Type I interferon induced by TLR2-TLR4-MyD88-TRIF-IRF3 controls Mycobacterium abscessus subsp. abscessus persistence in murine macrophages via nitric oxide.

Mycobacterium abscessus (MAB) is an emerging, rapidly growing non-tuberculous Mycobacterium causing therapy-resistant pulmonary disease especially in patients with cystic fibrosis (CF). Smooth and rough colony type MAB can be isolated from infected patients whereby rough colony type MAB are more often associated with severe disease. Disease severity is also associated with an alternated type I interferon (IFN-I) response of the MAB-infected patients. However the relevance of this response for the outcome of MAB infection is still unknown. In this study, we analyzed the IFNß expression of murine macrophages infected with a MAB rough colony strain (MAB-R) isolated from a patient with progressive CF and compared it to macrophages infected with the MAB smooth colony type reference strain (MAB-S). We found that MAB-R infected macrophages expressed significantly more IFNß mRNA and protein than MAB-S infected macrophages. Higher IFNß induction by MAB-R was associated with higher TNF expression and intracellular killing while low IFNß induction was associated with lower TNF expression and persistence of MAB-S. IFNß induction was independent of the intracellular cGAS-STING recognition pathway. MAB appeared to be recognized extracellularly and induced IFNß expression via TLR2-TLR4-MyD88-TRIF-IRF3 dependent pathways. By using macrophages lacking the IFN-I receptor we demonstrate that MAB induced IFN-I response essentially contributed to restricting MAB-R and MAB-S infections by activating macrophage Nos2 expression and nitric oxide production. Thus IFN-I seem to influence the intrinsic ability of macrophages to control MAB infections. As MAB persists over long time periods in susceptible patients, our findings suggest that virulence of MAB strains is promoted by an insufficient IFN-I response of the host.

Diagnostic performance of fluorescent light-emitting diode microscopy for tuberculous lymphadenitis in a high-burden setting.

OBJECTIVE: Diagnosis of tuberculous lymphadenitis using fine-needle aspiration cytology is a simple and safe but low-specificity method, whereas conventional smear microscopy has variable sensitivity due to low bacterial load. We evaluated the diagnostic performance of fluorescent light-emitting diode (LED) microscopy on routinely collected fine-needle aspirates from tuberculous lymphadenitis presumptive cases. METHODS: Fine-needle aspirates were collected from patients clinically suspected of having tuberculous lymphadenitis as part of routine diagnosis. Smear preparation was performed from the aspirate and processed for cytology, conventional Ziehl-Neelsen and LED microscopy. The remaining aspirate was processed for culture on Lowenstein-Jensen media. Capilia TB-Neo test was used to differentiate M. tuberculosis complex from non-tuberculous mycobacteria. RESULT: A total of 144 tuberculous lymphadenitis presumptive cases were included. 66.7% (96/144) were positive for M. tuberculosis complex on culture. Only one isolate was identified as non-tuberculous mycobacteria. The detection rates of Ziehl-Neelsen and LED microscopy were 18.8% (27/144) and 34% (49/144), respectively. As compared to culture, sensitivity was 25.0% [95% CI: 16.3-33.7] for Ziehl-Neelsen microscopy and 45.8% [95% CI: 35.9-55.8] for LED microscopy. The specificity was 93.8% [95% CI: 86.9-100] for Ziehl-Neelsen microscopy and 89.6% [95% CI: 80.9-98.2] for LED microscopy. LED microscopy showed a statistically significant increase in sensitivity and similar specificity compared to Ziehl-Neelsen microscopy. Mean reading time of positive slides was 2.62 min/slide for Ziehl-Neelsen and 1.60 min/slide for LED microscopy. Cytology showed sensitivity of 82.3% and specificity of 54.2%. LED microscopy detected TB bacilli in 33.3% of cases cytologically classified as suppurative abscess. CONCLUSION: The LED microscopy for tuberculous lymphadenitis had significantly higher sensitivity and shorter screening time than Ziehl-Neelsen microscopy. Use of LED microscopy among cases classified as suppurative abscess on fine-needle aspirate cytology improves evidence-based diagnosis of presumptive tuberculous lymphadenitis cases. Moreover, LED microscopy could be considered as an alternative approach in settings where fine-needle aspirate cytology is impractical.

