Molecular studies of meningococcal and pneumococcal meningitis patients in Ethiopia.

Neisseria meningitidis infections in sub-Saharan Africa usually present with distinct symptoms of meningitis but very rarely as fulminant septicemia when reaching hospitals. In Europe, development of persistent meningococcal shock and multiple organ failure occurs in up to 30% of patients and is associated with a bacterial load of >106/ml plasma or serum. We have prospectively studied 27 Ethiopian patients with meningococcal infection as diagnosed and quantified with real-time PCR in the cerebrospinal fluid (CSF) and serum. All presented with symptoms of meningitis and none with fulminant septicemia. The median N. meningitidis copy number (NmDNA) in serum was < 3.5 × 103/ml, never exceeded 1.8 × 105/ml, and was always 10-1000 times higher in CSF than in serum. The levels of LPS in CSF as determined by the limulus amebocyte lysate assay were positively correlated to NmDNA copy number ( r = 0.45, P = 0.030), levels of IL-1 receptor antagonist, ( r = 0.46, P = 0.017), and matrix metallopeptidase-9 (MMP-9; r = 0.009). We also compared the inflammatory profiles of 19 mediators in CSF of the 26 meningococcal patients (2 died and 2 had immediate severe sequelae) with 16 patients with Streptococcus pneumoniae meningitis (3 died and 3 with immediate severe sequelae). Of 19 inflammatory mediators tested, 9 were significantly higher in patients with pneumococcal meningitis and possibly linked to worse outcome.

Line-probe assay and molecular typing reveal a potential drug resistant clone of Mycobacterium tuberculosis in Ethiopia.

BACKGROUND: Antimicrobial resistance is a global concern of increasing significance. Multidrug resistant tuberculosis (MDR-TB) is spreading worldwide. It is important to monitor trends of antimycobacterial resistance. This is particularly true for high TB burden countries such as Ethiopia where disproportionally less drug sensitivity data are reported from. METHODS: The prevalence of drug resistance was assessed with the line probe assay GenoType MTBDRplus in a set of 161 M. tuberculosis strains that were selected from four common lineages and sub-lineages previously identified in Ethiopia. Most of the tested M. tuberculosis isolates had been genotyped by established Spoligotyping and MIRU-VNTR typing methods. RESULTS: The proportion of MDR-TB among the isolates was 3.1%. Mono-resistance was 1.2% to rifampicin and 4.3% to isoniazid, and resistance to either of the two first line drugs was 8.7%. Strains of Lineage 4 had the highest resistance rate (13.6%) followed by Lineage 3 (4.9%). None of the isolates representing Lineages 1 and Lineage 7 were drug resistant. Multidrug resistance among pulmonary TB and TB lymphadenitis clinical isolates was 2.8 and 3.7%, respectively. Drug resistance of strains carrying the most prevalent spoligotype in Ethiopia - SIT149 - was further explored. Stratification by MIRU-VNTR identified one genotype with a high rate of drug resistance against Rifampicin and Isoniazid and circulation of a potential MDR-TB clone is proposed. CONCLUSION: Although the strain selection was not fully randomized, the overall M. tuberculosis drug resistance rate in this strain set was 8.7% while the rate of MDR was 3.1%. In parallel, we identified a sub-lineage that showed a high rate of resistance to both rifampicin and isoniazid. These resistant strains may belong to a clone of M. tuberculosis that is circulating in the highlands of Ethiopia.

A multicentre, randomised, non-inferiority clinical trial comparing a nifurtimox-eflornithine combination to standard eflornithine monotherapy for late stage Trypanosoma brucei gambiense human African trypanosomiasis in Uganda.

