Ogawa Medium Research Articles

Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

Evaluation of the Use of Distilled Water as a Sodium Hydroxide Wash in Antimicrobial Cultures

Background: Respiratory specimens subjected to mycobacterial detection were initially pretreated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) to remove the mucus and normal flora.Next, they were washed and neutralized with a phosphate-buffered solution (PBS). The effectiveness of distilled water (DW) compared to PBS as a washing neutralizer during the identification of mycobacteria was evaluated in this study. Methods: We analyzed the results of the mycobacterial test conducted at a general hospital in Gwangju from October 2016 to September 2018. PBS and DW were used as a respiratory sample-washing agent for one year each. Results:The positive culture rate for the culture of mycobacteria was 12.7% (1,843/14,532) and 14.7% (2,095/14,291), when PBS and DW were used, respectively. The recovery rate of the mycobacteria growth indicator tubes (MGIT) and the separation rates of the Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) showed no significant change. However, in the 2% Ogawa medium, as the NTM culture increased from 47.4% (399/841) to 56.1% (630/1,122),the recovery rate increased from 45.6% (841/1,843) to 53.6% (1,122/2,095). The MGIT contamination rate decreased from 6.5% to 4.1%. Conclusion: DW as a washing agent for NALC-NaOH increased the recovery rate of Ogawa medium and reduced the contamination rate of MGIT. Therefore, the use of DW instead of PBS as a washing neutralizer during the identification of mycobacteria might be useful.

The fourth national tuberculosis prevalence survey in Myanmar.

Tuberculosis (TB) remains a significant cause of morbidity and mortality in Myanmar. The fourth National TB Prevalence Survey was conducted in 2017-2018 to determine the actual burden of TB not only at the national level but also for three subnational strata (the states, regions other than Yangon, and the Yangon region) and develop a more efficacious country strategy on TB care and control. One hundred and thirty eight clusters were selected by population proportionate sampling. Adult (≥15 years of age) residents having lived for 2 weeks or more in the households of the selected clusters were invited to participate in the survey. The survey participants were screened for TB by a questionnaire and digital chest X-ray (CXR) after providing written informed consent. Individuals with a positive symptom screen and/or chest X-ray suggestive of TB were asked to provide sputum samples to test for Mycobacterium tuberculosis (Mtb) by Ziehl-Neelsen direct light microscopy, Xpert MTB/RIF Ultra (Xpert), and culture (Ogawa media). Bacteriologically confirmed TB cases were defined by an expert panel. Of 75 676 eligible residents, 66 480 (88%) participated, and 10 082 (15%) screened positive for TB. Among these, 322 participants were defined as bacteriologically confirmed TB cases. Cough lasting for two weeks or longer, one of the criteria used for screening for symptoms, could detect only 14% (45/322) of the study cases. The estimated prevalence of bacteriologically confirmed adult pulmonary TB was 468 (95% CI: 391-546) per 100,000. The prevalence was much higher among males, the older age group, urban Yangon and remote villages. In-depth interview with the participants on TB treatment showed that none of them was diagnosed in a TB health centre (primary care facilities). The prevalence of TB in Myanmar is still high due to challenges such as uncontrolled urbanization, an ageing population, migration, and poor access to health facilities in remote areas. New screening and diagnostic tools might help to detect more TB patients. There is a need to lay greater emphasis on multisectoral approaches, decentralization and the integration of basic TB services into primary care facilities.

Substantial Improvement in Nontuberculous Mycobacterial Identification Using ASTA MicroIDSys Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with an Upgraded Database.

