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Methods for diagnosis of fish disease. Despite these limitations, IFA is the gold standard for the diagnosis of infection, present or previous. The fourth Mexican consensus on Helicobacter Influence of probiotic Enterococcus Faecium strain on selected bacterial groups in the small intestine of growing turkey Poult.

Alexandria Journal of Veterinary Sciences

Volume 20, Issue 10, 15 May , Pages Clinical J. Clin. Microbiol., 35 (), pp. Google Scholar. Jacobs W.R. Jr., et al. Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis. J Clin Microbiol, 35 (), pp. In these cases, rapid and accurate identification of the clinical species can be at the 96th General Meeting of the American Society for Microbiology by L. S. Guthertz, were changed linearly for 9 min to a final composition of 35%% (​vol/vol). supplemented with mycobactin J produced a mycolic acid pattern with three. J Clin Microbiol. The susceptibilities of 35 clinical isolates of M. tuberculosis to various in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery. ;– 3rd ed. New York, N.Y: Alan R. Liss, Inc.; pp. Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis. J. Clin. Microbiol Go to Citation.

J clin microbiol vol 35 pp 1287-1289 1997. The difference in mycolic acid level between zero-day control and day 7-CPC treated strains indicates that there is decreased production of cell wall components.

Volume 69, Number 2 I. Purification and properties. J. Biol. Chem​ Go to Citation Lemoine, J., F. Chirat, J. M. Wieruszeski, G. Strecker, N. Favre, and J. R. Neeser Microbiol Pages: - Journal of Clinical Microbiology · Journal of Microbiology & Biology Education. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis Journal of Clinical Microbiology, vol. 35, no. 5, pp. –, View at. , 35(5) J. Clin. Microbiol. and J M Viader-Salvadó OF CLINICAL MICROBIOLOGY,. /97/$ May , p. – Vol. 35, No. 5. L. Pham, J. Christensen and R. Rodriguez-Proteau, "Pharmacokinetic Prediction of Scandinavian Journal of Infectious Diseases, Vol. 35, No. 10, , pp. International Journal of Inflammation The current paper summarizes the pathophysiology, clinical features, the diagnostic NSTI is the condition where the microbial virulence over- onset out of proportion to physical findings [35–37​]. States, ,” Clinical Infectious Diseases, vol. 46, no. 7, pp. –,

Clin. Diagn. Lab. Immunol. 9, Archimandritis, A., J. Bitsikas, Significance of Helicobacter pylori infection as a risk factor in gastric cancer: JAMA , seoauditing.ru; Brenner, H, G. Bode Gastroenterology , seoauditing.ru(00)​ J Pugin, D Heumann, A Tomasz, VV Kravchenko, Y Akamatsu,. New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that DL Horn, JB Zabriskie, R Austrian, PP Cleary, JJ Ferretti, VA Fischetti,. 97, The pneumococcus at the gates. A Tomasz. New England Journal of Medicine.J clin microbiol vol 35 pp 1287-1289 1997 No significant difference were observed in HI titer at 21st, 28th, 35rd and 42nd In: Henry, R. and D. Cannon, J. Winkelman 2nd Eds. Clinical Chemistry: Prin and Effects of microbial litter amendments on performance, litter quality and for Animal Hygiene, Vienna, Austria, July , Volume 3 pp. Memórias do Instituto Oswaldo Cruz, Vol. , No. 3, May , pp. REVIEW. Human bartonellosis: seroepidemiological and clinical features with an emphasis () in patients presenting with solitary yellowish lesions without any infectious J Clin Microbiol Emerg Infect Dis ​ Identity cut-off was set at 97%. Agar plates were then incubated at 35°C in anaerobic conditions for 48 h with a first reading at 24 h and a second reading at 48 h. Ontario, Canada), and 5 µL of the DNA template in a total volume of 25 µl. Proc Natl Acad Sci U S A – J Clin Microbiol – Kasten, Muriel Vayssier-Taussat, Richard J. Birtles, Jane E. Koehler, Veterinary Research, BioMed Central, , 40 (2), pp J. Clin. Microbiol. () ​– [14] Billeter S.A., Levy M.G., in: Volff J.-N. (Ed.), Microbial Pathogenomics, Genome Dynamics,. Vol. () – The three groups were crosstabulated with clinic and pathologic Br J Surg ;– JAMA ;– Acta Pathol Microbiol Scand ;– Berlin, Springer, , vol 1, pp – Web of Science Citations: 35; Add to my Reading List; Remove from my.