BACKGROUND: While the combination of nifurtimox and eflornithine (NECT) is currently recommended for the treatment of the late stage human African trypansomiasis (HAT), single-agent eflornithine was still the treatment of choice when this trial commenced. This study intended to provide supportive evidence to complement previous trials. METHODS: A multi-centre randomised, open-label, non-inferiority trial was carried out in the Trypanosoma brucei gambiense endemic districts of North-Western Uganda to compare the efficacy and safety of NECT (200 mg/kg eflornithine infusions every 12 h for 7 days and 8 hourly oral nifurtimox at 5 mg/kg for 10 days) to the standard eflornithine regimen (6 hourly at 100 mg/kg for 14 days). The primary endpoint was the cure rate, determined as the proportion of patients alive and without laboratory signs of infection at 18 months post-treatment, with no demonstrated trypanosomes in the cerebrospinal fluid (CSF), blood or lymph node aspirates, and CSF white blood cell count < 20 /µl. The non-inferiority margin was set at 10%. RESULTS: One hundred and nine patients were enrolled; all contributed to the intent-to-treat (ITT), modified intent-to-treat (mITT) and safety populations, while 105 constituted the per-protocol population (PP). The cure rate was 90.9% for NECT and 88.9% for eflornithine in the ITT and mITT populations; the same was 90.6 and 88.5%, respectively in the PP population. Non-inferiority was demonstrated for NECT in all populations: differences in cure rates were 0.02 (95% CI: -0.07-0.11) and 0.02 (95% CI: -0.08-0.12) respectively. Two patients died while on treatment (1 in each arm), and 3 more during follow-up in the NECT arm. No difference was found between the two arms for the secondary efficacy and safety parameters. A meta-analysis involving several studies demonstrated non-inferiority of NECT to eflornithine monotherapy. CONCLUSIONS: These results confirm findings of earlier trials and support implementation of NECT as first-line treatment for late stage T. b. gambiense HAT. The overall risk difference for cure between NECT and eflornithine between this and two previous randomised controlled trials is 0.03 (95% CI: -0.02-0.08). The NECT regimen is simpler, safer, shorter and less expensive than single-agent DFMO. TRIAL REGISTRATION: ISRCTN ISRCTN03148609 (registered 18 April 2008).

Molecular epidemiology and drug sensitivity pattern of Mycobacterium tuberculosis strains isolated from pulmonary tuberculosis patients in and around Ambo Town, Central Ethiopia.

INTRODUCTION: Tuberculosis (TB) is caused by M. tuberculosis complex and remains a major global public health problem. The epidemic remains a threat to sub-Saharan Africa, including Ethiopia, with further emergence of drug resistant TB. We investigated the drug sensitivity pattern and molecular epidemiology of mycobacterial strains isolated from pulmonary TB patients in and around Ambo town in Oromia Region, Central Ethiopia. METHODS: A cross-sectional study was conducted involving 105 consecutive new smear positive pulmonary TB patients diagnosed at Ambo Hospital and surrounding Health Centers between May 2014 and March 2015 upon informed consent. Sputum samples were cultured on Löwenstein-Jensen (LJ) media using standard techniques to isolate mycobacteria. Region of difference 9 (RD9)-based polymerase chain reaction (PCR) and spoligotyping was employed for the identification of the isolates at species and strain levels. The spoligotype patterns were entered into the SITVIT database to determine Octal and SIT (Spoligotyping International Typing) numbers for each strain. The sensitivity of the isolates to isoniazid (INH), rifampicin (RIF), ethambutol (ETB) and streptomycin (STM) was evaluated on LJ-medium with the indirect proportion method. RESULTS: Cultures were positive in 86/105 (82%) of newly diagnosed smear positive pulmonary TB cases. All of the 86 isolates were confirmed as M. tuberculosis. The majority (76.7%) of them were clustered into seven groups while the rest (23.3%) appeared unique. The most predominant Spoligotypes were SIT53 and SIT149, consisting of 24.4% and 20.9% of the isolates, respectively. Assigning of the isolates to family using SPOTCLUST software revealed that 45.3% of the isolates belonged to T1, 23.3% to T3 and 13% to CAS family. The majority (76.7%) of the M. tuberculosis isolates were susceptible to all the four drugs. Any resistance to any one of the four drugs was detected in 23.3% of the isolates. The highest proportion of any resistance was observed against isoniazid (9.3%) and ethambutol (7%). There was only a single case (1.2%) of multidrug resistant/rifampicin resistant (MDR/RR) TB. CONCLUSION: The majority of the isolates were clustered suggesting on-going active transmission in the study area. Mono resistance is relatively prevalent while the magnitude of MDR/RR-TB was found to be lower than in previous studies.

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.

Ex-vivo characterization of regulatory T cells in pulmonary tuberculosis patients, latently infected persons, and healthy endemic controls.