Identifying Mycobacterium using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging. We evaluated the performance of MALDI-TOF MS in identifying nontuberculous mycobacteria (NTM) using the ASTA MicroIDSys system (ASTA Inc., Suwon, Korea) with the MycoDB v1.95s and upgraded MycoDB v2.0-beta databases. We tested 124 NTM isolates collected from Ogawa medium at Severance Hospital, Seoul, Korea, between January and April 2019. MicroIDSys scores were categorized into three groups: ≥140, reliable identification; 130–139, ambiguous identification; and <130, invalid identification. To validate the results, we used the reverse blot hybridization assay (Molecutech REBA MycoID, YD Diagnostics Corp., Korea). Initial analysis using MycoDB v1.95s resulted in 26.6% (33/124) reliable, 43.5% (54/124) ambiguous, and 29.8% (37/124) invalid identifications. Re-analysis using the upgraded MycoDB v2.0-beta database resulted in 94.4% (117/124) reliable, 4.0% (5/124) ambiguous, and 1.6% invalid (2/124) identifications. The percentage of reliable identifications that matched with the reference increased from 26.6% (33/124) with MycoDB v1.95s to 93.5% (116/124) with MycoDB v2.0-beta. The upgraded databases enable substantially improved NTM identification through deep learning in the inference algorithm and by considering more axes in the correlation analysis. MALDI-TOF MS using the upgraded database unambiguously identified most NTM species. Our study lays a foundation for applying MALDI-TOF MS for the simple and rapid identification of NTM isolated from solid media.

Multicenter evaluation of AYC.2.2 agar for the isolation of mycobacteria from clinical samples

PurposeThe aim of this multicenter study is to evaluate AYC.2.2 agar for the isolation of mycobacteria from clinical samples. MethodsTotally 5559 media were tested in 7 centers. AYC.2.2 agar media for the study were prepared by C1 and sent to other centers under appropriate conditions. Other media except AYC.2.2 agar were purchased commercially.The media were subjected to routine laboratory operations in the center where they were sent. After the samples received for routine processing (in all centers, samples were processed with the same method (NALC-NaOH)), they were cultivated on routine media and AYC.2.2 agar afterward. ResultsC1: Average growth time was determined as 12.74±3.74 days with MGIT 960 system; 24.42±4.75 days with LJ and 24.37±4.96 days with AYC.2.2 agar. C2: Average growth time was determined as 18.25±9.32 days with TK-Medium, 28.73±7.44 days with LJ, and 31.72±6.35 days with AYC.2.2 agar. C3: Average growth time was determined as 20.48±7.24 days with Ogawa medium, 20.74±7.12 days with LJ, and 20.26±7.43 days with AYC.2.2 agar. C4: Average growth time was determined as 15.27±6.37 days with MGIT 960 system, 22.14±9.1 days with LJ, and 22±8.45 days with AYC.2.2 agar. C5: Average growth time was determined as 13±4.24 days with MGIT 960 system, 32.16±6.23 days with LJ, and 33±5.73 days with AYC.2.2 agar. C6: Average growth time was determined as 9±3.11 days with MGIT 960 system, 18.68±5.32 days with LJ, and 18.34±4.63 days AYC.2.2 agar. C7: Average growth time was determined as 14.74±7.65 with MGIT 960 system, 26.01±8.21 days with LJ, and 26.24±7.88 days with AYC.2.2 agar. ConclusionsIn conclusion, similar results were obtained with LJ and Ogawa media and AYC.2.2 agar. Furthermore, more studies should be conducted for isolation of M. tuberculosis and performing antibiotic susceptibility tests using AYC.2.2 agar before it can be used as a routine media in the laboratories.

Mycobacteriosis in cultured koi carp Cyprinus carpio caused by Mycobacterium paragordonae and two Mycolicibacterium spp.

