Prevalence of non-tuberculosis mycobacteria among samples deposited from the National Tuberculous Reference Laboratory of Iran (2011-2018)

Objective: To investigate the prevalence of non-tuberculosis mycobacteria (NTM) among the samples deposited from the National Tuberculosis Reference Laboratory of Iran between 2011 and 2018. Methods: The study evaluated the prevalence of NTM among specimens from patients with pulmonary tuberculosis symptoms (n=15 771) deposited at the National Tuberculosis Reference Laboratory of Iran from 2011 to 2018. Detection of Mycobacterium (M.) tuberculosis was based on presence of a 190-bp amplicon from IS6110 insertion sequence using Tb1 and Tb2 primers, and amplicon-negative specimens were tested for NTM and M. tuberculosis (refractory to IS6110 amplification) using restriction fragment length polymorphism PCR of hsp65 amplicon fragment. Results: A total of 7 307 (46.33%) M. tuberculosis and 658 (4.17%) NTM specimens were found, the latter mainly comprising M. abscessus (10.18%), M. avium (2.28%), M. chelonae (8.97%), M. intracellulare (10.49%), M. kansasii (4.71%), and M. simiae (56.08%). Conclusions: As treatment for NTM differs from that for M. tuberculosis, accurate detection of Mycobacterium sp. is of public health significance.

Prevalence and Species Spectrum of Pulmonary Nontuberculous Mycobacteria Isolates at a Tertiary Care Center / 대한임상미생물학회지

BACKGROUND: Pulmonary infection with nontuberculous mycobacteria (NTM) is increasing in South Korea. Since treatment strategy differs by NTM species, accurate identification is necessary. In this study, using Mycobacterium pulmonary isolates recently recovered from a general hospital in Seoul, the prevalence of NTM isolates was investigated. METHODS: A total of 483 Mycobacterium pulmonary strains isolated between May and November 2018 from an 814-bed general hospital in South Korea were analyzed. Bacterial species were identified based on nucleotide sequences of the 16S–23S rDNA internal transcribed spacer and the rpoB gene. RESULTS: From a total of 1,209 pulmonary specimens from patients suspected to be infected with mycobacteria, 324 deduplicate strains were isolated, comprising 90 Mycobacterium tuberculosis and 229 NTM strains. Among the NTM isolates, 61.5% (n=144) were Mycobacterium avium complex (MAC), including 92 M. avium and 52 Mycobacterium intracellulare, while 8.1% (n=19) represented Mycobacterium abscessus, including 10 M. abscessus subsp. abscessus and 9 M. abscessus subsp. massiliense. In addition, 12 (5.1%) Mycobacterium lentiflavum, 12 (5.1%) Mycobacterium gordonae, 6 (2.6%) Mycobacterium kansasii, and 5 (2.1%) Mycobacterium fortuitum were identified. In addition, Mycobacterium mucogenicum (n=2), Mycobacterium septicum (n=1), Mycobacterium colombiens (n=1), Mycobacterium asiaticum (n=1), and Mycobacterium celatum (n=1) were identified. CONCLUSION: Among the recently recovered Mycobacterium pulmonary strains, more than half were identified as NTM, and MAC was the most prevalent NTM, followed by M. abcessuss.

Systemic disseminated Mycobacterium fortuitum infection:one case report and literature review / 中国感染控制杂志

Objective To explore the clinical characteristics of systemic disseminated infection caused by Mycobacterium fortuitum (M.fortuitum), and improve the diagnostic rate and understanding of the disease.Methods One case of systemic disseminated M.fortuituminfection was reported, and analyzed in combination with relevant literatures.Results Patient was with multiple systemic involvement (including lung, lymph node, skin, joint), lymph node tissue culture was positive for M.fortuitum, patient was given clarithromycin+levofloxacin+linezolid for treatment, disease was remitted.Conclusion Systemic disseminated M.fortuituminfection is rare, and patient with GATA2 deletion and IFN-γautoantibody may be a potential mechanism, diagnosis is mainly based on pathological morphology and microbiological detection, but positive rate is low, diagnosis is difficult.

Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis / 中华流行病学杂志

Objective To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows.Methods The milk sample was collected from a cow with mastitis,which was pretreated with 4％ NaOH and inoculated with L-J medium for Mycobacterium isolation.The positive cultures were initially identified by acid-fast staining and multi-loci PCR,then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA,hsp65,ITS and SodA genes.The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay.Results Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis,which were identified as non-tuberculosis mycobacterium by multi-loci PCR,and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis.The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics,including rifampicin and isoniazid,but they were sensitive to amikacin,moxifloxacin,levofloxacin,ethambutol,streptomycin,tobramycin,ciprofloxacin and linezolid.Conclusions Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique.The results of the study can be used as reference for the prevention and control the infection in cows.

Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis / 中华流行病学杂志

Objective To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows.Methods The milk sample was collected from a cow with mastitis,which was pretreated with 4％ NaOH and inoculated with L-J medium for Mycobacterium isolation.The positive cultures were initially identified by acid-fast staining and multi-loci PCR,then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA,hsp65,ITS and SodA genes.The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay.Results Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis,which were identified as non-tuberculosis mycobacterium by multi-loci PCR,and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis.The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics,including rifampicin and isoniazid,but they were sensitive to amikacin,moxifloxacin,levofloxacin,ethambutol,streptomycin,tobramycin,ciprofloxacin and linezolid.Conclusions Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique.The results of the study can be used as reference for the prevention and control the infection in cows.

Species identification on 43 clinical non-tuberculosis Mycobacteria isolates from Gansu Province, China / 中国人兽共患病学报

To understand pathogen spectrum of nontuberculosis Mycobacteria (NTM) and the dominant NTM in Gansu Province and provide the scientific basis for the effective prevention and treatment of NTM diseases,875 Mycobacteria isolates were collected from 2012 to 2014 in Lanzhou Pulmonary Hospital,NTM species were identified by means of PNB/TCH differentiate medium and 16S rRNA gene sequence analysis respectively.Forty-six isolats of NTM were identied from 875 PNB/TCH.Then with 16S rRNA gene sequence analysis,the NTM strains were identified to 3 strains of Nocadia and 43 strains of NTM,including M.intracellulare,M.kansasii,M.avium,M.senegalense,M.gordonae,M.szulgai,M.peregrinumand M.fortuitum.Among them,there were 31 strains of M.intracellulare,which accounted for 72.09％ of the total number of NTM strains.The dominant nontuberculosis Mycobacteria in Gansu Province were mainly M.intracellulare.The application of molecular biology can rapidly and accurately identify the species of nontuberculosis Mycobacteria,and can provide relevant evidence for clinical diagnosis and therapy.

Clinical application of genechip based detection system in diagnosing mycobacterium infection and TB drug resistance gene mutation / 国际检验医学杂志

Objective To investigate the clinical application value of genechip detection system in the mycobacterial species iden-tification and drug resistance analysis .Methods The specimens of sputum ,punctured pus ,pleural and abdominal ascites ,cerebro-spinal fluid and so on were performed the Mycobacterium DNA detection by using the gene chip technique .Then Mycobacterium tuberculosis positive samples were further performed the drug-resistant analysis .Meanwhile the Ziehl-Neelsen acid-fast staining was adopted to detect the sample .The positive rates were compared between the two groups .And TB-IGRA was used to examine the tubercle bacillus infection in partial patients .Results In 4402 samples ,137 cases (3 .36% ) of M ycobacterium tuberculosis (MTB) and 11 cases(7 .4% ) of non-tuberculosis mycobacterium(NTM) were detected .Puncture solution ,bronchoalveolar lavage fluid and tissue specimen had higher positive rate .In the 137 positive M TB samples by rifampicin-resistant gene rpoB ,isoniazide-re-sistant gene katG and inhA detection ,22 cases of resistance gene mutations were detected ;the positive rate of genechip for detecting sputum ,cerebrospinal fluid ,hydrothorax and ascites was higher than that of acid-fast staining .TB-IGRA detection had higher pos-itive rate of TB infection than genechip .Conclusion The genechip detection system can directly conduct Mycobacterium identifica-tion and drug resistance analysis ,which is especially suitable for sputum ,cerebrospinal fluid ,hydrothorax and ascites samples ,and which is simple and rapid with higher sensitivity and good specificity .