BACKGROUND: Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. METHOD: We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. RESULTS: The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection.

Efficacy and Safety of 'Fixed Dose' versus 'Loose' Drug Regimens for Treatment of Pulmonary Tuberculosis in Two High TB-Burden African Countries: A Randomized Controlled Trial.

BACKGROUND: There are limited data on the performance of the use of fixed-dose combination (FDC) TB drugs when used under programmatic settings in high TB-endemic countries. We evaluated the efficacy and safety of FDC versus loose formulation (LF) TB treatment regimens for treatment of pulmonary TB (PTB) in the context of actual medical practice in prevailing conditions within programmatic settings in five sites in two high TB-burden African countries. METHODS: A two-arm, single-blind, randomized clinical trial comparing FDCs with separate LFs involving 1000 adults newly diagnosed with culture positive PTB was conducted at five sites in two African countries between 2007 and 2011. Participants were randomized to receive daily treatment with anti-TB drugs given as either FDC or separate LFs for 24 weeks (intensive phase- 8 weeks of isoniazid, rifampicin, ethambutol and pyrazinamide; continuation phase- 16 weeks of rifampicin and isoniazid). Primary outcome measures were microbiological cure and safety at the end of six months' treatment; pre-specified non-inferiority margin for difference in cure rate was 4%. The primary efficacy analysis was based on the modified intent to treat (mITT) cohort comprising all randomized patients with a positive baseline culture result for TB and who received at least one dose of study treatment. Patients missing end of treatment culture results were considered failures. Further analyses were done in which mITT patients without an end of treatment (EOT) culture were excluded in a complete case analysis (mITTcc) and a per protocol cohort analysis defined as mITTcc patients who received at least 95% of their intended doses and had an EOT culture result. RESULTS: In the mITT analysis, the cure rate in the FDC group was 86.7% (398/459) and in the LF group 85.2% (396/465) (difference 1.5-% (90% confidence interval (CI) (-2.2%- 5.3%)). Per Protocol analysis showed similar results: FDC 98.9% (359/363) versus LF 96.9% (345/356), (difference 2.0% (90% CI: 0.1%- 3.8%)). The two arms showed no significant differences in terms of safety, early culture conversion and patient adherence to treatment. INTERPRETATION: The comparison of the two drug regimens satisfied the pre-specified non-inferiority criterion. Our results support the WHO recommendations for the use of FDC in the context of actual medical practice within health services in high TB-endemic countries. TRIAL REGISTRATION: ISRCTN Registry 95204603.

Performance of general health workers in leprosy control activities at public health facilities in Amhara and Oromia States, Ethiopia.

BACKGROUND: Leprosy is a chronic infectious disease of public health importance and one of the leading causes of permanent physical disability. Nevertheless, the drop in prevalence following multidrug therapy has resulted in the neglect of leprosy. The annual incidence of leprosy has remained the same in Ethiopia since decades with more than 76% of the reported new cases coming from Oromia and Amhara Regional States. This study was aimed to assess the knowledge, attitude and skill of general health workers in leprosy control activities at public health facilities in Oromia and Amhara Regional States. METHODS: A cross-sectional study was conducted from September 2011 to February 2012 at different public health facilities in selected eight zones in Oromia and Amhara Regional States. A multistage sampling method was used to obtain representative samples. High and low endemic zones for leprosy were included in the study in both regional states. Data were collected from general health workers through a structured self-administered questionnaire and at on-site assessment of their performance. Baseline socio-demographic data, health workers' attitude towards leprosy and their knowledge and skill in the management of leprosy were assessed. Bloom's cut off point was used to describe the knowledge and practical skills of the respondents while Likert's scale was used for attitude assessment. RESULT: A total of 601 general health workers responsible for leprosy control activities at public health facilities were included in knowledge and attitude assessment and 83 of them were subjected to practical evaluation, with on-site observation of how they handle leprosy patients. These included medical doctors (4%), health officers and nurses with Bachelor degree in Science (27%), clinical nurses with diploma (66%) and health assistants (2.8%). The median age of the respondents was 26.0 years and females made up of 45%. Generally the knowledge and skills of the respondents were found to be poor while attitude towards leprosy was positive for the majority of the respondents. The result showed that 519 (86.3%) had poor knowledge. Overall 155 (25.8%) of the respondents had positive attitude towards leprosy while 205 (34.1%) had intermediate (mixed) attitude and 241 (40.1%) had negative attitude to the disease. Among 83 respondents assessed for diagnosis of leprosy only 15(18.0%) diagnosed leprosy correctly. Variation in knowledge and attitude indicated a significant difference (p < 0.05) among different health institutions, professions, gender, in-service training and years of experience. CONCLUSION: The current finding underlines that although leprosy control activities are integrated to the general health services in the country, the knowledge and skills of leprosy diagnosis, treatment and management by health workers was unsatisfactory. Hence, attention should be given to develop training strategies that can improve health worker knowledge and promote better leprosy management at public health facilities. This could be achieved through pre-service and in-service training and giving adequate emphasis to leprosy related practical work and continuous follow- up.