Mycobacteriosis has caused economic losses in fish production of koi carp in Japan since 1981. In this study, we performed histopathological analyses, bacterial identification and challenge test for characterization of mycobacteriosis in koi carp. Spontaneously diseased koi carp externally showing lethargy and emaciation with pin-head body shape were obtained from culture ponds in Niigata, Japan. We isolated bacteria on 1% Ogawa medium at 30 °C and obtained 14 mycobacterial isolates. Chromogenicity, growth rate and biochemical properties were analyzed using the bacterial isolates. Phylogenetic analyses using 16S rRNA and RpoB-HSP65 concatenated sequence were also performed with Neighbor-Joining method. To fulfil the Koch's postulates, common carp were challenged with five representative isolates. Among the visceral organs, the peritoneal fibroplasia accompanied many macrophage aggregations were observed in the diseased fish. Granulomas consisted of central macrophages with melanin pigment were found in the trunk kidney of the diseased fish. Ziehl-Neelsen positive bacilli were detected in mesentery of the posterior chamber of the swim bladder and in the trunk kidney. The bacterial isolates were classified into three groups based on the colony morphology: yellow/smooth colonies (Y/S-isolates), white/rough colonies (W/R-isolate), and white/smooth colonies (W/S-isolates). The results in biochemical tests and phylogenetic analyses revealed that Y/S-isolates were identified as M. paragordonae. W/R- and W/S-isolates were classified into rapid-growing mycobacterium (Runyon IV) based on the morphological and biochemical characteristics. Although W/R- and W/S-isolates were not identified in species level, the former was closely related to Mycolicibacterium peregrinum and the latter was classified into Mycolicibacterium smegmatis clade in the phylogenetic tree for 16S rRNA. The cumulative mortality of fish challenged with Y/S-, W/R- and W/S-isolates were 30%–50% in 100 days post-infection and the dead fish showed the typical symptoms. These data revealed that mycobacteriosis of koi carp cultured in Japan was caused by several mycobacterial species including M. paragordonae and two unidentified Mycolicibacterium spp.

FREEZE-DRIED, VIABLE, DISPERSED AND STABLE FORMULATION OF THE “LIQUID INTRA-VESICAL IMMUNOTHERAPY BCG MOREAU® FINLAY”

The current study designed to obtain, execute and test a freeze-drying protocol for the preparation and a better storage conservation of the live attenuate Mycobacterium bovis BCG-Moreau-Finlay, fundamental element for the immunotherapy against bladder carcinoma, passed successfully all trials. The new lyophilized formulation also reduces the in vivo toxicity compared to present BCG liquid preparation by means of lowering dose needed for the treatment. The second re-seeding of the Moreau Rio de Janeiro sub-strain of Mycobacterium bovis BCG (pBCG Moreau); was sub-cultured in the Sauton Synthetic Medium (SSMII), previously maintained in Seed Lot Systems and harvested during the logarithmic growth phase. Basic Bacterial Suspension (BBS) was then prepared, homogenized and stabilized by freeze-drying in vehicle A, compare to vehicle B. By means of Thin Layer Chromatography (TLC) was realized the comparative quality control of the (BBS) preparation before and after lyophilisation procedure of the final product. A high (%) colony count measure in Ogawa medium and average survival of 50.4% (Heat stability over the time). Additional values as (0.75 – 0.95 %) were obtained for the ranks of dispersion using Zieln Nielsen and the Dubos Methods. The integrity/vitality oscillated from 67.8 to 68.5 (%) measured by oxygen consumption, the residual humidity indicators from 73 to 74 (%)[BBS] and the accepts organoleptic properties designed all aftermaths concern the in vitro assays required by World Health Organization (WHO) standards for BCG intra-Vesical final product. A freeze-dried “Onco BCG Moreau - Finlay” formulation was successfully produced and has many “in vitro and in vivo test” practical and theoretical advantages when it compared to the current liquid preparation, such as the best preserving BCG biological properties and reducing the dose of product required for treatment.

Performance of Xpert MTB/RIF for the Diagnosis of Extrapulmonary Tuberculosis.