Empirical treatment of nontuberculosis mycobacterium infection after liver transplantation: one case report and literature review / 器官移植

Objective To summarize the clinical experience of diagnosis and treatment of nontuberculosismycobacterium (NTM) infection after liver transplantation. Methods Clinical experience of effective treatment of 1 case with NTM at 7th month after liver transplantation at the Shanghai Changzheng Hospital affiliated to the Second Military Medical University was summarized and literature review was performed. Results Following liver transplantation, the NTMpatient was clinically manifested with fever in the afternoon. CT scan prompted the progression of the disease. The lesions were enlarged and fused with thin-walled cavity in the right upper lung. The diagnosis of NTM infection was validated by fiberoptic bronchoscopy (brush or lavage approach), spot test of T cells infected with mycobacterium tuberculosis (T-SPOT. TB), multiple phlegm culture and empirical anti-tuberculosis therapy. The patient was effectively treated and successfullydischarged after diagnostic quadruple anti-tuberculosis therapy. The patient was followed up until the day of manuscript submission. The patient was physically stable without the symptoms of fever and cough with asthma. The liver function was normal. Conclusions The incidence of NTM infection is rare and inneglectable after liver transplantation. Application of fibrobronchoscopy via brush or lavage approach can enhance the positive diagnostic rate. Diagnostic quadruple antituberculosis therapy is efficacious for NTM infection.

Antimicrobial Susceptibility Testing and Molecular Characterization of Mycobacterium fortuitum Isolates in China / 生物医学与环境科学（英文）

We performed molecular identification of clinical isolates of Mycobacterium fortuitum (M. fortuitum) and conducted drug susceptibility testing to analyze the in vitro susceptibility of clinical M. fortuitum isolates and potential molecular mechanism conferring resistance to fluoroquinolone and macrolide drugs. The results showed that moxifloxacin had the highest in vitro activity against M. fortuitum, and most M. fortuitum isolates were resistant to clarithromycin and linezolid in China. The loss of genetic mutation in clarithromycin- and amikacin-resistant isolates indicates that some other intrinsic mechanism conferring clarithromycin and amikacin resistance plays an essential role in M. fortuitum infection.

First Report in China on the Identification and Drug Sensitivity of Mycobacterium elephantis Isolated from the Milk of a Cow with Mastitis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>In this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study.</p><p><b>METHODS</b>Milk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay.</p><p><b>RESULTS</b>Smooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline.</p><p><b>CONCLUSION</b>To the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.</p>

Identification of predominant prevailing nontuberculosis mycobacteria in Hangzhou and analysis of their predisposing factors and drug resistance / 中国人兽共患病学报

This study is aim to determine the predominant prevailing nontuberculosis mycobacteria (NTM) in Hangzhou and to understand the predisposing factors and drug resistance of NTM.PNB/TCH growth tests were used to preliminarily identify the NTM in the mycobacterium-positive culture samples from 1 972 patients.The species of NTM isolates were con firmed using mycobacterium molecular linear probe method and sequencing of PCR product of 16S rRNA gene.The influence of sex and age on predisposing factors of NTM was subsequently analyzed.Concentration proportional method was applied to de tect the susceptibility of NTM isolates to armazide,rifampicin,streptomycin,ethambutol,ofloxacin and kanamycin.Results of PNB/TCH growth tests showed that 9.8％ (193/1 972) of the 1 972 mycobacterium-positive culture samples were positive.The mycobacterium molecular linear probe hybridization and PCR product sequencing confirmed that in the 193 samples with positive results of PNB/TCH growth tests,66.3％,18.1％,8.3％ and 7.3％ were infected with NTM alone,two mycobacteria,Mycobacterium tuberculosis and bacteria that not belonging to the genus of Mycobacteria,respectively.In the 173 NTM isolates (128 with single infection and 45 with mixed infection),57.8％ were identified as Mycobacterium intracellulare,followed by Mycobacterium abscessus (12.1％),Mycobacterium kansasi i (9.8％),Mycobacterium chelonei (9.8％) and Mycobacterium avium (5.8％).In the 35 mixed infection samples,28.5％ and 20.0％ were the co-infection of M.intracellulare and M.chelonei with M.tuberculosis,respectively.Male patients infected with NTM were more than female patients (1.67 ∶ 1) and the infection rate (80.4％) of populations with age over 50 years old was significantly high than that with lower than 50 years old (P＜0.01).Pulmonary infection proportion (95.1 ％) of NTM was significantly higher than other positions of body (P＜0.01).100％ of NTM isolates were resistant to armazide while the resistance rates of the isolates against the other 5 antituberculosis drugs were as high as 70.3 ％ 90.6 ％.As the conclusion of this study,M.intracellulare is the predominant prevailing NTM species in the recent years in Hangzhou,NTM mainly causes pulmonary infection and middle-aged and old people are easily infected by NTM.Moreover,NTM isolates have high resistance against commonly used antituberculosis drugs.