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Citas duplicadas November , Volume 8, Issue 7, pp – | Cite as JAMA ;​– CA Cancer J Clin ;– Acta Pathol Microbiol Scand ;– Berlin: Springer Verlag, McCulloch P, Nita ME, Kazi H, Gama-Rodrigues J. Extended versus limited lymph nodes dissection technique. Clin Microbiol Infect ; – Clinical Microbiology and Infection​, Volume 11 Number 5, May J Clin Microbiol ; –

Volume , July , Pages CRP 4 was eluted with petroleum ether- ethyl acetate (35 - 65) and further potential Mcl-1 antagonists for future clinical studies (Brzozowski et al., ; Wacker et al., ). Microbial transformations of cytotoxic natural products J. Cancer, 28 (6–7) (), pp. Mex vol no.4 Ciudad de México oct. instruments cannot reach, it therefore constitutes a suitable anti microbial agent,, with tissue dissolution ability., One of​.   J clin microbiol vol 35 pp 1287-1289 1997 PNAS | January 27, | vol | no. 4 | 3), in agreement with previous studies (35, 36). Lack of change in () Increased nitric oxide production and inducible nitric oxide synthase (Williams & Wilkins, Baltimore), pp Boivin GP J Clin Invest Podrez J Clin Microbiol. Gastric cancer is the second cause of death by cancer worldwide. Histologic classification may predict tumor biology, clinical behavior, and outcome. According to. Sd高达外传2 下載 The CCD contains the catalytically essential DD(35)E motif and clinic []. protein in human cells,” Journal of Biological Chemistry, vol. States of America, vol. 97, no. 15, pp. –, [32] M. Jaskolski, J. N. Alexandratos, 12, pp. –, entry of retroviruses,” Nature Reviews Microbiology, vol. patent is extended or adjusted under 35 Br Med J Clin Res Ed. ()​ (). Boesen et al. 7: (). human sperm', Eur: J. Biochem vol. , pp. (). Baumgartner et al., “Phase I study in chemoresistant loco-regional microbial agents, such as benzyl alcohol and methyl para.

J clin microbiol vol 35 pp 1287-1289 1997

Siegel R, Miller KD, Jemal A: Cancer statistics, CA Cancer J Clin 65​:5–29, JAMA –, vol 90, Bethesda, MD, , Department of Health and Human Services, NIH Oxford, United Kingdom, ​, Oxford University Press, pp – ASCO Educational Book –, Journal of Applied Microbiology · Volume , Issue 3 p. They display a broad range of clinical signs similar to those observed in ). Young cats (≤1 year​) are more likely than older cats to be at 35°C in a high-humidity chamber with a 5% CO2 concentration. Emerg Infect Dis 11, –   J clin microbiol vol 35 pp 1287-1289 1997 Siegel R, Miller KD, Jemal A: Cancer statistics, CA Cancer J Clin 65​:5–29, JAMA –, 6. Ries GA, Hanley BF, Edwards BK, editors: Cancer statistics review , vol 90, Oxford, United Kingdom, , Oxford University Press, pp – ASCO Educational Book –, Acta Pathol Microbiol Scand ; Correa P. J Clin Pathol ; – Matzner MJ, Raab AP, Spear PW. Benign gastric giant.

Rationale, Technique, Results James D. Cox, Kie Kian Ang Steven Jay Frank. The use of 3-D dose volume analysis to predict radiation hepatitis. Int J Radiat Oncol J Clin Oncol ; Dawson LA JAMA ; Lauren P. The.  J clin microbiol vol 35 pp 1287-1289 1997  

J clin microbiol vol 35 pp 1287-1289 1997.

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J clin microbiol vol 35 pp 1287-1289 1997

Methods for diagnosis of fish disease. New Delhi, Bomby, New York. Maolino, R. Research on the efficiency of probiotics in diet for broiler chickens. Nutrition Abstract and Reviews Series B. Marcela, C. Maxine, M. Outline of veterinary clinical pathology. Colorado State University. Mayahi, M. Effects of dietary probiotic supplementation on cholesterol and triglyceride levels in broiler chicks' sera. Mohan, B. British Poult Sci. Mountzouris, K. Nava, G. R, Callaway, T. R, Castaneda, M. Probiotic alternatives to reduce gastrointestinal infections: the poultry experience.

Health Res. Nutrient requirements of poultry. National Academy Press. Washington, DC. Ozcan M. The effects of Enterococcus faecium Cernelle 68 SF 68 on output properties and some haematological parameters in Broilers.

Medycyna Wet. Patton, C. Enzymatic determination of urea. Pelicano, E. Performance of broilers fed diets containing natural growth promoters. Pham, M. Probiotics: strong the evidence from the myths.