Mycobacterial burden is low in extrapulmonary specimens, making diagnosis and treatment difficult. Xpert MTB/RIF is a real-time PCR assay for the detection of Mycobacterium tuberculosis and rifampin resistance. This study evaluated the performance of the Xpert MTB/RIF assay in extrapulmonary specimens. Acid-Fast Bacilli (AFB) smear, culture, and Xpert MTB/RIF were performed on extrapulmonary specimens. Mycobacterial culture was performed on BACTEC MGIT liquid for 6 weeks and 2% Ogawa medium for 8 weeks. Overall sensitivity and specificity of Xpert MTB/RIF was estimated using culture as a gold standard. Xpert MTB/RIF sensitivity and cycle-threshold (Ct) values according to AFB smear grade were evaluated. The sensitivity, specificity, and concordance of rifampin resistance compared to the phenotypic drug sensitivity test were evaluated. A total of 1,289 specimens were included in the study. The overall sensitivity and specificity of the Xpert MTB/RIF assay were 59.4% (41/69, 95% CI 46.9 - 70.9%) and 99.3% (1,212/1,220, 95% CI 98.7 - 99.7), respectively. Positive predictive value of Xpert MTB/RIF was 83.7% (41/49, 95% CI 69.8 - 92.2) and negative predictive value was 97.7% (1,212/1,240, 95% CI 96.7 - 98.5%). Xpert MTB/RIF assay sensitivity significantly increased with increases in AFB smear grade (p < 0.001). AFB smear grades and Xpert MTB/RIF Ct values were negatively correlated. Rifampin resistance results of Xpert MTB/RIF and culture showed a concordance rate of 97.2%. The Xpert MTB/RIF assay could be used to replace the AFB smear for the diagnosis of extrapulmonary tuberculosis, and has high specificity for the detection of rifampin resistance.

ISOLATION AND ANTIMICROBIAL SUSCEPTIBILITIES OF NONTUBERCULOUS MYCOBACTERIA FROM WILDLIFE IN JAPAN.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens of humans and animals and are transmitted among the environment, wildlife, livestock, and humans. The aim of this study was to investigate the rate of isolation and antimicrobial susceptibility of NTM in wildlife. In total, 178 samples of feces (n=131) and tissues (n=47) were collected from 11 wildlife species in Gifu Prefecture and Mie Prefecture, Japan, between June 2016 and October 2018. We isolated NTM from 15.3% (20/ 131) of fecal samples using Ogawa medium, and isolates were identified by sequencing the rpoB and hsp65 genes. The rpoB sequences were compared with those from other strains of human and environmental origin. The NTM isolates were obtained from sika deer (Cervus nippon), wild boar (Sus scrofa), Japanese monkey (Macaca fuscata), raccoon dog (Nyctereutes procyonoides), masked palm civet (Paguma larvata), and Japanese weasel (Mustela itatsi) and were classified as rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). The 12 RGM identified were Mycolicibacterium peregrinum (n=5), Mycolicibacterium fortuitum (n=3), Mycolicibacterium septicum (n=3), and Mycolicibacterium thermoresistibile (n=1), and the eight SGM were Mycobacterium paraense (n=4), Mycolicibacter arupensis (n=2), Mycolicibacter virginiensis (n=1), and Mycobacterium nebraskense (n=1). The NTM from wildlife showed ≥99% similarity with strains from different sources including humans. The RGM were susceptible to the antimicrobial agents tested except for M. fortuitum, which was resistant to azithromycin and clarithromycin. The SGM showed multiple drug resistance qualities but were susceptible to amikacin, clarithromycin, and rifabutin. These results indicate that wildlife may be reservoir hosts of NTM in Japan. The presence of antimicrobial-resistant NTM in wildlife suggests that the trends of NTM antimicrobial susceptibility in wildlife should be monitored.