A Randomized Controlled Double Blind Trial of Ciclosporin versus Prednisolone in the Management of Leprosy Patients with New Type 1 Reaction, in Ethiopia.

BACKGROUND: Leprosy Type 1 (T1R) reactions are immune-mediated events leading to nerve damage and preventable disability affecting hands, feet and eyes. Type 1 Reactions are treated with oral corticosteroids. There is little evidence on alternative treatments for patients who do not respond to steroids or experience steroid adverse effects. We report the results of a randomized controlled trial testing the efficacy and adverse effect profile of ciclosporin and prednisolone (CnP) in comparison to prednisolone only (P) in patients with new T1R in Ethiopia. Ciclosporin is a potent immunosuppressant. Outcomes were measured using a clinical severity score, recurrence rate, adverse events and quality of life. RESULTS: Seventy three patients with new T1R were randomized to receive CnP or P for 20 weeks. Recovery rates in skin signs was similar in both groups (91% vs 88%). Improvements in nerve function both, new and old, sensory (66% vs 49%) and motor (75% vs 74%) loss were higher (but not significantly so) in the patients on CnP. Recurrences rates of T1R (85%) were high in both groups, and recurrences occurred significantly earlier (8 weeks) in patients CnP, who needed 10% more additional prednisolone. Serious major and minor adverse events rates were similar in patients in the two treatment arms of the study. Both groups had a significant improvement in their quality of life after the study, measured by the SF-36. CONCLUSIONS: This is the first double-blind RCT assessing ciclosporin, in the management of T1R in Africa. Ciclosporin could be a safe alternative second-line drug for patients with T1R who are not improving with prednisolone or are experiencing adverse events related to prednisolone. This study illustrates the difficulty in switching off leprosy inflammation. Better treatment agents for leprosy patients with reactions and nerve damage are needed.

Comparison of Efficacy and Safety of Ciclosporin to Prednisolone in the Treatment of Erythema Nodosum Leprosum: Two Randomised, Double Blind, Controlled Pilot Studies in Ethiopia.

BACKGROUND: Erythema Nodosum Leprosum (ENL) is a serious complication of leprosy. It is normally treated with high dose steroids, but its recurrent nature leads to prolonged steroid usage and associated side effects. There is little evidence on the efficacy of alternative treatments for ENL, especially for patients who have become steroid resistant or have steroid side effects. These two pilot studies compare the efficacy and side effect profile of ciclosporin plus prednisolone against prednisolone alone in the treatment of patients with either new ENL or chronic and recurrent ENL. METHODS AND RESULTS: Thirteen patients with new ENL and twenty patients with chronic ENL were recruited into two double-blinded randomised controlled trials. Patients were randomised to receive ciclosporin and prednisolone or prednisolone treatment only. Patients with acute ENL had a delay of 16 weeks in the occurrence of ENL flare-up episode, with less severe flare-ups and decreased requirements for additional prednisolone. Patients with chronic ENL on ciclosporin had the first episode of ENL flare-up 4 weeks earlier than those on prednisolone, as well as more severe ENL flare-ups requiring 2.5 times more additional prednisolone. Adverse events attributable to prednisolone were more common that those attributable to ciclosporin. CONCLUSIONS: This is the first clinical trial on ENL management set in the African context, and also the first trial in leprosy to use patients' assessment of outcomes. Patients on ciclosporin showed promising results in the management of acute ENL in this small pilot study. But ciclosporin, did not appear to have a significant steroid-sparing effects in patients with chronic ENL, which may have been due to the prolonged use of steroids in these patients in combination with a too rapid decrease of steroids in patients given ciclosporin. Further research is needed to determine whether the promising results of ciclosporin in acute ENL can be reproduced on a larger scale.