MSCT findings and diagnosis of pulmonary non-tuberculosis mycobacteria disease / 实用放射学杂志

Objective To explore CT findings of pulmonary non‐tuberculosis mycobacteria (NTM ) disease .Methods Forty‐two patients with pulmonary NTM disease confirmed by the biphasic medium flora identification (NTM group) ,and 60 patients with lung tuberculosis (TB group) confirmed by the tubercle bacillus cultivation and flora identification in our hospital were included in the ret‐rospective analysis .9 CT signs and distribution features of the lesions were analyzed and compared between the two groups .The difference was statistically significant if P<0 .05 .Results Pulmonary NTM disease was more common in female patients (χ2=5 .500 ,P=0 .019) ,and the mean age was significantly older than that of the tuberculosis (t=3 .456 ,P=0 .001) .The detection rate in the right middle lobe and left tongue section was high in NTM group than in TB group (χ2 =8 .361 ,P=0 .004) .Logistic regression showed that bronchiectasis and bronchial stenosis or occlusion were independent risk factors for the NTM disease .They were important signs for the differential diagnosis from tuberculosis .Conclusion MSCT findings of pulmonary NTM disease have certain characteristic , which are helpful for the diagnosis .

Endobronchial Mycobacterium avium Infection in an Immunocompetent Patient / 감염과화학요법

Although Mycobacterium avium complex (MAC) is the most common pathogen in nontuberculous mycobacterial (NTM) pulmonary diseases, endobronchial lesions caused by MAC infections are very rare even in an immunocompromised host. Herein, we describe the case of a 59-year-old, HIV-negative and non-immunocompromised woman who developed multifocal pulmonary infiltrations with endobronchial lesion caused by M. avium. Bronchoscopic examination revealed white- and yellow-colored irregular mucosal lesions in the bronchus of the left lingular division. M. avium was identified using sputum culture and bronchial washing fluid culture. Following the recommendations of the American Thoracic Society and Infectious Diseases Society of America (ATS/IDSA), the patient was begun on treatment with antimycobacterial drugs. After treatment, pneumonic infiltration decreased.

Rapid identification and differentiation of the species of the Mycobacterium chelonae /abscessus complex by hsp65 and rpoB PCR-RFLP / 中国人兽共患病学报

The Mycobacterium chelonae/abscessus (M.chelonae/abscessus) complex belongs to the rapidly growing genus Mycobacterium (RGM).It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks.It includes members of M.chelonae,M.immnunogenum,M.abscessus,M.massiliense,and M.bolletii.In order to investigate the epidemiological characteristics of the M.chelonae/abscessus complex in China and to conduct the molecular methods for species identification of M.chelonae/abscessus,we collected clinical M.chelonae/abscessus complex strains identified by phenotypic tests.Members were verified by sequencing of 16S rRNA,Species and subspecies were identified by hsp65 and rpoB PCR RFLP methods.In total,27 clinical specimens were identified as Mycobacterium chelonae/abscessus complex by phenotypic tests.16s rRNA gene sequence analysis of all 27 clinical samples shared over 99.7％ similarity with M.chelonae and M.abscessus.Species identification with hsp65 PCR-RFLP and rpoB PCR-RFLP revealed that 18 specimens were M.abscessus and 4 were M.absecces.The remaining 5 samples displayed a pattern that failed to match any previously reported pattern.Thus,this might represent a novel species that is part of the Mycobacterium chelonae/abscessus complex.We identified that a majority of the chronic lung infection in China is caused by the M.chelonae/abscessus complex.Specifically,the M.abscessus species might be the most infectious,while other species in the complex can still cause infection.Interestingly,there may be a novel or previously unidentified species that is a part of the complex.Finally,we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR-RFLP.

