Molecular typing and drug sensitivity testing of Mycobacterium tuberculosis isolated by a community-based survey in Ethiopia.

BACKGROUND: The identification of circulating TB strains in the community and drug sensitivity patterns is essential for the tuberculosis control program. This study was undertaken to identify M. tuberculosis strains circulating in selected communities in Ethiopia as well as to evaluate the drug sensitivity pattern of these strains. METHOD: This study was a continuation of the Ethiopian National TB Prevalence Survey that was conducted between 2010 and 2011. Culture-positive isolates of M. tuberculosis from previous study were typed using region of difference (RD) 9-based polymerase chain reaction (PCR) and spoligotyping. Drug sensitivity testing was conducted using the indirect proportion method on Lowenstein-Jensen media. RESULT: All 92 isolates were confirmed as M. tuberculosis by RD9-based PCR and spoligotyping of 91 of these isolates leds to the identification of 41 spoligotype patterns. Spoligotype revealed higher diversity (45 %) and among this 65.8 % (27/41) were not previously reported. The strains were grouped into 14 clusters consisting of 2-15 isolates. The dominant strains were SIT53, SIT149 and SIT37 consisting of 15, 11, and 9 isolates, respectively. Our study reveals 70 % (64/91) clustered strains and only 39.1 % (25/64) occurred within the same Kebele. Further assignment of the strains to the lineages showed that 74.7 % (68/91) belonged to Euro-American lineage, 18.6 % (17/91) to East Africa Indian lineage and the remaining 6.5 % (6/91) belonged to Indo-oceanic lineage. Valid drug susceptibility test results were available for 90 of the 92 isolates. Mono-resistance was observed in 27.7 % (25/90) and poly-resistance in 5.5 % (5/90) of the isolates. Moreover, multi-drug resistance (MDR-TB) was detected in 4.4 % of the isolates whilst the rest (60/90) were susceptible to all drugs. The highest level of mono-resistance, 26.6 % (24/90), was observed for streptomycin with majority (91.1 %) of streptomycin mono-resistant strains belonging to the Euro-American lineage. CONCLUSION: In this study, the strains of M. tuberculosis circulating in selected sites of Ethiopia were identified along with the drug sensitivity patterns. Thus, these findings are useful for the TB Control Program of the country.

Toxicity study and evaluation of biochemical markers towards the identification of the causative agent for an outbreak of liver disease in Tahtay Koraro Woreda, Tigray.

BACKGROUND: A team of experts of the Faculty of Medicine, Addis Ababa University reported the emergence of unidentified fatal liver disease in Tahtay Koraro Woreda, Tigray in the mid of December 2005. The EHNRI has been then instructed to investigate the possible etiological agent that are likely to be responsible in triggering the health problem and a field survey team consisting of experts were went to the affected area to investigate the situations surrounding the disease. OBJECTIVES: This investigation was conducted to determine the possible etiological agent(s) for the stated health problem in the affected village. METHOD: Acute toxicity study was performed on animal model for the various samples used in human consumption, which was followed by histopathological examination of the liver of the sacrificed laboratory animals. In order to facilitate the elucidation of the causative agent for the alleged health problem further tests for clinical markers and antigens were also performed on the serum collected from affected persons. RESULT: Neither death nor toxic symptoms manifestations were observed on laboratory animals when feeding the consumable samples for a period of two weeks, however histopathological examination of the liver of the sacrificed animals that were given the unprotected pond water and Tela samples from the affected village as a drink revealed severe hepatoic necrosis. Biochemical test results of the serum samples revealed raised level of some clinical markers that are highly significant for detecting liver abnormality of toxic origin. Serological test for surface antigen ruled out the possible causes of infectious origin such as viral hepatitis. CONCLUSION: The overall results confirmed that the causative agent for the outbreak of the liver disease was of toxic origin rather than due to infectious agent and this was found to be associated with consumption of contaminated water as well as Tela.

Training laboratory technicians from the Ethiopian periphery in the MODS technique enables rapid and low-cost diagnosis of Mycobacterium tuberculosis infection.

Tuberculosis (TB) is a leading cause of morbidity and mortality and is frequently complicated by emergence of drug-resistant strains. Diagnosis of TB in developing countries is often based on the relatively insensitive acid-fast staining that does not enable susceptibility profiling. Microscopic observation drug susceptibility assay (MODS) is an inexpensive, simple method that enables rapid TB culture coupled with susceptibility testing. A 3-week MODS training of three Ethiopian laboratory technicians was conducted at Hadassah-Hebrew University Medical Center, Israel. Results of the trainee readings were blindly assessed by an experienced instructor. Two hundred fifty-five (255) trainee culture readings were evaluated throughout the course. The sensitivity and specificity were 75-100% and 31.5-100%, respectively. Multivariate analysis revealed that sensitivity and duration of incubation were positively correlated, although specificity was positively correlated with the length of training. MODS can be reliably performed by laboratory technicians inexperienced in culture techniques in developing countries, with high sensitivity and specificity reached after a brief learning period.

