghibe River Valley and permethrin resistance in Gorgora. Bioassay susceptibility tests also indicated the presence of cross-resistance between DDT and permethrin, but not between DDT and deltamethrin. The knockdown resistance (kdr) mutation of leucine to phenylalanine in the sodium ion channel gene was detected in populations from Gorgora and the Ghibe River Valley. CONCLUSION: Since An. arabiensis shows high levels of resistance to DDT in all villages tested and varying pyrethroid resistance in Gorgora and the Ghibe River valley, precautionary measures should be taken in future vector control operations. Moreover, the status of resistance in other locations in Ethiopia and the spread of resistant gene (s) should be investigated.

Serodiagnosis of tuberculous lymphadenitis using a combination of antigens.

BACKGROUND: The diagnosis of extra-pulmonary tuberculosis (EPTB) by conventional methods such as culture and microscopy has low sensitivity and requires an invasive procedure. A simple rapid serological test would be of great value. METHODS: Six antigens (ESAT-6, Ag85A, TB10.4, Rv3881c, lipoarabinomannan (LAM) and Ara6-BSA) were tested in an ELISA to detect antigen specific IgG and IgM antibodies in sera from 54 culture and histology-confirmed tuberculous lymphadenitis (TBLN) patients, among whom four were HIV seropositive, sera from 25 smear positive pulmonary tuberculosis (PTB) patients, 15 culture and histology-negative lymphadenitis (non-TBLN) patients (n=15) and 22 healthy controls (HCs). RESULTS: The sensitivities of the antigens for the detection of IgG in sera of TBLN patients ranged from 4 to 30 %. Specificities ranged from 91 to 100 % with sera from HCs. Sensitivities of the antigens for detection of IgM ranged from 0 to 15 % and specificities ranged from 91 to 100 %. LAM was the most potent antigen followed by ESAT-6 and Rv3881c for detection of IgG. However, the sensitivity for antigen specific IgG antibody detection was improved when LAM was combined with ESAT-6 and Rv3881c.The sensitivity was 54 % and the specificity 91 %. CONCLUSIONS: The study suggests that the combined use of LAM, ESAT-6 and Rv3881c for the detection of IgG in sera of TBLN patients could be a supplement to microscopy of fine- needle aspirate (FNA) to diagnose EPTB.

Comparison of immunogenicity of five MSP1-based malaria vaccine candidate antigens in rabbits.

A number of laboratories around the world are producing Plasmodium falciparum erythrocyte-stage vaccine candidates in the pursuit of a vaccine against clinical malaria disease. These candidates are often based on the same parasite protein. Rigorous clinical development and testing of multiple candidates is limited by available resources, which underscores the need to conduct comparative studies of the different vaccine candidates. The purpose of this study was to compare five different candidate proteins all based on P. falciparum merozoite surface protein-1 (MSP1). After investigators submitted their candidates, basic protein profiles were evaluated in a blinded fashion by an independent laboratory, and groups of rabbits were immunized with the proteins. Sera obtained from the rabbits were compared for antibody titers by ELISA and for functional activity by an in vitro parasite growth inhibition assay (GIA) activity, again in a blinded fashion. In selected cases the fine specificity of the antibodies was assessed. Significant differences in immunogenicity as well as the functional activity of antibodies induced by the various vaccine candidates were noted. Data from this study can assist in making decisions for further clinical development of MSP1-based candidates, and this process sets a precedent for future comparisons of malaria vaccine candidates.