Mulualem Tadesse

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<Ct<38). Resistance to rifampicin was identified in 4.7% (4/86) of Xpert-positive cases. Conclusion Xpert test showed a high sensitivity and specificity for the diagnosis of TBL on concentrated FNA samples. In addition, Xpert offered rapid detection of rifampicin-resistant M. tuberculosis strains from lymph node aspirates.

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

BackgroundThe 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.MethodsTwo 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.ResultsCytomorphology 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.ConclusionFNA 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.

Increased detection of smear-negative pulmonary tuberculosis by GeneXpert MTB/RIF® assay after bleach concentration.

Objective/background: The GeneXpert MTB/RIF assay (Xpert) was endorsed as the initial diagnostic tool in people suspected of human immunodeficiency virus-associated or drug-resistant tuberculosis (TB). However, information regarding the performance of Xpert for diagnosing smear-negative TB in high burden settings remains limited. We evaluated the diagnostic accuracy of Xpert and the impact of bleach concentration on the performance of Xpert using smear-negative sputum samples from human immunodeficiency virus-negative patients. Methods: One spot and one morning smear-negative sputum samples per patient were examined using Xpert and culture at the Mycobacteriology Research Center of Jimma University, Ethiopia. The sputum culture on both Löwenstein–Jensen and/or Mycobacteria Growth Indicator Tube was the gold-standard. Results: Of 185 smear-negative presumptive pulmonary TB cases, 19 (10.3%) had culture-proven TB. The sensitivity of Xpert on spot and morning sputum was similar (63.2%). Testing two specimens per patient insignificantly increased the sensitivity of Xpert. Bleach concentration and pelleting improved the sensitivity of Xpert over unprocessed sputum in paired samples (73.8% vs. 63.2%) without affecting the specificity (95%). Bleach concentration and pelleting allowed an additional seven cases of TB (missed on the first and second direct Xperts) to be detected, five of which were from culture-negative cases. Conclusion: Testing of a single sputum sample by Xpert can reach reasonable sensitivity and results would be available on the same day, avoiding loss of patients and treatment delay. The sensitivity of Xpert was improved after bleach concentration and pelleting, although its added value needs further study on a larger scale.

The predominance of Ethiopian specific Mycobacterium tuberculosis families and minimal contribution of Mycobacterium bovis in tuberculous lymphadenitis patients in Southwest Ethiopia

BACKGROUND Ethiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages responsible for TBLN in Southwest Ethiopia. METHODS A total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) (n=167) and qPCR-based single nucleotide polymorphism (n=38). Isolates were classified into main phylogenetic lineages and families by using the reference strain collections and identification tools available at MIRU-VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF. RESULTS The majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2 (n=44), Ethiopia_3 (n=34) and Ethiopia_H37Rv-like (n=30). Other sub-lineages included URAL (n=18), S (n=17), Uganda I (n=16), LAM (n=13), X (n=5), TUR (n=5), Uganda II (n=4) and unknown (n=19). Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2 (Beijing) were represented by 3 and 1 isolates respectively. M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance. CONCLUSIONS In TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The contribution of M. bovis in TBLN infection is minimal.

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.

Mother to Child HIV Transmission and Its Predictors among HIV-Exposed Infants: A Retrospective Follow-Up Study in Southwest Ethiopia

Despite the marked progress in coverage of prevention of mother to child HIV transmission (PMTCT) programs, mother to child HIV transmission (MTCT) rate is not well documented in Southwest Ethiopia. A retrospective follow up study was carried at Jimma University Specialized Hospital PMTCT clinic to quantify MTCT rate and its predictors among HIV-exposed infants. Data were extracted from medical records of HIV-infected women and exposed infants between September 2010 and December 2012. Univariate and multivariate logistic regression analyses were carried out to identify potential factors predicting MTCT. A total of 146 infants born to HIV-infected mothers were included in the analysis. Out of 146 infants, 25 (17%, 95% CI: 11%-23.2%) were HIV positive. In the adjusted multivariate logistic regression analysis, mothers being on late AIDS stage (AOR=5.8; 95% CI: 1.6-16.5), absence of maternal PMTCT interventions (AOR=4.9; 95% CI: 1.4-16.5), home delivery (AOR=8.1; 95% CI: 2.1-31.9) and mixed infant feeding (AOR=5.6; 95% CI: 1.4-41.2) were independently associated with MTCT. We documented a high rate of MTCT among exposed infants in Southwest Ethiopia. All pregnant HIV positive mothers should be enrolled in PMTCT programs at earlier stage and exclusive breast feeding should be encouraged so as to decrease MTCT.