Improved tuberculosis smear detection in resource-limited settings: Combined bleach concentration and LED fluorescence microscopy.

Improved diagnostics for tuberculosis is a high priority in resource-limited settings (RLS). Sputum concentration and fluorescence microscopy (FM) are standard techniques in developed countries where appropriate biosafety precautions are possible. Recently, inexpensive fluorescent lenses using LED light sources have made auramine-based FM more feasible in RLS. Sterilization of sputum with bleach protects lab personnel and, combined with concentration, increases the sensitivity of microscopic detection. We compared the effect of both bleach concentration and FM with LED based lenses to culture for the detection of tuberculosis in military medical hospitals in Addis Ababa, Ethiopia. Three sputum specimens were obtained from 409 patients (1227 total). Standard Ziehl-Neelsen (ZN) or auramine staining were compared with direct or bleach-concentrated specimens. The prevalence by culture was 26%. Sensitivity of microscopic diagnosis was increased both by bleach concentration (14%) and auramine staining (5%). The overall yield of smear positivity varied from 21% for direct ZN to 27% for auramine after concentration (P<0.00001, Cochran test for matched proportions). Twenty-nine HIV+ patients were diagnosed with TB, but ten (34%), would have been missed with direct ZN staining. Bleach concentration and auramine staining with new LED fluorescent systems are cost-effective and safe methods to increase the diagnostic yield of smears, including in HIV-infected patients.

Human herpesvirus 8 infection in HIV-1-infected and uninfected pregnant women in Ethiopia.

BACKGROUND: Human Herpes Virus (HHV-8) is related to Kaposi Saracoma, an opportunistic infection occurring with HIV infection. Little is known about the seroepidemiology of Human Herpesvirus 8 (HHV-8) infection among Ethiopian women, even though women are a major HIV risk group in Ethiopia. OBJECTIVES: This study aimed at determining the seroprevalence of HHV-8 infection in HIV-1-infected and uninfected pregnant women in five selected regions of Ethiopia. METHODS: A cross-sectional study was conducted from December 2006 to June 2007 where pregnant women were recruited after age-matching in groups. A total of 400 pregnant women were enrolled, with 200 being HIV-infected and 200 being HIV-uninfected Sera were screened for IgG lytic antibody to HHV-8 using an Indirect Fluorescence Assay (IFA) in Virology Unit of Ethiopian Health and Nutrition Research Institute (EHNR1). RESULTS: Of 400 pregnant women attending antenatal clinic (ANC) testing sites of five regions in Ethiopia, 212 (53.0%) were positive for HHV-8 IgG lytic antibody. There was a high prevalence of HHV-8 infection among HIV-1-infected pregnant women (138, 69.0%) as compared with HIV-1-uninfected pregnant women (74, 37.0%). CONCLUSION: The study shows a high prevalence of HHV-8 infection among HIV-1-infected pregnant women as compared with HIV-1-uninfected pregnant women. Therefore, creating awareness and educating women on safe sexual practice and avoiding deep kissing may be a fundamental ways to limit the roots of transmission. Moreover, initiating strong antiretroviral therapy (ART) for HIV infected women would be best treatment prior to the development of Kaposi's sarcoma (KS).

Prevalence of smear negative pulmonary tuberculosis among patients visiting St. Peter's Tuberculosis Specialized Hospital, Addis Ababa, Ethiopia.

BACKGROUND: Smear negative pulmonary tuberculosis is an increasing clinical and epidemiological problem, particularly in areas that are affected by the dual tuberculosis/Human immunodeficiency Virus infections. OBJECTIVE: This study was initiated to investigate the value of clinical parameters, chest x-ray findings and culture in the diagnosis of smear negative pulmonary tuberculosis. DESIGN: A cross sectional study was conducted among suspected pulmonary tuberculosis patients visiting St. Peter Tuberculosis Specialized Hospital, Addis Ababa, Ethiopia between November 15, 2004 and October 30, 2005. METHODS: A total of 297 informed and consented patients with suspected pulmonary tuberculosis were screened for acid fact bacilli by direct smear microscopy. All smear negative pooled sputum samples were further processed for culture using conventional Lowenstein-Jensen solid medium and automated BACTEC MGIT 960 system liquid medium at the Ethiopian Health and Nutrition Research Institute. RESULTS: 247/297 (83.2%) patents with suspected pulmonary tuberculosis have had a negative smear results for acid fast bacilli. Abnormal chest x-ray findings were observed in 196 (79.4%) patients. 43/247 (17.4%) patients whose smears were negative for acid fast bacilli found to be positive for mycobacterial culture. The Mycobacterium species identified were M. tuberculosis (n = 40) (93%) and non-tuberculous mycobacteria (n = 3) (7%). Significant difference was not demonstrated statistically between BACTEC MGIT 960 and Lowenstein-Jensen medium in terms of mycobacterial recovery rate (p > 0.05). CONCLUSIONS: The present study showed 82.6% smear negative pulmonary tuberculosis cases were still etiologically unexplained by culture. Therefore, there is a need to develop a scheme to determine the most cost-effective approaches for the diagnosis of smear negative pulmonary tuberculosis in the Ethiopian setting, such as improving the screening method patients with tuberculosis and other chronic pulmonary diseases, chest-x-ray readings and interpretation, specimen collection and processing, smear microscopy, culture and applying laboratory quality control schemes in parallel.