Drug resistance patterns of Mycobacterium tuberculosis complex and associated factors among retreatment cases around Jimma, Southwest Ethiopia.

BACKGROUND: Information on the pattern of drug resistant tuberculosis (TB) among re-treatment cases is crucial to develop appropriate control strategies. Therefore, we conducted this study to assess the drug resistance pattern of M. tuberculosis complex (MTBC) isolates and associated factors among re-treatment cases in Jimma area, Southwest Ethiopia. METHODS: Health facility-based cross-sectional study was conducted between March 2012 and April 2013 in Jimma area, Southwest Ethiopia. We included 79 re-treatment cases selected conveniently. Socio demographic and clinical data were collected using structured questionnaire. Sputum sample processing, mycobacterial culture, isolation and drug susceptibility testing (DST) were done at Mycobacteriology Research Centre (MRC) of Jimma University. All data were registered and entered in to SPSS version 20. Crude odds ratio (COR) and adjusted odds ratios (AOR) were calculated. P-values less than 0.05 were considered statistically significant. RESULTS: Seventy-nine re-treatment cases included in the study; 48 (60.8%) were males. Forty-seven (59.5%) study participants were from rural area with the mean age of 31.67 ± 10.02 SD. DST results were available for 70 MTBC isolates. Majority (58.6% (41/70)) isolates were resistant to at least one of the four first line drugs. The prevalence of multidrug-resistant TB (MDR-TB) was 31.4% (22/70). Place of residence (AOR = 3.44 (95 % CI: 1.12, 10.60), duration of illness (AOR = 3.00 (95 % CI: 1.17, 10.69) and frequency of prior TB therapy (AOR = 2.99, (95 % CI: 1.01, 8.86) were significant factors for any drug resistance. Moreover, history of treatment failure was found to be associated with MDR-TB (AOR = 3.43 (95 % CI: 1.14, 10.28). CONCLUSION: The overall prevalence of MDR-TB among re-treatment cases around Jimma was high. The rate of MDR-TB was higher in patients with the history of anti-TB treatment failure. Timely identification and referral of patients with the history of treatment failure for culture and DST need to be strengthened.

Bacteriological methods as add on tests to fine-needle aspiration cytology in diagnosis of tuberculous lymphadenitis: can they reduce the diagnostic dilemma?

BACKGROUND: The diagnostic accuracy of fine-needle aspiration (FNA) cytology for the diagnosis of tuberculous lymphadenitis (TBLN) is confounded by mimicking cytomorphologic disorders. The objective of this study was to determine whether supplementing FNA cytology with bacteriological methods improves the overall accuracy of TBLN diagnosis. METHODS: Two hundred presumptive TBLN cases were included in the study. FNA specimens were collected and examined for cytomorphologic changes, for acid-fast bacilli (AFB) by microscopy and for mycobacterial growth on culture. Culture was done using Lowenstein-Jensen (LJ) medium and mycobacteria growth indicator tube (BACTEC MGIT 960 TB detection system). Differentiation between M. tuberculosis complex (MTBc) and non-tuberculous mycobacteria (NTM) was done by using 500 µg/ml para-nitrobenzoic acid (PNB) susceptibility testing. RESULTS: Cytomorphology detected TBLN among 80% (160/200) of the presumptive cases. Culture results were available for 188 cases. Twelve samples were excluded due to contamination on both culture methods. Culture confirmed cases accounted for 78% (147/188) of which MTBc constituted 97.3% (143/147). Among presumptive cases, classified by FNA cytology as 'abscess', 11 were culture positive. Microscopy detected 31.3% (46/147) of culture confirmed mycobacterial lymphadenitis of which 11% (4/37) were diagnosed non-suggestive for tuberculosis (TB) by FNA cytology. Compared to culture (LJ & BACTEC MGIT 960) and AFB microscopy as composite gold standard, FNA cytology had a sensitivity of 88.4% and a specificity of 48.8%. The positive predictive value was 86.1% while the negative predictive value was 54.1%. The confirming power and the ROC curve area was 1.73 and 0.69, respectively. CONCLUSION: FNA cytology showed a relatively high sensitivity but a low specificity. Combining bacteriological methods with FNA cytology in an endemic region like Ethiopia improves the overall accuracy of the diagnosis of mycobacterial lymphadenitis, which in turn may lead to better patient management.