Prevalence and antimicrobial susceptibility of pulmonary tuberculosis infections among the outdoor patients in Tertiary Care Hospitals of Quetta city, Balochistan

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, responsible for killing millions of people throughout the world. This study was designed to evaluate the prevalence and antimicrobial susceptibility of pulmonary TB infections among the outdoor patients in Tertiary Care Hospitals of Quetta city. A total of 250 TB suspects were examined through Ziehl–Neelsen staining and culturing on Lowenstein–Jenson and modified Ogawa medium. Sociodemographical data of the patients were collected using prestructured questionnaire. Most of the suspects in this study were females (64%) with clinical signs and symptoms of TB. Furthermore, 35–50-year age group was most affected, with percentage prevalence of 51%, as compared with 15–35-year age group. Most patients were uneducated (78.4%) and with poor socioeconomic status (74.4%). Ten sputum smear samples were acid fast bacilli (AFB) positive 10/250 (4%) on Ziehl–Neelsen staining. All the sputum samples were inoculated (direct method) on solid Lowenstein–Jenson and modified Ogawa medium, and 9.2% and 8% of the samples, respectively, were positive. However, on drug susceptibility testing, no mycobacterium tuberculosis (MTB)-resistant strains were detected.

BIAKAN METODE TETRAZOLIUM MICROPLATE ASSAY TERKAIT DAHAK PASIEN TERDUGA TUBERKULOSIS PARU

The definitive diagnosis of pulmonary tuberculosis is the discovery of Mycobacterium tuberculosis from sputum culture, but theconventional culture methods using Ogawa media require between 3−10 weeks detection time. Therefore it is needed a prompt diagnostictools to shorten the detection time. Tetrazolium microplate assay (TEMA) that used tetrazolium bromide as a growth indicator also usemitochondrial dehydrogenate enzymes in the mitochondria of living M. tuberculosis may reduce yellow tetrazolium bromide into purpleformazan crystals. The aim of this study was to know the validity and speed of time detection of M. tuberculosis growth by analyzingit. This study was carried out from November 2012 up to February 2013, which obtained 105 subjects conducted in the Departmentof Clinical Pathology at Dr. Hasan Sadikin Hospital with a cross sectional study design. The subjects consisting of sputum sample frompatients who suspected pulmonary TB which is examined for culture of M. tuberculosis with TEMA method using Ogawa media. Statisticalanalysis was used a 2í—2 table to test the validity and Mann Whitney test for the differences in growth detection time. The validity testof TEMA method got the sensitivity of 90.4% and specificity of 96.2%. The detection time of M. tuberculosis growth in TEMA methodswas found fastest in the third day while from the Ogawa media cultur was found on the 13th day with the M. tuberculosis growth mediausing TEMA methods detected in 12 days. While for those cultured on Ogawa's media the mean duration is 22 days (p&lt;0.001). Basedon this study, can be concluded the examination of M. tuberculosis culture from sputum patient suffer of pulmonary TB with with TEMAmethod has given high validity and faster in the time detection for the diagnosis of pulmonary TB.

Case of Mycobacterium haemophilum misdiagnosed as Mycobacterium intracellulare due to one base insertion in the bacterial genome

Mycobacterium haemophilum is a slow-growing, non-tuberculous mycobacteria that causes cutaneous infection. We describe a case of cutaneous infection in a 68-year-old Japanese man with polymyositis. This was caused by M. haemophilum harboring one base insertion in gene sequence. At first, the causal microorganism was misidentified as M. intracellulare by COBAS® TaqMan® MAI test. However, poor growth on Ogawa media and growth enhancement on 7H11C agar around a hemin-containing disk prompted us to reinvestigate the causal microorganisms, which were revealed to be M. haemophilum. Amplified polymerase chain reaction products were sequenced, and the 16S rRNA gene, rpoB, hsp65 and internal transcribed spacer region sequences showed a 100%, 100%, 99.66% and 99.7% match, respectively, with the corresponding regions of M. haemophilum, but it harbored a novel gene sequence in hsp65. The sequences determined by gene analysis of the M. haemophilum strain were deposited into the International Nucleotide Sequence Database. Although numerous cases of M. haemophilum infection have been reported in other countries, only six cases have been reported in Japan to date. It could be possible that this novel mutation lead to misdiagnosis. As M. haemophilum prefers a lower growth temperature (30-32°C) and it requires iron in the culture medium, M. haemophilum could be misidentified or overlooked. Accordingly, a M. haemophilum infection should be considered in cases of cutaneous infection of the body sites, of which surface temperature is low.