Survival Analysis of Loss to Follow-Up Treatment among Tuberculosis Patients at Jimma University Specialized Hospital, Jimma, Southwest Ethiopia

Background. Tuberculosis (TB) patients who do not complete treatment pose a potential public health risk through disease reactivation, increased transmission, and development of drug-resistance. This study is aimed at analyzing the time to loss to follow-up treatment and risk factors among TB patients. Methods. This was a retrospective cohort study based on record review of 510 TB patients enrolled in Jimma University Specialized Hospital. The Cox’s proportional hazard model and Kaplan-Meier curves were used to model the outcome of interest. Loss to follow-up was used as an outcome measure. Results. Out of 510 TB patients, 69 (13.5%) were lost to follow-up (LTFU) treatment. The median times of survival starting from the date of treatment initiation were 5.7 months. The majority of LTFU patients interrupted treatment during continuation phase. Treatment LTFU has an association with HIV status, weight, and residence. However, living in the rural area has a cause for LTFU patients on multivariate analysis (HR 4.4, 95% CI 1.58–12.19). Conclusions. High rate of LTFU was observed among TB patients in Southwest Ethiopia. Treatment LTFU was more frequently observed among patients who came from rural areas. This underlines the need for distributing TB treatment to the rural area.

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.

Xpert MTB/RIF for rapid detection of rifampicin-resistant Mycobacterium tuberculosis from pulmonary tuberculosis patients in Southwest Ethiopia.

Objective/background: Accurate and rapid detection of drug-resistant strains of tuberculosis (TB) is critical for early initiation of treatment and for limiting the transmission of drug-resistant TB. Here, we investigated the accuracy of Xpert MTB/RIF for detection of rifampicin (RIF) resistance, and whether this detection predicts the presence of multidrug resistant (MDR) TB in Southwest Ethiopia. Methods: Smear- or culture-positive sputa obtained from TB patients with increased suspicion of drug resistance were included in this study. GenoType MTBDRplus line-probe assays (LPAs) and Xpert MTB/RIF tests were performed on smear-positive sputum specimens and on cultured isolates for smear-negative specimens. We performed routine drug-susceptibility testing using LPA as the reference standard for confirmation of RIF and isoniazid (INH) resistance. Results: First-line drug-susceptibility results were available for 67 Mycobacterium tuberculosis complex-positive sputum specimens using the LPA test, with our preliminary results indicating that 30% (20/67) were MDR-TB, 3% (2/67) were RIF monoresistant, 6% (4/67) were INH monoresistant, and 61% (41/67) were susceptible to both RIF and INH. Relative to routine RIF-susceptibility testing (LPA), Xpert MTB/RIF detected all RIF resistance correctly, with 100% sensitivity and 97.8% specificity and a positive-predictive value of 95.7%. Of the 23 RIF-resistant strains according to Xpert MTB/RIF, 87% (20/23) were resistant to both RIF and INH (MDR), 8.7% (2/23) were RIF monoresistant, and 4.3% (1/23) were sensitive to RIF according to the LPA test. A high proportion of RIF resistance was documented among patients previously categorized as failure cases (50%, 10/20), followed by relapse cases (31.6%, 6/19) and defaulters (28.6%, 2/7). Conclusion: Xpert MTB/RIF was highly effective at identifying RIF-resistant strains in smear- or culture-positive samples. RIF resistance based on Xpert MTB/RIF results could be used to estimate MDR and allow rapid initiation of MDR-TB treatment in regions with high levels of drug-resistant TB.

Risk Factors for Mortality among Adult HIV/AIDS Patients Following Antiretroviral Therapy in Southwestern Ethiopia: An Assessment through Survival Models

Introduction: Efforts have been made to reduce HIV/AIDS-related mortality by delivering antiretroviral therapy (ART) treatment. However, HIV patients in resource-poor settings are still dying, even if they are on ART treatment. This study aimed to explore the factors associated with HIV/AIDS-related mortality in Southwestern Ethiopia. Method: A non-concurrent retrospective cohort study which collected data from the clinical records of adult HIV/AIDS patients, who initiated ART treatment and were followed between January 2006 and December 2010, was conducted, to explore the factors associated with HIV/AIDS-related mortality at Jimma University Specialized Hospital (JUSH). Survival times (i.e., the time from the onset of ART treatment to the death or censoring) and different characteristics of patients were retrospectively examined. A best-fit model was chosen for the survival data, after the comparison between native semi-parametric Cox regression and parametric survival models (i.e., exponential, Weibull, and log-logistic). Result: A total of 456 HIV patients were included in the study, mostly females (312, 68.4%), with a median age of 30 years (inter-quartile range (IQR): 23–37 years). Estimated follow-up until December 2010 accounted for 1245 person-years at risk (PYAR) and resulted in 66 (14.5%) deaths and 390 censored individuals, representing a median survival time of 34.0 months ( IQR: 22.8–42.0 months). The overall mortality rate was 5.3/100 PYAR: 6.5/100 PYAR for males and 4.8/100 PYAR for females. The Weibull survival model was the best model for fitting the data (lowest AIC). The main factors associated with mortality were: baseline age (>35 years old, AHR = 3.8, 95% CI: 1.6–9.1), baseline weight (AHR = 0.93, 95% CI: 0.90–0.97), baseline WHO stage IV (AHR = 6.2, 95% CI: 2.2–14.2), and low adherence to ART treatment (AHR = 4.2, 95% CI: 2.5–7.1). Conclusion: An effective reduction in HIV/AIDS mortality could be achieved through timely ART treatment onset and maintaining high levels of treatment adherence.