Relevance of inducible nitric oxide synthase for immune control of Mycobacterium avium subspecies paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD), an incurable chronic intestinal bowel disease in ruminants. JD occurs worldwide and causes enormous economic burden in dairy industry. Research on JD pathobiology is hampered by its complexity which cannot completely be mimicked by small animal models. As a model the mouse allows dissecting some pathogenicity features of MAP. However, for unknown reasons MAP exhibits reduced growth in granulomas of infected mice compared to other Mycobacterium avium subspecies. Here, we characterized immune reactions of MAP-infected C57BL/6 mice. After infection, mice appeared fully immunocompetent. A strong antigen-specific T cell response was elicited indicated by IFNγ production of splenic T cells re-stimulated with MAP antigens. Function of splenic dendritic cells and proliferation of adoptively transferred antigen-specific CD4+ T cells was unaltered. Isolated splenic myeloid cells from infected mice revealed that MAP resides in CD11b+ macrophages. Importantly, sorted CD11b+CD11c- cells expressed high level of type 2 nitric oxide synthase (NOS2) but only low levels of pro- and anti-inflammatory cytokines. Correspondingly, MAP-infected MAC2 expressing myeloid cells in spleen and liver granuloma displayed strong expression of NOS2. In livers of infected Nos2-/-mice higher bacterial loads, more granuloma and larger areas of tissue damage were observed 5 weeks post infection compared to wild type mice. In vitro, MAP was sensitive to NO released by a NO-donor. Thus, a strong T cell response and concomitant NOS2/NO activity appears to control MAP infection, but allows development of chronicity and pathogen persistence. A similar mechanism might explain persistence of MAP in ruminants.

Spoligotype-based population structure of Mycobacterium tuberculosis in the Jimma Zone, southwest Ethiopia.

BACKGROUND: To understand the population dynamics and propose more effective preventive strategies, defining the population structure of the circulating Mycobacterium tuberculosis strains is important. METHODS: A total of 177 M. tuberculosis complex isolates from pulmonary tuberculosis (TB) cases in southwest Ethiopia were genotyped by spoligotyping. Of the strains included in this study, 126 were pan-susceptible strains while the remaining 51 isolates were resistant to one or more first-line anti-TB drugs. The genotyping results were compared to the international spoligotyping (SITVIT) database of the Pasteur Institute of Guadeloupe and the newly revised publicly available international multi-marker database (SITVITWEB/SPOLDB4). An online tool Run TB-Lineage was also used to predict the major lineages using a conformal Bayesian network analysis. RESULTS: The spoligotyping of the 177 isolates resulted in 69 different spoligotype patterns of which 127 (71.8%) were clustered into 19 spoligoclusters (with clustering rate of 61.02%). Each cluster contains 2-29 isolates. Of the isolates with corresponding SIT in SITVIT/SDB4, the predominant strains identified were SIT 37 of the T3 subfamily with 29 isolates followed by SIT 53 of the T1 subfamily with 20 isolates. SIT 777 of the H4 subfamily and SIT 25 of the CAS1_DELHI subfamily each consisting of six isolates were identified. Eighty spoligotype patterns were orphan as they were not recorded in the SITVIT2/SPDB4 database. Further classification of the isolates on the basis of major lineages showed that 82.5% and 14.1% of the isolates belonged to Euro-American and East African Indian lineages, respectively, while 2.8% of the isolates belonged to Mycobacterium africanum and 0.6% to Indo-Oceanic. CONCLUSION: The ill-defined T and H clades were predominant around Jimma. The substantial number of orphans recorded in the study area warrants for additional studies with genotyping methods with better resolution and covering whole areas of southwest Ethiopia.

