Gezahegne Mamo

Pathology of Camel Tuberculosis and Molecular Characterization of Its Causative Agents in Pastoral Regions of Ethiopia

A cross sectional study was conducted on 906 apparently healthy camels slaughtered at Akaki and Metehara abattoirs to investigate the pathology of camel tuberculosis (TB) and characterize its causative agents using postmortem examination, mycobacteriological culturing, and multiplex polymerase chain reaction (PCR), region of difference-4 (RD4)-based PCR and spoligotyping. The prevalence of camel TB was 10.04% (91/906) on the basis of pathology and it was significantly higher in females (χ2 = 4.789; P = 0.029). The tropism of TB lesions was significantly different among the lymph nodes (χ2 = 22.697; P = 0.002) and lung lobes (χ2 = 17.901; P = 0.006). Mycobacterial growth was observed in 34% (31/91) of camels with grossly suspicious TB lesions. Upon further molecular characterization using multiplex PCR, 68% (21/31) of the colonies showed a positive signal for the genus Mycobacterium, of which two were confirmed Mycobacterium bovis (M. bovis) by RD4 deletion typing. Further characterization of the two M. bovis at strains level revealed that one of the strains was SB0133 while the other strain was new and had not been reported to the M. bovis database prior to this study. Hence, it has now been reported to the database, and designated as SB1953. In conclusion, the results of the present study have shown that the majority of camel TB lesions are caused by mycobacteria other than Mycobacterium tuberculosis complex. And hence further identification and characterization of these species would be useful towards the efforts made to control TB in camels.

Community-based cross-sectional survey of latent tuberculosis infection in Afar pastoralists, Ethiopia, using QuantiFERON-TB Gold In-Tube and tuberculin skin test

BackgroundThere is little information concerning community-based prevalence of latent tuberculosis infection (LTBI) using T-cell based interferon-γ (IFN-γ) release assays (IGRAs), particularly in TB endemic settings. In this study, the prevalence of LTBI in the Afar pastoral community was assessed using QuantiFERON-TB Gold In-Tube (QFTGIT) and tuberculin skin tests (TST).MethodsA community-based cross-sectional survey of LTBI involving 652 apparently healthy adult pastoralists was undertaken in the pastoral community of Amibara District of the Afar Region between April and June 2010.ResultsThe prevalence of LTBI was estimated as 63.7% (363/570) using QFTGIT at the cut-off point recommended by the manufacturer (≥ 0.35 IU/ml IFN-γ), while it was 74.9% (427/570) using a cut-off point ≥ 0.1 IU/ml IFN-γ. The QFTGIT-based prevalence of LTBI was not significantly associated with the gender or age of the study participants. However, the prevalence of LTBI was 31.2% (183/587) using TST at a cut-off point ≥ 10 mm of skin indurations, and it was higher in males than females (36.8% vs. 23.5%, X2 = 11.76; p < 0.001). There was poor agreement between the results of the tests (k = 0.098, 95% CI, 0.08 - 0.13). However, there was a positive trend between QFTGIT and TST positivity (X2 = 96.76, P < 0.001). Furthermore, individuals with skin indurations ≥ 10 mm were 13.6 times more likely to have positive results using QFTGIT than individuals with skin indurations of 0 mm (adjusted OR = 13.6; 95%CI, 7.5 to 24.7, p < 0.001).ConclusionsThere is currently no agreed gold standard for diagnosis of LTBI. However, the higher prevalence of LTBI detected using QFTGIT rather than TST suggests that QFTGIT could be used for epidemiological studies concerning LTBI at the community level, even in a population unreactive to TST. Further studies of adults and children will be required to assess the effects of factors such as malnutrition, non-tuberculosis mycobacterial infections, HIV and parasitic infections on the performance of QFTGIT.

Performance of QuantiFERON-TB Gold In-Tube (QFTGIT) for the diagnosis of Mycobacterium tuberculosis (Mtb) infection in Afar Pastoralists, Ethiopia

BackgroundCurrently, T-cell based gamma interferon (IFNγ) release assays (IGRAs) are acknowledged as the best methods available for the screening of latent tuberculosis infection (LTBI) and also as aid for the diagnosis of active tuberculosis (TB). To our information, the performance of these diagnostic tests has not been evaluated in Ethiopia. Therefore, the intent of this study was to evaluate the performance of QuantiFERON-TB Gold In-Tube (QFTGIT) in patients clinically suspected of active pulmonary TB (PTB) as well as in healthy subjects prior to its utilization for the epidemiological study of active TB and LTBI in Afar pastoralists.MethodsThe sensitivity of QFTGIT was evaluated in 140 subjects who were clinically suspected of PTB using the cut-off value recommended by the manufacturer (≥ 0.35 IU/ml) and disease-specific cut-off value. Sputum culture result was used as a gold standard. The specificity of the test was evaluated both in patients and in 55 tuberculin skin test (TST) negative healthy subjects.ResultsOut of the 140 study participants, 37 (26.4%) were positive for active PTB by culture. Out of the 37 subjects who had positive results by culture, 6 individuals were HIV-seropositive. Out of the 103 subjects who were negative by culture, 6 subjects had indeterminate results and 21 were HIV-seropositive. The performance of the test was assessed using data from 107 (31 culture positive and 76 culture negative) individuals who were clinically suspected of PTB and HIV-seronegatives. Using the manufacturer recommended cut-off value, the sensitivity of the test was 64.5% (20/31), while its specificity was 36.8% (28/76). The sensitivity of the test was increased to 77.4%, while the specificity was reduced to 23.7% using a cut-off value ≥ 0.1 IU/ml of IFNγ as disease-specific cut-off value. In TST negative healthy subjects, the specificity of the test was 58.2%.ConclusionOur findings revealed a low sensitivity of QFTGIT in the diagnosis of Mycobacterium tuberculosis (Mtb) infection in the present study area using the cut-off value recommended by the manufacturer. Nevertheless, the sensitivity increased from 64.5% to 77.4% by lowering the cut-off value recommended by the manufacturer to ≥ 0.1 IU/ml of IFNγ level. Hence, it is of practical importance to evaluate the performance of QFTGIT in population under different settings prior to its application either for the diagnosis of active TB or LTBI.

