A Study On Scraping Materials From Positive Acid-Fast Stained Sputum Smear For The Gene Amplification Of Mycobacterium

BackgroundTuberculosis is one of the most challenging public health problems worldwide.With the universal implementation of the DOTS, the TB epidemic has declined. Inrecent years, the emergence of drug-resistant TB, TB with HIV and populationmigration have complicated the management and control of tuberculosis.Nontuberculous mycobacterium (NTM) is an environmental pathogen that is widelydistributed in nature, including those mycobacterium species that are not the membersof mycobacterium tuberculosis complex (MTC) and mycobacterium leprae. NTM caninfect susceptible hosts by various ways. It can cause not only local or systemiclesions but also death. A growing number of researches show that the prevalence ofNTM infection and related diseases is on rise.It is simple，fast, and effective for the Ziehl Neelsen’s (ZN) smear microscopy todetect TB patients in high incidence of TB areas. However, its limited specificity(identifies only acid fast bacilli) would make more and more smear-positive patientsto be misdiagnosed and received inappropriate or unnecessary empiricalantituberculous treatment. The traditional species identification method ofmycobacteria can distinguish the two, but it is tedious and time-consuming. What’smore, most of the basic mycobacterium laboratories in developing countries couldn’tculture the mycobacterium.ObjectivesTo understand the prevalence and epidemiological characteristics of NTM.Inaddition，to evaluate the clinical application value of IS6110fluorescence quantitativePCR for rapid species identification of mycobacteria in positive sputum smear. Thelast but not the least, to explore the amplification of mycobacterium rpoB gene with scraping materials from positive sputum smear.Part I Prevalence of nontuberculous mycobacteria in Yuexiu and Haizhuregion, GuangzhouSubjects and methodsTB suspects were selected fromYuexiu and Haizhu TB Control Institutes from2010to2012, and collected their experimental data of culturing sputummycobacterium. Multiple positive culture and species identification from the samecase during the study period were only calculated on1case, and selected the earliestexperimental results for the statistical analysis.The deep sputum specimens were colleted in the morning for mycobacteriumculture, and the isolated mycobacterium strains were detected by PNB growthinhibition test to differentiate MTC from NTM. While the NTM species wereidentified by culture characteristics and a series of biochemical test.Results1. The recovery rate of MTC and NTM isolates from sputum specimens of TBsuspects was80.9%and19.1%respectively. The annual recovery rate of NTMisolates was17.6%in2010,17.1%in2011and21.2%in2012.2. The sex ratio for cases who obtained NTM isolates from sputum specimenswas1.56:1. The cases over60years old only accounted for26%, the45~59and15~29were peak ages for the isolation of NTM.3. The proportion of rapid and slow growth mycobacterium was50.6%and49.4%in those NTM isolates whose species were confirmed. MycobacteriumChelonae-abscessus complex and Mycobacterium avium-intracellulare complex werethe most frequently isolated organisms, their proportion was40.5%and24.1%respectively.Part Ⅱ The prevalence of mycobacteria in Patients with AFB smear-positiveSubjects and methods331cases with positive sputum AFB smear and culture in the same period wereselected from Yuexiu and Haizhu TB Control Institutes from2012to2013.238casesare male,93cases are female (M/F=2.56). Age ranges from15to87years old, the mean age is39.225cases are first-treatment patients,106cases are re-treatmentpatients.Deep sputum specimens from the patients were colleted in the morning for AFBsmear examination, mycobacterium culture and species identification.Results1. The recovery rate of MTC and NTM isolates from sputum specimens ofPatients with AFB smear-positive was81%and19%respectively. The annualrecovery rate of NTM isolates was14.1%in2012and25.2%in2013.2. The recovery rate of NTM isolates from first-treatment and re-treatmentcases was12%and34%respectively.3.The proportion of positive smears with a score no more than1+was55.97%in MTC group and82.54%in NTM group,while the proportion of positive smearswith a score more than1+was44.03%in MTC and17.46%in NTM group.Part Ⅲ Rapid identification of mycobacterium tuberculosis in positive acid-faststained smear by IS6110-fluorescence quantitative PCRSubjects and methods331positive AFB smear slides were selected from the cases of part Ⅱ, including25slides with score of1-9bacteria per300microscope fields,177slides with score of1+,66slides with score of2+,30slides with score of3+and33slides with score of4+. All slides were divided into two groups according to the result of the identificationtest,268slides in the MTC group and63slides in NTM group.Mycobacterium DNA from scraping materials of smear slides were detected byfluorescence quantitative PCR with the diagnostic kit for TB DNA.Results1. The sensitivity and specificity of IS6110-FQ PCR for preliminaryidentification of mycobacteria in positive acid-fast stained smears was69.40%and76.19%respectively.2. IS6110-FQ PCR method had a poor consistency with the traditionalmycobacterium identification test in the differenciation of the mycobacterium insmear-positive sputum specimens (kappa=0.324). 3. The detection rate of IS6110-FQ PCR for different positive grades smearslides in MTC group was26.67%,63.70%,68.97%,92.86%and93.75%respectively.4. A positive correlation between positive degree of acid-fast stained smear andtarget DNA copies was found (r=0.563).Part Ⅳ A study of the amplification of mycobacterium rpoB gene with thescraping materials from positive acid-fast stained sputum smearSubjects and methods18mycobacterium tuberculosis clinical isolates were chose from Guangzhoumycobacteria strains laboratory of Guangzhou chest hospital.80positive acid-fastsputum smears (3+~4+) were selected from the patients with pulmonary tuberculosisof Yuexiu TB Control Institute.Two pairs of primers were designed. DNA extractes from scraping material ofstained smear slides and clinical isolates were used for amplificating rpoB gene.ThePCR products were detected by gel electrophoresis.Results1. The amplifications of rpoB gene with the genome of18clinical TB isolates allshowed bright target band and were confirmed by sequencing.2. The amplifications of rpoB gene with the DNA extracts from scrapingmaterials of stained smear slides by different template quantities,different annealingtemperature and different PCR methods had no target band. Further FQ-PCR detectedthat the copies of DNA from smear by two different extraction methods were between102-103copies/ul and104-105copies/ul, respectively.3. Genomic DNA from clinical isolates was multiple diluted from100to107foramplification of rpoB gene, the detection limit of the two sets of primers wasrespectively104and103times dilution in regular PCR, While in FQ-PCR was106copies/ul and107copies/ul.Conclusions1. The recovery rate of NTM from sputum specimens of TB suspects is19.1%.2. The majority of NTM infected people are young and middle-aged indivisuals.3. Rapid growth mycobacterium is the major isolates of NTM. 4. NTM are recovered from19%of patients with smear-positive sputum.Therecovery rate of NTM in2013is significantly higher than that in2012.5. The re-treatment patients are more likely to be NTM isolation indivisuals thanthe first-treatment patients.6. The positive grade of sputum smear with NTM is often less than1+.7. There is a positive correlation between positive degree of acid-fast stainedsmear and target DNA copies.8. IS6110-fluorescence quantitative PCR could be an alternative method forrapid and preliminary identification of mycobacteria in positive acid-fast stainedsmear with a score not less than3+.9. The DNA extracted from scraping materials of positive smear slides is notenough what contributes to the fail of rpoB amplification.