Presence of Infected Gr-1intCD11bhiCD11cint Monocytic Myeloid Derived Suppressor Cells Subverts T Cell Response and Is Associated With Impaired Dendritic Cell Function in Mycobacterium avium-Infected Mice.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

cGAS-STING-TBK1-IRF3/7 induced interferon-ß contributes to the clearing of non tuberculous mycobacterial infection in mice.

Type I interferons (IFN-I), such as IFN-α and IFN-ß are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-ß than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-ß in macrophages. Both bacteria induced IFN-ß via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-ß activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-ß impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.

Drug resistance-conferring mutations in Mycobacterium tuberculosis from pulmonary tuberculosis patients in Southwest Ethiopia.

OBJECTIVE/BACKGROUND: The nature and frequency of mutations in rifampicin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis isolates vary considerably according to geographic locations. However, information regarding specific mutational patterns in Ethiopia remains limited. METHODS: A cross-sectional prospective study was carried out among confirmed pulmonary tuberculosis cases in Southwest Ethiopia. Mutations associated with RIF and INH resistances were studied using GenoType MTBDRplus line probe assay in 112 M. tuberculosis isolates. Culture (MGIT960) and identification tests were performed at the Mycobacteriology Research Center of Jimma University, Jimma, Ethiopia. RESULTS: Mutations conferring resistance to INH, RIF, and multidrug resistance were detected in 36.6% (41/112), 30.4% (34/112), and 27.7% (31/112) of M. tuberculosis isolates respectively. Among 34 RIF-resistant isolates, 82.4% (28/34) had rpoB gene mutations at S531L, 2.9% (1/34) at H526D, and 14.7% (5/34) had mutations only at wild type probes. Of 41 INH-resistant strains, 87.8% (36/41) had mutations in the katG gene at Ser315Thr1 and 9.8% (4/41) had mutations in the inhA gene at C15T. Mutations in inhA promoter region were strongly associated with INH monoresistance. CONCLUSION: A high rate of drug resistance was commonly observed among failure cases. The most frequent gene mutations associated with the resistance to INH and RIF were observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. Further studies on mutations in different geographic regions using DNA sequencing techniques are warranted to improve the kit by including more specific mutation probes in the kit.

GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculous Lymphadenitis on Concentrated Fine Needle Aspirates in High Tuberculosis Burden Settings.

INTRODUCTION: The diagnosis of tuberculous lymphadenitis (TBL) remains challenging. The routinely used methods (cytology and smear microscopy) have sub-optimal sensitivity. Recently, WHO recommends GeneXpert to be used as the initial diagnostic test in patients suspected of having extra-pulmonary tuberculosis (EPTB). However, this was a conditional recommendation due to very low-quality evidence available and more studies are needed. In this study we evaluated the performance of Xpert for the diagnosis of TBL on concentrated fine needle aspirates (FNA) in Southwest Ethiopia. METHODS: FNA was collected from presumptive TBL cases. Two smears were prepared from each aspirate and processed for cytology and conventional microscopy. The remaining aspirate was treated with N-acetyl-L-cysteine-NaOH and centrifuged for 15minutes at 3000g. The concentrated sediment was used for culture and Xpert test. Capilia TB-Neo test was used to differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM). Composite bacteriological methods (culture and/or smear microscopy) were considered as a reference standard. RESULT: Out of 143 enrolled suspects, 64.3% (92/143) were confirmed TBL cases by the composite reference standard (CRS). Xpert detected M. tuberculosis complex (MTBC) in 60.1% (86/143) of the presumptive TBL cases. The sensitivity of Xpert compared to CRS was 87.8% [95% CI: 81.0-94.5] and specificity 91.1% [95% CI: 82.8-99.4]. The sensitivity was 27.8% for smear microscopy and 80% for cytology compared to CRS. Cytology showed the lowest specificity (57.8%). Xpert was positive in 4 out of 45 culture- and smear-negative cases. Among 47 cytomorphologically non-TBL cases, 15 were positive on Xpert. More than half of Xpert-positive cases were in the range of very low cut-off threshold values (28

Concentration of lymph node aspirate improves the sensitivity of acid fast smear microscopy for the diagnosis of tuberculous lymphadenitis in Jimma, southwest Ethiopia.