IgA Response to ESAT-6/CFP-10 and Rv2031 Antigens Varies in Patients With Culture-Confirmed Pulmonary Tuberculosis, Healthy Mycobacterium tuberculosis- Infected and Non-Infected Individuals in a Tuberculosis Endemic Setting, Ethiopia

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT‐6/CFP‐10 and Rv2031c antigens in sera of patients with culture‐confirmed pulmonary tuberculosis (PTB), healthy Mtb‐infected and non‐infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme‐linked immunosorbent assay (ELISA). QuantiFERON‐TB Gold In‐Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT‐6/CFP‐10 and Rv2031 were significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.001). The mean OD values of IgG against ESAT‐6/CFP‐10 and Rv2031 were also significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb‐infected cases compared with non‐infected individuals. There were positive correlations (P < 0.05) between the level of IFN‐γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb‐infected subjects. This study shows the potential of IgA response against ESAT‐6/CFP‐10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb‐infected and non‐infected cases. Nevertheless, further well‐designed cohort study is needed to fully realize the full potential of this diagnostic marker.

Prevalence of bovine tuberculosis and assessment of Cattle owners awareness on its public health implication in and around Mekelle, Northern Ethiopia

A cross-sectional study was conducted from September, 2010 to July, 2011 on 480 cattle in and around Mekelle town, northern Ethiopia, to determine the prevalence of bovine tuberculosis (BTB) using comparative intradermal tuberculin (CIDT) test and to assess cattle owners’ awareness on its public health implication using a questionnaire survey. The individual animal and herd bovine tuberculin positivity prevalence were 54/480 (11.3%) (95% CI: 8.4 to 14.1%) and 24/120 (20%) (95% CI: 12.7 to 27.3%) at cut-off > 4 mm, respectively. Cattle kept in intensive type of production (odds ratio (OR) = 3.7), in larger herds with more than 10 cattle (OR = 11.3) and under poor management condition (OR = 4.3), were more likely to be infected with bovine tuberculosis. On the basis of animal characteristics, female (OR = 4.8), exotic (OR = 6.1) and cross bred (OR = 6.6), and cattle with poor body condition (OR = 2.7) were more reactive to tuberculin test than male, Zebu breed and good body conditioned animals, respectively. Out of the 54 CIDT positive cattle, 4 were slaughtered and tuberculous lesions were detected from the organs and carcasses of those cattle. One hundred and twenty household cattle owners or members of these households were interviewed, of these only 37 (30.8%) and 18 (15%) respondents had recognized or had heard about BTB and aware of zoonotic importance of BTB, respectively. The result of this study revealed poor awareness of cattle owners on BTB and its transmission; likewise, the study suggests that the prevalence of BTB in the study area is moderate and strong tuberculoid like lesions found from CIDT tested slaughtered animals. Further molecular characterization of cattle TB isolates present in the area are warranted. Key words: Bovine tuberculosis, cattle, comparative intradermal tuberculin (CIDT), Ethiopia.

Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia

The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power.

Molecular characterization of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Felege Hiwot Referral Hospital, northwest Ethiopia

BACKGROUND Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), is a serious infection in humans and animals. Ethiopia is one of the countries in Sub-Saharan Africa with the highest burden of TB. However, limited information is available on the genotypic characteristics of M. tuberculosis strains infecting humans. The objective of the present study was to characterize the mycobacterial species isolated from pulmonary TB patients using molecular typing. MATERIALS AND METHODS A cross-sectional study was conducted on 123 patients with smear-positive pulmonary TB, using Ziehl Neelsen staining and bacteriological culturing. Molecular characterizations of the mycobacterial isolates were performed using region of difference 9 (RD9) deletion typing and spoligotyping methods. RESULTS The proportion of culture positivity was 95.9% (118/123). All the 118 isolates were confirmed to be M. tuberculosis by polymerase chain reaction-based RD9 deletion typing. Further characterization of all isolates using spoligotyping resulted in the identification of 36 different spoligotype patterns. Out of these, 32 (88.9%) patterns have already been reported in the SpolDB database, whereas the remaining four (11.1%) patterns were new and not registered in the database. The isolates were further grouped into 17 clustered (99 isolates) and 19 nonclustered patterns. The most predominant spoligotypes were SIT25 and SIT53, consisting of 22 isolates and 14 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN SPOTCLUST showed that the dominant lineages identified in the present study were Euro-American and Central Asian genotypes consisting of 64 isolates and 37 isolates, respectively. CONCLUSION This study confirmed the presence of known M. tuberculosis strains and revealed new strains circulating in northwest Ethiopia and the distribution of the major phylogenetic families. It thus contributes to a better understanding of the genotypic profile of M. tuberculosis strains circulating in Ethiopia.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity

Significance Middle East respiratory syndrome (MERS) is a zoonotic disease of global health concern, and dromedary camels are the source of human infection. Although Africa has the largest number of dromedary camels, and MERS-coronavirus (MERS-CoV) is endemic in these camels, locally acquired zoonotic MERS is not reported from Africa. However, little is known of the genetic or phenotypic characterization of MERS-CoV from Africa. In this study we characterize MERS-CoV from Burkina Faso, Nigeria, Morocco, and Ethiopia. We demonstrate viral genetic and phenotypic differences in viruses from West Africa, which may be relevant to differences in zoonotic potential, highlighting the need for studies of MERS-CoV at the animal–human interface. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b. Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal–human interface.

Association of the level of IFN-γ produced by T cells in response to Mycobacterium tuberculosis-specific antigens with the size of skin test indurations among individuals with latent tuberculosis in a highly tuberculosis-endemic setting

There is growing evidence showing the potential of T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) for predicting the risk of progression of Mycobacterium tuberculosis (Mtb) infection, though there is little information from tuberculosis (TB)-endemic settings. In this study, we assessed the association between the level of IFN-γ produced by T cells in response to Mtb-specific antigens and the size of skin test indurations in 505 adult individuals who were screened for latent tuberculosis infection (LTBI) using the QuantiFERON-TB Gold In Tube (QFTGIT) assay and tuberculin skin test (TST). There was a strong positive correlation between the level of IFN-γ induced by the specific antigens and the diameter of the skin indurations (Spearmans rho = 0.6, P < 0.001). Body mass index and parasitic infection were not associated with the level of IFN-γ production or the TST reaction. In linear regression analysis, the size of the skin test indurations was significantly associated with the mean level of IFN-γ [coefficient, 0.65; 95% confidence interval (CI), 0.47 to 0.82, P < 0.001]. Similarly, results from logistic regression analysis demonstrated that individuals who had skin test indurations ≥ 10 mm were 6.82 times more likely than individuals who had skin test indurations < 10 mm to have high levels of IFN-γ (i.e. positive QFTGIT result) (adjusted odd ratio = 6.82; 95% CI, 3.67 to 12.69, P < 0.001). In conclusion, the results of this study could provide indirect evidence for the prognostic use of the QFTGIT assay for progression of Mtb infection, though prospective follow-up studies are needed to provide direct evidence.

Molecular typing of Mycobacterium tuberculosis complex isolated from pulmonary tuberculosis patients in central Ethiopia

BackgroundIdentification of the types of strains of Mycobacterium tuberculosis (M. tuberculosis) complex causing tuberculosis (TB) could contribute to TB control program of specific geographic region as well as it could add knowledge onto the existing literature on TB worldwide. The objective of the present study was to identify the species and strains of M. tuberculosis complex causing pulmonary tuberculosis in central Ethiopia.MethodsA health institution- based cross-sectional study was conducted on 338 smear positive TB cases visiting three hospitals between October 2012 and September 2013. Morning and spot sputum samples were collected before the starting of treatment regimens. Thus, a total of 338 pooled sputum samples collected from these cases. Samples were cultured on Löwenstein Jensen media and the isolates were identified by the region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping.ResultOf the total isolates 98.6% of the isolates were identified to be M. tuberculosis while the remaining 1.4% were identified as M. africanum. Further, typing of M. tuberculosis using spoligotyping lead to the identification of 90 different strains of M. tuberculosis. Of these strains, 32 were clustered consisting of more than one isolate while the remaining 58 strains were unique consisting of single isolate. Thus, 79.3% (223/281) of the isolates were found in the clustered while only 20.6% (58/281) of the strains were unique. Forty-five of the spolgotyping patterns were registeredin the SITVIT2 or SpolDB4 database in while the remaining 45 were notfound in the database and hence were orphan strains. The dominant strains were SIT53, SIT149, and SIT54, consisting of 43, 37 and 34 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN TB-Lineage showed that 86.8, 6.4, 5, 1.4% ofthe isolatesbelonged to the Euro-American lineage, East-African-Indian, Indo-oceanic and M. africanum, respectively.ConclusionThe identification of clustered and new strains using spolygotyping in present study does not give conclusive finding as spoligotyping has low discriminatory power. Thus, further identification of these isolates using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VENTR) and or whole genome sequencing (WGS) recommended.