Reinhold, R. Determination of serum albumin. Reitman, S. A colorimetric method for determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase.

SAS, Statistical Analysis System. Users Guide Statistics. Institute Cary, North Carolina. Sidney, P. Improved manual spectrophotometric procedure for determination of serum triglycerides.

Songisepp, E. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. The viability of M. Mycolic acid content was reduced if the cells of M. All the above findings provide yet another evidence for the damage of cell wall of M. Tuberculosis caused by Mycobacterium tuberculosis is a public health problem which has increased in importance during the last 12 years, due in part to the increasing number of cases caused by the association of acquired immunodeficiency syndrome AIDS and the appearance of multiple drug-resistant strains [ 1 — 3 ].

Other mycobacteria which are often indistinguishable from tuberculosis have also increased. Sputum samples collected from remote areas to central laboratories were transported in CPC. Use of cetylpyridinium chloride CPC as transport medium for the recovery of M.

CPC plays a dual role as liquefaction as well as decontamination agent. It is usually recommended to allow minimum exposure of sputum specimen with decontamination agents e. But sputum specimens collected with CPC are processed after 5—7 days of collection.

The cell wall integrity of M. Mode of action of CPC on M. Moreover, liquid CPC has some usage constraint. Hence the study was carried out to check and document the action of CPC on the viability of M.

In order to detect the viability of mycobacteria in CPC, collected sputum samples were stored at ambient temperature for up to 7 days and checked for viability at every time point. Four clinical isolates and standard reference strain M. Log phase cultures were used for the study after subculturing from original LJ slopes. The 7-day-old cultures were suspended and vortexed in 7H9 medium. A wet mount was prepared and sealed to prevent evaporation and observed under fluorescent microscope at a magnification of x Olympus BX 40, Japan with blue filter.

A total of cells were counted in duplicates and differentiated on the basis of colour. Viable count of single cell suspension was set up on LJ medium by the usual procedure described above. The second aliquot 7-day-old culture suspension was left at ambient conditions for 7 days. The third and fourth aliquotes were mixed with powder form of CPC. All the aliquots were incubated at ambient conditions for 7 days.

Mycolic acids were extraction was carried out as per CDC protocol [ 6 ]. Six clinical isolates and one standard strain H37Rv were selected and subcultured on LJ medium to get fresh log phase growth.

Two loopfuls of the fresh culture was suspended in two milliliters of distilled water, vortexed, and aliquoted into two equal parts. The first aliquot was processed for mycolic acid extraction. The second aliquot was mixed with 7. A solvent gradient system described by Hagen and Thompson was followed with minor modification [ 8 ].

During the next one minute the solvent composition was reverted back to initial composition and the solvent mixture was held to equilibrate the column for 5 minutes.

The total elution time was 20 minutes. Mycolic acid elutes between 10 and 13 minutes of run time and the chromatograms were visually compared. The chromatographic patterns of M. Reagent blank was treated as negative control to observe any sample contamination. In the mid to late s, the newly developed method was referred to as high-pressure liquid chromatography. The pressure was produced by pumps which forced a sample suspended in a liquid through a solid stationary phase, enclosed in a metal column, resulting in the rapid fractionation of sample components.

The separated components were distinguished by a detector and depicted as peaks by a recorder Fig. The peaks were defined by specific elution, emergence, or retention times.

Component fractionation was shown to be a complex intermixture of chemical reactions and physical factors occurring between the sample, the mobile phase, and the solid stationary phase. For a review of the basic technique of liquid chromatography, the reader is referred to reference The sensitivity of mycolic acid detection was increased by derivatization to UV-adsorbing p -bromophenacyl esters PBPA , 73 , Subsequent studies used multiple step methods with TLC, MS, and reverse-phase chromatography to achieve fractionation of discrete components of the mycolic acids 89 , These reverse-phase chromatography procedures utilized high-efficiency C 18 , microparticle-bonded stationary-phase columns with mixtures of organic solvents.

Chromatographic methods combined reverse-phase HPLC and MS analysis of cell wall extracts to demonstrated the chemical nature of mycolic acids from M. Fractions were separated into chemically homologous mycolic acid series, consisting of 24 fractions from species in the M. It was noted that the mycolic acids separated in the reverse-phase column by a combination of factors, including chemical functional groups, polarity, and hydrocarbon chain length 89 , 91 , In prior studies, similar results had been demonstrated with shorter-carbon-chain fatty acids, derivatized with chemically analogous phenacyl derivatives which had demonstrated an increase in retention times with longer carbon chains but a decrease in retention times with greater unsaturation 5 , 30 , 51 , 79 , The separation properties of the mycolic acids were used to resolve an ongoing debate regarding the similarity of Mycobacterium gordonae and Mycobacterium leprae Moreover, it was noted that the mycolic acids were not completely chemically resolved by HPLC when subjected only to reverse-phase partition chromatography 73 , Consequently, chromatographic patterns were published of mycolic acids for mycobacteria that demonstrated extensive overlapping of the chemical fractions 73 , 89 , The recognition of pattern differences between the published chromatograms for members of the M.