Identificación de micobacterias no tuberculosas: comparación de métodos bioquímicos y moleculares / Identification of non-tuberculosis mycobacteria: comparison between biochemical and molecular methods

Las infecciones causadas por micobacterias no tuberculosas (MNT) o atípicas constituyen en la actualidad un grave problema de salud, especialmente en pacientes inmunocomprometidos. Estas micobacterias presentan patrones de susceptibilidad a antibióticos particulares y distintos a M. tuberculosis, por lo que la administración del tratamiento adecuado requiere de un método rápido, sencillo y sensible de identificación. La técnica de PRA (Análisis de Restricción de Productos de PCR), basada en la digestión enzimática del producto de amplificación del gen hsp65, ha mostrado ser un método adecuado de identificación de micobacterias. En el presente trabajo se comparó la técnica de PRA con el estándar de identificación de micobacterias representado por las pruebas bioquímicas en 30 aislados provenientes del Laboratorio de Tuberculosis del Instituto de Biomedicina. La técnica de PRA permitió identificar 96% de las cepas analizadas, en comparación con 92.% de cepas identificadas por las técnicas bioquímicas. Los resultados obtenidos fueron idénticos en 18 de 22 cepas, correspondiendo al 82% de los resultados. Se concluye que el PRA es un método rápido, sencillo y económico que produce resultados concordantes con las técnicas tradicionales, con un menor grado de error. Basados en estos resultados se recomienda el uso del PRA en los laboratorios clínicos como método de identificación de rutina para micobacterias.

Infections caused by atypical mycobacteria at present constitute a serious health problem, especially in immunocompromised patients. These mycobacteria present particular susceptibility patterns, different from M. tuberculosis, due to which the administration of an adequate treatment requires a fast, simple and sensitive identification method. The PRA technique (PCR Restriction), based on the enzymatic digestion of the amplification product of the hsp65 gene has shown to be an adequate method for the identification of mycobacteria. In this study we compared the PRA technique with the standard mycobacterial identification method, represented by biochemical tests, in 30 isolates from the Tuberculosis Laboratory of the Instituto de Biomedicina. The PRA technique allowed the identification of 96% of the strains analyzed, as compared with 92% of strains identified through biochemical methods. The results obtained were identical in 18 of 22 strains, corresponding to 82% of the results. It is concluded that the PRA technique is a fast, simple and economical method that produces results in concord with traditional techniques, with a lesser degree of error. Based in these results, the use of PRA as routine identification technique for mycobacteria is recommended for clinical laboratories.

Application of multiplex PCR in rapid identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria / 解放军医学杂志

Objective To investigate the feasibility of multiplex PCR for rapid identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria. Methods According to MTP40 gene sequence of Mycobacterium tuberculosis, 32kD gene sequence of Mycobacterium and IS6110 insertion sequence gene sequence of Mycobacterium tuberculosis complex, three specific pairs of primers (PT1-PT2, MT1-MT2 and IS5-IS6) for Mycobacterium were designed, and the target DNA for MTP40, 32kD and IS6110 was 396bp, 506bp and 984bp, respectively. The genome of 92 Mycobacterium tuberculosis clinically isolated strains and 5 non-tuberculosis Mycobacteria clinical strains were amplified in the same system, and the results were compared with reference strains. Results Among 92 clinical strains of Mycobacterium tuberculosis, the DNA fragments of 396bp, 506bp and 984bp were found in 90 Mycobacterium tuberculosis clinical strains, as well as in the reference strain H37Rv; the sensitivity of multiplex PCR for Mycobacterium tuberculosis was 97.8%, and the specificity was 100.0%. The DNA fragments of 506bp were all found in 5 non-tuberculosis Mycobacteria clinical strains, the sensitivity and specificity for non-tuberculosis Mycobacterium were both 100.0%. Conclusion The multiplex PCR is a rapid, sensitive and specific method for identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, and it may provide an effective way for clinical diagnosis of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, therefore useful in clinical application.