Intensified tuberculosis case finding among HIV-Infected persons from a voluntary counseling and testing center in Addis Ababa, Ethiopia.

OBJECTIVE: To evaluate commonly available screening tests for pulmonary tuberculosis (TB), using sputum bacteriology as a gold standard, in HIV-infected persons attending an urban voluntary counseling and testing clinic in Addis Ababa, Ethiopia. DESIGN: Prospective enrollment of HIV-infected persons, all of whom underwent TB screening, regardless of symptoms, with: (1) symptom screening and physical examination, (2) 3 sputum specimens for smear microscopy, and (3) chest radiograph. One sputum was also sent for concentrated smear microscopy and mycobacterial culture. Chest radiographs were reviewed by 2 independent radiologists. A confirmed TB diagnosis was defined as 1 positive sputum smear and/or 1 positive sputum culture. RESULTS: We enrolled 438 HIV-infected persons: 265 (61%) females, median age 34 years (range: 18-65), median CD4 cell count 181 cells per cubic millimeter (range: 2-1185). Overall, 32 (7%) persons were diagnosed with TB, of whom 5 (16%) were asymptomatic but culture-confirmed TB cases. Screening for cough >2 weeks would have detected only 12 (38%) confirmed TB cases; screening for cough or fever, of any duration, would have detected 24 (75%) cases, with specificity of 64%. Negative predictive value of screening for these 2 symptoms was 97%. Simulation of the current Ethiopian national guidelines had a sensitivity of 63% and specificity of 83% for diagnosing TB disease among study patients. CONCLUSIONS: Traditional symptom screening is insufficient for detecting TB disease among HIV-infected persons but may serve to exclude TB disease. More sensitive, rapid, and low-cost diagnostic tests are needed to meet the demand of resource-limited settings.

Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean.

BACKGROUND: Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. METHODOLOGY/PRINCIPAL FINDINGS: We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9,250-8,160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. CONCLUSIONS/SIGNIFICANCE: Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

Genetic characterization and progression of B3 measles genotype in Ethiopia: a study of five measles outbreak cases.

BACKGROUND: One of the countries where measles remains endemic is Ethiopia. Previously, sequence data from Measles Viruses (MV) circulating in Ethiopia were obtained from clinical specimens. Now the Ethiopian Health and Nutrition Research Institute (ENHRI) has implemented cell culture techniques to isolate measles virus and molecular epidemiologic studies can be generated more easily. OBJECTIVE: To characterize the strains of Measles Virus circulating in Ethiopia during measles outbreaks in 2006 using viral isolates, and compare the results to previously identified Ethiopian strains. METHODS: A case study and convenience sampling method were conducted on five measles outbreak cases tb identify the circulating measles virus genotype in Addis Ababa and Amhara regions of Ethiopia in 2006. RESULTS: Three isolates were obtained from five specimens collected in two regions (1 from Amhara: Bahir Dar, and 2 from Addis Ababa: Addis Ketema and Kolefe Keranio subcities) in Ethiopia during 2006. The viral isolates were analyzed using standard genotyping protocols and were classified as genotype B3, identical to the strain circulating widely in West Africa and imported into Europe (Britain, Netherlands, Germany) and America (Mexico, USA, Canada). CONCLUSION: The conserved sequences among three isolates, covering a 3-month period, suggest that this B3 strain was circulating in Addis Ababa, Bahir Dar and possibly elsewhere in Ethiopia. To interrupt the transmission and circulation of MV, Ethiopia needs a strong national program of epidemiological surveillance, with characterization of circulating MV performed in a timely manner.

Evaluation of a measles vaccine campaign in Ethiopia using oral-fluid antibody surveys.