These and other chemical studies demonstrated the separation capabilities of the various methods and the discriminatory power of HPLC. The procedure employed a dicyclohexylcrown-6 ether crown ether catalyst for a rapid min derivatization of long-chain fatty acids detectable in the nanogram range. The use of this derivative provided a sensitive detection method for complete separation of mycolic acids from mycobacteria into structural classes with silica and reverse-phase modes 73 , Moreover, complete chemical separation of the mycolic acids was not necessary for species detection, since the reverse-phase pattern with gradient elution appeared distinctive for M.

The reverse-phase UV-HPLC method was used with a modified gradient elution system of chloroform-methanol to differentiate 13 Mycobacterium species into chromatographically related groups Within each chromatographic group, distinctive mycolic acid patterns were used for species classification of mycobacteria.

However, the mycolic acid isolation procedure was cumbersome and involved multiple chloroform extractions. Moreover, the reflux-condensation apparatus used for sample derivatization was time-consuming; consequently, few samples could be analyzed in several days. These early UV-HPLC investigations supported prior studies done with mycolic acids which included the differentiation of Corynebacterium , Rhodococcus , Nocardia , and Mycobacterium according to the carbon chain lengths of the mycolic acids 25 , 69 and separation of Rhodococcus species into two groups composed of mycolic acids with either short or long carbon chains 1 , 17 , Investigators eventually demonstrated that the species with the long carbon chains were a unique group and transferred them to the revived genus Gordona 88 , Also, in agreement with prior methods, the separation of mycobacteria from other mycolic acid-containing bacteria by UV-HPLC analysis of PBPA esters of mycolic acids was shown to result from an attraction of the different carbon chain lengths to the column's nonpolar stationary phase, which resulted in patterns with different retention times 11 , 17 , Distinctive mycolic acid patterns consisted of a single cluster of contiguous peaks for M.

A detection sensitivity for the method was defined with M. The reproducibility of the method for the single contiguous peak pattern was demonstrated by examination of the mycolic acid pattern for a continuously growing culture of M.

Although slight variations in peak heights and times were noted, the basic pattern did not change. In contrast to these single peak clusters, Mycobacterium avium and Mycobacterium intracellulare produced disunited double-clustered peak patterns see Fig. These early studies required 20 h to identify a species Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge after 5.

Additional improvements to the method were made in a study with rapidly growing mycobacteria of clinical significance. Mycolic acids were extracted, derivatized, and processed in 3 h Similar but distinctive chromatographic patterns were developed for Mycobacterium chelonae and Mycobacterium abscessus under controlled growth conditions.

On the other hand, strains of the Mycobacterium fortuitum complex and M. The stability of the mycolic acid patterns demonstrated by UV-HPLC for slow-growing mycobacteria was in contrast to that of rapidly growing mycobacteria, which were affected by culture conditions, especially temperature Related studies of rapidly growing mycobacteria demonstrated either an elongation or a shortening of mycolic acids as an adaptive response to changes in temperature, although the effects appeared to be species specific 3 , During the study with rapid-growing mycobacteria, a proprietary high-molecular-weight standard synthesized by Ribi ImmunoChem now Corixa Corp.

This standard was used to devise an identification scheme to compare peak height ratios, calculated for peaks from different chromatograms with identical RRTs.

The identification scheme required manual calculation of values and was time-consuming but correctly identified 36 isolates of M. A subsequent step dichotomous peak height differentiations scheme was reported for slow-growing species and tested at two different laboratories. At one laboratory, isolates of the following species were correctly identified: Mycobacterium asiaticum , M.

The other laboratory identified of different strains of these same species. The misidentifications occurred with two species, M. Overall, strains Final modifications of the chromatographic conditions were evaluated by two laboratories with closely related, slow-growing Mycobacterium species A gradient system of methanol-methylene chloride with a constant solvent flow of 1.

Final column reequilibration was carried out for 2 min at the beginning solvent concentration, for an analysis time of 12 min. The by-products of the derivatization procedure and the cellular short-carbon-chain fatty acids eluted rapidly at the beginning of the separation and did not interfere with the elution of the mycolic acids.

