Fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in Ethiopia.

BACKGROUND: Despite its low sensitivity, microscopy remains the main method for the diagnosis of pulmonary tuberculosis in most laboratories in Ethiopia. Few studies have evaluated the performance of light-emitting diode fluorescent microscopy (LED-FM) in bleach-concentrated smear-negative sputum specimens. OBJECTIVE: This study aimed to evaluate the diagnostic performance of LED-FM for smear-negative pulmonary tuberculosis in Ethiopia. METHODS: A total of 194 adult patients with a cough lasting for more than two weeks, and who had three direct smear-negative sputum tests for Mycobacterium tuberculosis by Ziehl-Neelsen light microscopy, were included. All direct Ziehl-Neelsen-stained smear-negative sputum samples were cultured and were also visualised by LED-FM. Smears for LED-FM were performed from bleach-concentrated sputum sediment. The diagnostic performance of the LED-FM was compared to the culture method (the reference standard). RESULTS: Of the 194 smear-negative sputum specimens analysed, 28 (14.4%) were culture-positive and 21 (10.8%) were LED-FM-positive for M. tuberculosis. However, only 11 of the 21 (52.4%) LED-FM-positive patients had a confirmed tuberculosis diagnosis by culture. Light-emitting diode fluorescence microscopy (FM) had a sensitivity of 39.3% (95% confidence interval: 21.2-57.4) and specificity of 93.9% (95% confidence interval: 90.4-97.6). Ten LED-FM-positive specimens were culture-negative, and all of these specimens had scanty grading (1-19 bacilli per 40 fields on LED-FM). CONCLUSION: This study showed that implementation of LED-FM on bleach pre-treated and concentrated sputum can significantly improve the diagnosis of smear-negative pulmonary tuberculosis. However, all scanty grade, positive smears by LED-FM need to be confirmed by reference culture method.

Nasopharyngeal Carriage and Antimicrobial Susceptibility Patterns of Streptococcus pneumoniae among Children under Five in Southwest Ethiopia.

Nasopharyngeal carriage of Streptococcus pneumoniae is found to play an important role in the development and transmission of pneumococcal diseases. In this study, we assessed the nasopharyngeal carriage, antimicrobial susceptibility patterns and associated risk factors of S. pneumoniae among children under five. A total of 361 children under five attending the outpatient department of Shanan Gibe Hospital in Jimma, Southwest Ethiopia were enrolled from June to September 2014. Nasopharyngeal specimens were collected using sterile plastic applicator rayon tipped swab and inoculated on tryptone soy agar supplemented with 5% sheep blood and 5 µg/mL gentamycin. Antimicrobial susceptibility testing was performed using the modified disk diffusion method. The overall prevalence of S. pneumoniae carriage was 43.8% (158/361) among children under five. Resistance to tetracycline, cotrimoxazole, penicillin, chloramphenicol and erythromycin was observed in 53.2% (84/158), 43.7% (69/158), 36.1% (57/158), 13.3% (21/158) and 8.9% (14/158) of isolates respectively. Multidrug resistance was seen in 17.7% (28/158) of isolates. In multivariate logistic regression analysis, children living with sibling(s) < 5 years old (adjusted odds ratio (AOR) = 1.798; 95% confidence interval (CI), 1.169-2.766) and malnutrition (AOR = 2.065; 95% CI, 1.239-3.443) were significantly associated with S. pneumoniae carriage. A high nasopharyngeal carriage of S. pneumoniae was observed among children under five in Southwest Ethiopia. There should be a strategy to prevent S. pneumoniae nasopharyngeal colonization and identify the appropriate antibiotic to the individual child.

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.

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.

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.

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

Fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in Ethiopia

Objective: This study aimed to evaluate the diagnostic performance of LED-FM for smear-negative pulmonary tuberculosis in Ethiopia.Methods: A total of 194 adult patients with a cough lasting for more than two weeks, and who had three direct smear-negative sputum tests for Mycobacterium tuberculosis by Ziehl-Neelsen light microscopy, were included. All direct Ziehl-Neelsen-stained smear-negative sputum samples were cultured and were also visualised by LED-FM. Smears for LED-FM were performed from bleach-concentrated sputum sediment. The diagnostic performance of the LED-FM was compared to the culture method (the reference standard).Results: Of the 194 smear-negative sputum specimens analysed, 28 (14.4%) were culture-positive and 21 (10.8%) were LED-FM-positive for M. tuberculosis. However, only 11 of the 21 (52.4%) LED-FM-positive patients had a confirmed tuberculosis diagnosis by culture. Light-emitting diode fluorescence microscopy (FM) had a sensitivity of 39.3% (95% confidence interval: 21.2­57.4) and specificity of 93.9% (95% confidence interval: 90.4­97.6). Ten LED-FM-positive specimens were culture-negative, and all of these specimens had scanty grading (1­19 bacilli per 40 fields on LED-FM).Conclusion: This study showed that implementation of LED-FM on bleach pre-treated and concentrated sputum can significantly improve the diagnosis of smear-negative pulmonary tuberculosis. However, all scanty grade, positive smears by LED-FM need to be confirmed by reference culture method