We undertook a study to demonstrate the potential contribution of oral-fluid (OF) antibody prevalence surveys in evaluating measles vaccine campaigns. In Asela town, southern Ethiopia, oral fluids were collected from 1928 children aged 9 months to 5 years attending for campaign immunization in December 1999 and 6 months later, from 745 individuals aged 9 months to 19 years, in the same location. Measles antibody status was determined by microimmune measles specific IgG enzyme immunoassay (EIA). Antibody prevalence was estimated at 48% in children attending for vaccination (pre-campaign), and 85% post-campaign in the comparable age group. The estimated reduction in the susceptible proportion was 75%. In older children the proportion antibody negative post-campaign was 28% in 7-9 year olds, and 13% in 10-14 year olds levels of susceptibility which raise concern over continued measles transmission. This is the first evaluation of a measles vaccine campaign based on oral-fluid seroprevalence surveys and it demonstrates the merit of oral-fluid surveys in informing health authorities about vaccination strategy refinement.

Attempts to revive Mycobacterium tuberculosis from 300-year-old human mummies.

Environmental persistence of Mycobacterium tuberculosis is subject to speculation. However, the reality that infected postmortem tissues can be a danger to pathologists and embalmers has worrisome implications. A few experimental studies have demonstrated the organism's ability to withstand exposure to embalming fluid and formalin. Recently, a failure was reported in an attempt to resuscitate an original isolate of Robert Koch to determine the lifetime of the tubercle bacillus. The present study also considers a historical approach to determine persistence under favorable environmental conditions. It asks whether acid-fast forms observed in tissues of 300-year-old Hungarian mummies can be resuscitated. Finding organisms before the advent of antibiotics and pasteurization may yield valuable genetic information. Using various media modifications, as well as guinea pig inoculation, an attempt was made to culture these tissues for M. tuberculosis. In addition, a resuscitation-promoting factor, known to increase colony counts in high G+C bacteria, was applied to the cultures. Although an occasional PCR-positive sample was detected, no colonies of M. tuberculosis were obtained. Our results may indicate that the life span of the tubercle bacillus is less than a few hundred years, even though in the short run it can survive harsh chemical treatment.

Colorimetric phage-based assay for detection of rifampin-resistant Mycobacterium tuberculosis.

Tests based on bacteriophage replication enable rapid screening of Mycobacterium tuberculosis for drug resistance. We describe a novel broth-based colorimetric method for detecting phage replication. When clinical isolates were tested by this novel method, high concordance was observed with both the traditional phage assay and gene mutation analysis for detection of resistance to rifampin.

Evaluation of microscopic observation drug susceptibility assay for detection of multidrug-resistant Mycobacterium tuberculosis.

Early detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is of primary importance for both patient management and infection control. Optimal methods for identifying drug-resistant Mycobacterium tuberculosis in a timely and affordable way in resource-limited settings are not yet available. This study prospectively evaluated a low-technology but rapid drug susceptibility testing method, the microscopic observation drug susceptibility assay (MODS), in the concurrent detection of M. tuberculosis and its susceptibilities to isoniazid and rifampin (two drugs defining multidrug-resistant M. tuberculosis) directly from sputum specimens. Sputum samples were collected from 262 smear-positive TB patients in Addis Ababa, Ethiopia. To undertake MODS, 100 mul of decontaminated samples was inoculated into a 24-well plate containing 1 ml of 7H9 broth with and without appropriate drugs. The assay uses an inverted-light microscope to detect characteristic mycobacterial growth in liquid culture. Of 262 smear-positive patients, MODS detected 254 (96.9%) and culture in Löwenstein-Jensen medium detected 247 (94.3%) (P = 0.016). For the 247 cultures, the sensitivity, specificity, and accuracy of MODS for detecting MDR-TB were 92.0, 99.5, and 98.8%, respectively, using the method of proportion as a reference (concordance, 98.8%; kappa value, 0.932). Results for MODS were obtained in a median time of 9 days. MODS is an optimal alternative method for identifying MDR-TB in a timely and affordable way in resource-limited settings.

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria.

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S-23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five different restriction enzymes, namely CfoI, HaeIII, RsaI, MspI and TaqI. Restriction digestion of the PCR product using CfoI enabled differentiation between 9 of the 13 mycobacterial species, whereas the remaining four enzymes differentiated between 7 of these 13 species. None of the five enzymes distinguished between different isolates of Mycobacterium tuberculosis or between species within the M. tuberculosis complex i.e., M. tuberculosis, M. bovis, M. bovis BCG and M. africanum. Although ARDRA analysis of the 16S-23S rDNA does not seem to have a potential for intraspecies differentiation, it has proven to be a rapid and technically relatively simple method to recognise strains belonging to the M. tuberculosis complex as well as to identify mycobacterial species outside this complex.