BACKGROUND: Tuberculous lymphadenitis (TBLN) is the most common form of extrapulmonary tuberculosis. The cytomorphological features of lymph node smears have reduced specificity for the diagnosis of tuberculosis. The diagnosis of TBLN with direct smear microscopy lacks sensitivity due to the limited number of bacilli in lymph node aspirate. Therefore, we aimed to assess whether the concentration of lymph node aspirate improves the sensitivity of acid fast smear microscopy for the diagnosis of tuberculous lymphadenitis. METHODS: A cross-sectional comparative study was conducted on 200 patients clinically suspected for tuberculous lymphadenitis in Jimma, Ethiopia. Lymph node aspirate was collected. The first two drops were used for cytomorphological study and direct acid fast staining. The remaining aspirate was treated with N-acetyl-L-cysteine (NALC) and concentrated by centrifugation at 3000 g for 15 minutes. The sediment was used for acid fast staining and culture. Differentiation of M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) was done by para-nitrobenzoic acid susceptibility test. RESULT: Complete data were available for 187 study subjects. 68% (127/187) were positive for M. tuberculosis on culture. Four isolates, 2.1% (4/187), were identified as NTM. The detection rate of direct smear microscopy was 25.1% and that of the concentration method 49.7%. Cytomorphologically, 79.7% of cases were classified as TBLN. The sensitivity of direct smear microscopy was 34.6%, for concentrated smear microscopy 66.1%, and for cytomorphology 89.8%. Two AFB positive cases on concentration method were non-tuberculosis mycobacteria (NTM). The concentration method yielded a positive result from seven cases diagnosed as suppurative abscess by cytology. Both for the direct and concentration methods the highest rate of AFB positivity was observed in smears showing caseous necrosis alone. Smear positivity rate decreased with the appearance of epithelioid cell aggregates. CONCLUSION: The concentration of lymph node aspirates for acid fast smear microscopy had significantly higher sensitivity than direct microscopy.

Relatively low primary drug resistant tuberculosis in southwestern Ethiopia.

BACKGROUND: The prevalence of drug resistant tuberculosis (TB) in Ethiopia in general, and Jimma area in particular, is not well documented. We conducted a study at Jimma University specialized hospital in southwest Ethiopia among new cases of smear positive TB patients to determine the pattern of resistance to first-line drugs. METHODS: A health institution based cross sectional study was conducted from November 2010 to September 2011. Any newly diagnosed smear positive TB patient 18 years and above was included in the study. Demographic and related data were collected by trained personnel using a pretested structured questionnaire. Mycobacterial drug susceptibility testing (DST) to the first line drugs isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STM) was performed on cultures using the indirect proportion method. M. tuberculosis complex (MTBC) was identified with the Capilia TB-Neo test. RESULTS: 136 patients were enrolled in the study. Resistance to at least one drug was identified in 18.4%. The highest prevalence of resistance to any drug was identified against INH (13.2%) followed by STM (8.1%). There was no statistically significant difference in the proportion of any resistance by sex, age, HIV status and history of being imprisoned. The highest mono resistance was observed against INH (7.4%). Mono resistance to streptomycin was associated with HIV infection (crude OR 15.63, 95%CI: 1.31, 187). Multidrug-resistance TB (MDR-TB) was observed in two patients (1.5%). CONCLUSION: Resistance to at least one drug was 18.4% (INH-13.2% and STM-8.1%). STM resistance was associated with HIV positivity. There was relatively low prevalence of MDR-TB yet INH resistance was common around Jimma. The capacity of laboratories for TB culture and DST should be strengthened, in order to correctly manage TB patients and avoid amplification of drug resistance.