Isolation and genetic characterization of Nocardia seriolae from snubnose pompano Trachinotus blochii in Vietnam.

A total of 480 cage-cultured fish were collected from 4 coastal provinces in central Vietnam to investigate the causative agent of nocardiosis. Fish displayed unique characteristics such as paleness and lethargy and exhibited haemorrhages and ulcers on the skin. Prominent white nodules varying in size were observed in the spleen, kidney, and liver. Furthermore, histopathological sections showed typical granulomatous lesions in these organs. Using the Ziehl-Neelsen staining method, isolated bacteria exhibited acid-fast, bead-like filament morphology when cultured in brain-heart infusion medium or Ogawa medium. Phylogenetic analysis of 16S rDNA confirmed that the isolated bacterium was Nocardia seriolae. This study demonstrates for the first time an outbreak of N. seriolae in snubnose pompano in central Vietnam.

Possible role of anaerobes in the pathogenesis of nontuberculous mycobacterial infection.

Recent advances in cultivation-independent molecular biological modalities for detecting bacterial species have indicated that several bacterial species may play a role in the pathogenesis of certain infectious diseases. The aim of this study was to evaluate the role of bacterial flora in the pathogenesis of nontuberculous mycobacteriosis (NTM) using a bacterial floral analysis of bronchoalveolar lavage fluid (BALF) with 16S rRNA gene sequencing in patients with bronchiectasis. Fifty-eight patients with bronchiectasis evaluated using chest computed tomography were enrolled. BALF obtained from the most affected lung lesions was evaluated using culture and culture-independent methodologies. Approximately 600 bp of the bacterial 16S rRNA gene (E341F-E907R) was amplified via polymerase chain reaction using universal primers, and clone libraries were constructed. Nucleotide sequences of 96 randomly chosen clones for each specimen were determined, and the homology was searched using a basic local alignment search tool to determine the bacterial phylotypes and their proportions (bacterial floral analysis) in each specimen. Twenty-nine patients with bronchiectasis were diagnosed with NTM based on culture-based methods using Ogawa medium. The molecular method showed a significantly high rate of anaerobes among the patients with NTM compared with that observed in the bronchiectasis patients without NTM. In addition, findings of collapse/consolidation were significantly related to the proportion of Prevotella species in the BALF samples determined using the molecular method (P < 0.001). Given the results of the present study, anaerobes may play an important role in the pathogenesis of bronchiectasis in patients with NTM.

Detection in Tetrazolium Microplate Assay Culture Methods from Pulmonary Tuberculosis Suspected Sputum

The definitive diagnosis of pulmonary tuberculosis is the discovery of Mycobacterium tuberculosis from sputum culture, but the conventional culture methods using Ogawa media require between 3−10 weeks detection time. Therefore it is needed a prompt diagnostic tools to shorten the detection time. Tetrazolium microplate assay (TEMA) that used tetrazolium bromide as a growth indicator also use mitochondrial dehydrogenate enzymes in the mitochondria of living M. tuberculosis may reduce yellow tetrazolium bromide into purple formazan crystals. The aim of this study was to know the validity and speed of time detection of M. tuberculosis growth by analyzing it. This study was carried out from November 2012 up to February 2013, which obtained 105 subjects conducted in the Department of Clinical Pathology at Dr. Hasan Sadikin Hospital with a cross sectional study design. The subjects consisting of sputum sample from patients who suspected pulmonary TB which is examined for culture of M. tuberculosis with TEMA method using Ogawa media. Statistical analysis was used a 2×2 table to test the validity and Mann Whitney test for the differences in growth detection time. The validity test of TEMA method got the sensitivity of 90.4% and specificity of 96.2%. The detection time of M. tuberculosis growth in TEMA methods was found fastest in the third day while from the Ogawa media cultur was found on the 13th day with the M. tuberculosis growth media using TEMA methods detected in 12 days. While for those cultured on Ogawa’s media the mean duration is 22 days (p<0.001). Based on this study, can be concluded the examination of M. tuberculosis culture from sputum patient suffer of pulmonary TB with with TEMA method has given high validity and faster in the time detection for the diagnosis of pulmonary TB.