Surveillance of Bacterial Meningitis, Ethiopia, 2012-2013.

Among 139 patients with suspected bacterial meningitis in Ethiopia, 2012-2013, meningococci (19.4%) and pneumococci (12.9%) were the major disease-causing organisms. Meningococcal serogroups detected were A (n = 11), W (n = 7), C (n = 1), and X (n = 1). Affordable, multivalent meningitis vaccines for the African meningitis belt are urgently needed.

A Randomized, Placebo-Controlled Trial Evaluating Safety and Immunogenicity of the Killed, Bivalent, Whole-Cell Oral Cholera Vaccine in Ethiopia.

Killed whole-cell oral cholera vaccine (OCV) has been a key component of a comprehensive package including water and sanitation measures for recent cholera epidemics. The vaccine, given in a two-dose regimen, has been evaluated in a large number of human volunteers in India, Vietnam, and Bangladesh, where it has demonstrated safety, immunogenicity, and clinical efficacy. We conducted a double-blind randomized placebo-controlled trial in Ethiopia, where we evaluated the safety and immunogenicity of the vaccine in 216 healthy adults and children. OCV was found to be safe and elicited a robust immunological response against Vibrio cholerae O1, with 81% adults and 77% children demonstrating seroconversion 14 days after the second dose of vaccine. This is the first study to evaluate safety and immunogenicity of the vaccine in a population outside Asia using a placebo-controlled, double-blind, randomized study design.

Investigation of the high rates of extrapulmonary tuberculosis in Ethiopia reveals no single driving factor and minimal evidence for zoonotic transmission of Mycobacterium bovis infection.

BACKGROUND: Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extrapulmonary TB in Ethiopia. METHODS: Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis. RESULTS: No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3. CONCLUSIONS: The study suggests a complex role for multiple interacting factors in the epidemiology of extrapulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.

A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting.

BACKGROUND: Early detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas. RESULTS: Baseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks. CONCLUSIONS: The 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for surveillance as well as for the detection of second line drug resistance in targeted groups such as in those who fail empirical MDR treatment.

Human immunodeficiency virus infection among new smear positive pulmonary tuberculosis patients in Addis Ababa, Ethiopia.

BACKGROUND: It has been few years since the launching of provider-initiated HIV counselling and testing (PICT) for all tuberculosis (TB) suspected patients and patients presenting with signs and symptoms of TB. However, little is known about the prevalence of HIV in new smear positive confirmed TB cases in Addis Ababa. OBJECTIVE: To determine the proportion of HIV among newly diagnosed smear positive TB cases, who were screened between February 2007 and July 2010 in Addis Ababa. METHODS: A total of 418 pulmonary TB patients and 188 HIV positive non-TB cases were recruited from different health centres in Addis Ababa. All TB patients were tested for HIV. RESULTS: Of the total 418 new smear positive TB patients tested for HIV, 97 (23.2%) were HIV positive. The occurrence of HIV among TB patients was significantly higher in females, 50/182 (27.7%) compared to males, 47/236 (19.7%) (P < 0.05). The mean CD4 lymphocyte count among HIV positive active TB cases was significantly lower (P < 0.05) (210 +/- 23.9 cells/microL) compared to the counts among non-TB HIV positive cases (407.01 +/- 31.3 cells/microL). The proportion of HIV was significantly higher in the age group 31-40 (46.3%) and > 41 (42.2%) year (p < 0.001) compared to younger, 18-20 (3.75%) and 21-30 (17.8%) years of age groups. CONCLUSION: The occurrence of HIV in smear positive TB cases is high, with a higher proportion seen among females compared to males.

Characterization of the etiological agents of tuberculous lymphadenitis in Dera Woreda, North Showa, Ethiopia.

BACKGROUND: Tuberculous lymphadenitis (TBLN) is a common form of extra pulmonary tuberculosis where lymph nodes particularly from cervical, axillary and inguinal sites are mostly involved, however, its diagnosis poses a major challenge in resource limited settings. OBJECTIVE: To identify the etiological species of Mycobacteria responsible for TBLN in Derra area, a rural district in Ethiopia, where the status of TBLN is unknown. MATERIALS AND METHODS: A total of 153 patients who were clinically suspected for TBLN, between the periods of August 2004 and February 2005 were included in the study. Fine needle aspirates (FNA) were collected and processed from 145 participants and further analyzed using Ziehl Neelsen staining, culture, cytology and polymerase chain reaction (PCR) using sets of primers, targeting the IS6110 insertion sequence and the pncA gene allelic variation at position 169. RESULTS: Out of the 145 FNA samples, 66 (45.5%) demonstrated growth on Lowenstein-Jensen (LJ) medium and 115 (79.3%) cases were confirmed as TBLN by a combination of results from acid fast bacilli (AFB) smear examination, culture, cytology and PCR. From the 145 clinically suspected TBLN cases, 108 (75%) were identified by PCR at complex level of which 107/108 (99.1%) were positive for M. tuberculosis and 1/108 (0.9%) was positive for M. bovis using pncA primers. CONCLUSION: The study indicates that M. tuberculosis is the major cause of tuberculous lymphadenitis in Dera area.

Tuberculosis among Addis Ababa city bus drivers and cash collectors.

BACKGROUND: The risk of transmission of tuberculosis is high in crowded conditions. Addis Ababa has a relatively high burden of tuberculosis and its city buses are often filled with commuters. Tuberculosis is a potential occupational hazard for city bus drivers and cash collectors. OBJECTIVES: The objective of this study was to estimate the proportion of tuberculosis among city bus drivers and cash collectors. METHODS: We estimated the proportion of tuberculosis of all forms among city bus drivers and cash collectors of the Addis Ababa City Bus Organization, from January 2003 to December 2004. Study participants were screened with clinical, bacteriological and chest X-ray examination from January 2003 to December 2004. RESULTS: Sixteen cases of tuberculosis were identified among 903 study participants (466 city bus drivers and 437 cash collectors) examined, of whom 12 were already on anti-TB treatment and 4 were diagnosed during the study. The proportion of all forms of tuberculosis, smear-positive pulmonary tuberculosis and extra-pulmonary tuberculosis was 16/903 (1772/100,000), 4/903 (443/100,000) and 3/903(332/100,000), respectively. CONCLUSION AND RECOMMENDATIONS: The proportion of all forms of tuberculosis among the study population was higher than the notification case rates for Addis Ababa reported in 2003, which indicates the need for a routine screening of city bus drivers and cash collectors. Measures such as opening windows and reducing crowding in buses are recommended as a means of reducing the risk of exposure to bus drivers and cash collectors.

Recent trends of cutaneous leishmaniasis in Alert Hospital, Addis Ababa.

BACKGROUND: Cutaneous leishmaniasis is endemic to many parts of the world and has re-emerged in a number of endemic countries in recent years. Environmental changes, immune status of the host and treatment failure are the three most important risk factors associated with the re-emerging and spread of Leishmaniasis. Cutaneous leishmaniasis (CL) ranges from localized, self-healing type to the disfiguring mucocutaneous and diffuse cutaneous type. OBJECTIVE: To access the trend of CL patient flow in ALERT Hospital, Addis Ababa, Ethiopia. METHODS: Patients' clinical and laboratory records were collected retrospectively for 1651 leishmaniasis suspected individuals from ALERT Hospital, from January 1, 2007 to December 30, 2010. RESULTS: From the suspected individuals, 234 cases were positive for Leishmania species with Giemsa stain and/or histopathology and confirmed for CL, of whom 30 (12.8%) were diagnosed in 2007, 29 (12.4%) in 2008, 75 (32.1%) in 2009, and 100 (42.7%) were in 2010. CONCLUSIONS AND RECOMMENDATIONS: The overall proportion of cases with leishmaniasis among the suspected cases was 234/1651 (14.2%). The distribution of CL reports was higher for patients coming from Addis Ababa surrounding areas and Oromia region, 96/234 (41.03%) and 71/234 (30.34%), respectively. In general, the trend of leishmaniasis in and around Addis Ababa seems to be increasing, which calls for further detailed epidemiological studies, including vector and reservoir host studies, to help in the prevention and control of the disease.

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.