Tuberculosis diagnostics in Fiji: how reliable is culture?

Acid-fast bacilli (AFB) smear microscopy and Mycobacterium tuberculosis culture are the first-line diagnostic tests for tuberculosis (TB). The contamination of TB cultures significantly reduces the reliability of TB diagnosis. To investigate factors associated with TB culture contamination in Fiji, and the relative diagnostic performance of culture compared to microscopy. All tests performed at the Daulakao Mycobacterium Reference Laboratory (DMRL) in Fiji from 2010 to 2012 were reviewed. Study variables included AFB smear and TB culture results, age and type of specimen, referring TB testing centre and patient age. Of 5708 specimens reviewed, 70% had both AFB smear and culture results recorded; 421 specimens were contaminated; 2.7% of specimens were either degraded or had no result recorded. There was moderate agreement (κ = 0.577) between the two tests. Culture was more likely to be positive at higher AFB smear scores. Culture contamination was associated with distance from the DMRL, sample age and operator-associated factors. Increases in the speed of referral from TB testing centres or the addition of preservatives to sputum specimens may results in less culture contamination. The planned introduction of liquid culture techniques in combination with culture on Ogawa media is likely to increase the sensitivity of TB diagnosis in Fiji.

Validity of Acid-Fast Smear of Gastric Aspirates for the Diagnosis of Childhood Pulmonary Tuberculosis among Human Immunodeficiency Virus-Infected Children

Background/Aims: Gastric aspirate specimen is accepted as an alternative specimen of choice to sputum in the diagnosis of childhood pulmonary tuberculosis (TB); and specimen microscopy is usually the only available bacteriologic confirmatory test for streaming cases into the National TB Control Programme treatment protocol. Doubts expressed about the continued relevance of this test among the HIV-infected are based on observations that the bacteriologic yield of acid-fast smears of gastric aspirate specimens from these patients is markedly reduced. This study is aimed at determining the validity of acid-fast smear of gastric aspirates among the HIV-infected and compare with those of the HIV-uninfected. Design, Place and Duration of Study: Diagnostic study. Suspected tuberculosis patients registered in the paediatrics department of University of Benin Teaching Hospital Original Research Article International Journal of TROPICAL DISEASE & Health, 4(1): 111-122, 2014 112 were prospectively recruited from January 2010 to March, 2011. Methodology: A total of 263 children were recruited. Voluntary counseling and testing for HIV was done for each child. Two to three gastric aspirate specimens were also collected from each child using standard gastric aspirate (GA) collection protocols. On each specimen, Zeihl-Neelsen (ZN) staining and culture on Ogawa medium were done. After two or more weeks of incubation, typical acid-fast bacilli isolates on Ogawa medium, which failed to grow on para-nitrobenzoic acid-Ogawa (PNB-Ogawa) media were taken as positive culture for Mycobacterium tuberculosis complex (MTBC). MTBC Culture was taken as the “Reference test” for calculations of sensitivity and specificity. Results: Of the 263 children surveyed 116 (44.0%) were HIV-infected while 147 (56.0%) of them were HIV-uninfected. Among HIV-infected patients, the sensitivity was 38.3% [95%CI:24.4 – 52.2] while the specificity was 95.7% [95% CI:90.9 – 100.0]. Among the HIV-uninfected patients, the sensitivity was 22.0%[95%CI:10.5 – 33.5] while the specificity was 99.0%[95% CI:96.9 -100.0]. The sensitivity of the acid-fast smear in the HIV-infected group of patients was significantly higher than that of the HIV-uninfected group (p = 0.0401).The difference in specificity between the two groups was not statistically significant (p=0.496). There was also no significant difference in their respective positive-predictive values (85.7 vs 91.7)(p = 0.60). Conclusion: The sensitivity of GA acid-fast smear was significantly higher among the HIV infected group.

Gastric aspirate smear microscopy as a diagnostic tool for childhood pulmonary tuberculosis

Background: Most tuberculosis (TB) control programs use positive sputum Ziehl-Neelsen (Z-N) smear microscopy results as a basis for TB case confirmation, valid enough to warrant commencement of treatment. Children do not only produce poor quality sputum a proportion cannot produce sputum, at all. Gastric aspirate (GA) smear microscopy, a recommended alternative, is the only available bacteriologic test in most health-care institutions. Objectives: This study was aimed to determine the usefulness of acid fast smears of GA for the diagnosis of pulmonary TB in children. Materials and Methods: A total of 263 GA samples from children with suspected pulmonary TB underwent Z-N staining and culture on Ogawa medium. Acid-fast isolates on Ogawa medium were sub-cultured on Ogawa medium containing para-nitrobenzoic acid (PNBA-Ogawa medium). Lack of growth on PNBA-Ogawa confirmed Mycobacterium tuberculosis complex (MTBC) and ruled out non-tuberculous mycobacteria. Validity parameters were calculated with GA culture as Gold standard. Results: GA samples from 33 (12.5%) of the 263 patients were smearing positive. However, GA culture on Ogawa medium was positive for MTBC in 97 (37%) of the 263 patients. Twenty-nine of the 97 culture-positive GA were also Z-N smear-positive, giving a GA Z-N smear sensitivity of 29.9%. Of the166 culture-negative cases, 162 were Z-N smear-negative, giving a GA Z-N smear specificity of 97.6%. Positive- and negative-predictive values were 88% and 70% respectively. Conclusion: GA smear microscopy is highly specific for diagnosis of pulmonary TB in children. The test can be useful in culture facility-limited settings as acceptable sensitivity can be achieved if specimen collection/handling protocols are strictly adhered to.

Usefulness of the BACTEC MGIT 960 System for Mycobacterial Culture and TB Ag MPT64 Immunochromatographic Assay to IdentifyMycobacterium tuberculosis

Background: This study was conducted to evaluate the usefulness of the BACTEC MGIT (Mycobacterium Growth Indicator Tube) 960 system for mycobacteria culture and immunochromatographic assay to identify Mycobacterium tuberculosis (MTB) in positive MGIT culture. Methods: Mycobacteria-culture-positive cases were retrospectively analyzed from December 2010 to July 2011. The detection rates and the recovery times of the mycobacteria between the Ogawa media and the MGIT were compared. An immunochromatographic assay (ICA) (SD BIO-LINE) was also performed in the positive MGIT culture for identification, and the results were compared with those of the Ogawa media in the Korea National Tuberculosis Association. Results: Among the 261 patients (M:F, 168:93; mean age, 61.6±17.16 yrs), 450 specimens (sputa, 365; bronchial washing, 61; and pleural effusion, 24) were found positive with mycobacteria. Mycobacteria were grown both on the MGIT and Ogawa media in 310 cases (68.9%); only on the MGIT in 115 cases (22.6%); and only on the Ogawa media in 25 cases (5.5%) (p<0.05).The recovery time was 28.2±8.9 days in the Ogawa media and 11.1±5.8 days in the MGIT (p<0.05). Among the 127 cases from the positive MGIT culture, all 92 cases that were confirmed as MTB cases bythe Korea National Tuberculosis Association were identified as MTB by ICA, with 100% sensitivity. Conclusion: MGIT increases the detection rate and shortens the recovery time of mycobacteria in clinical respi- ratory specimens, and the TB Ag MPT64 kit using ICA is useful in identifying MTB in a positive MGIT culture.

市販結核菌用培地(小川培地)の比較検討

市販結核菌用培地(小川培地)の比較検討