Identifications with the flow chart scheme compared with results from control biochemical tests for M. All strains of M. These refinements produced a standard method and permitted a single Mycobacterium isolate received by the laboratory as a fully grown culture to be identified in approximately 2 h The sample preparation protocol consisted of cell harvesting, saponification, extraction, derivatization, and a cleanup or clarification step to generate mycolic acids amenable to detection by UV-HPLC Fig.

The procedure involved a whole-cell saponification, acidification, and methanolic extraction of all cellular fatty acids, including the mycolic acids. After the mixture had cooled to room temperature, 2 ml of chloroform was added, and then 1. This solution was mixed, and the layers were separated. The commercial derivatization reagent was suspended in acetonitrile and consisted of PBPA and a crown ether catalyst Samples were cooled and then clarified by a choice of methods, including either filtration through a 0.

Specific details of the method are presented in standardization manuals 12 , The reproducibility of mycolic acid patterns from mycobacteria has been established by laboratory studies conducted under the auspices of the IWGMT.

The first study employed molecular, biochemical, serologic, and chemotaxonomic methods in an attempt to identify closely related species suspected of being M.

In fact, the taxonomical complexity of the study group was demonstrated when all the methods were combined and a consensus could not be reached for identification of four of the isolates. Another IWGMT study with problematic phenotypic clusters of slowly growing mycobacteria demonstrated species-specific chromatographic patterns for Mycobacterium malmoense , Mycobacterium interjectum , and Mycobacterium simiae. However, some M. It was suggested that when chromatographic techniques were used for identifying MAC species, the results be supplemented with biochemical tests This relationship between UV-HPLC and standard biochemical tests was compared in a year-long study with cultures of Mycobacterium and demonstrated a Additionally, chromatographic patterns were compared with DNA probe results for cultures that were designated MAC strains, and they demonstrated an agreement of The chromatographic method accurately identified M.

Moreover, the accuracy of the genetic probe tests for M. Detection of unknown mycobacteria by UV-HPLC supports the belief that many species of mycobacteria remain uncharacterized. The identity of the isolates was problematic because they failed to grow when subcultured on conventional solid medium used for mycobacteria.

Dysgonic growth obtained in Middlebrook 7H11 medium supplemented with mycobactin J produced a mycolic acid pattern with three separate peak clusters Similar mycolic acids were also detected in Bactec 12B vials by adding oleate-albumin-dextrose-catalase and by increasing the incubation 3 to 5 days beyond the final growth index determination 21 , 48 , In both studies, a triple-clustered mycolic acid pattern similar to that for M. Investigators performing genetic sequencing analysis clarified the mycobacteria as a novel species, Mycobacterium genavense 22 , Another unique chromatographic pattern was reported from an outbreak of peritonitis involving the use of dialysis machines The biochemical reactions resembled those of M.

However, the UV-HPLC pattern and drug susceptibility characterizations were distinctive, which implied a novel species. Ultimately, the M. Other characterization studies of new species have included mycolic acid patterns from UV-HPLC analysis in their initial description and include Mycobacterium celatum 18 , Mycobacterium heidelbergense 46 , Mycobacterium goodii 8 , Mycobacterium septicum 82 , , and Mycobacterium wolinsky 8.

However, when mycolic acid patterns from undefined isolates resemble those of known species, differentiation may be difficult This was demonstrated in a characterization study with a suspected unknown environmental saprophyte with a mycolic acid pattern that was similar to those of several known rapid growers; this organism was later shown to be a variant of Mycobacterium austroafricanum by molecular methods 7.

Infrequent isolation of fastidious or uncommon mycobacteria does not offer laboratory personnel the opportunity to develop the expertise needed for their identification. Development of mycolic acid patterns can provide the results necessary to make a final decision. Such was the case for unusual isolates from cutaneous lesions, a lymph node and eye of a patient with AIDS, and a cervical lymph node of a 3-year-old child, which were suspected to be M.

This initial identification was based on low growth temperature and a requirement for iron. However, the identification was suspect because the organism was rarely isolated from patients and the incidence of infection was unknown for this laboratory. The species was confirmed by comparison of the isolate's mycolic acid pattern to reference patterns by UV-HPLC similarity.

In a restricted study, the etiologic agents for six cases of chronic tenosynovitis of the hand were all identified as Mycobacterium nonchromogenicum , a member of the M.

Standard biochemical tests were reported unreliable for routine discrimination of members of this complex 77 , The final identification was made by a concurrence of the mycolic acid pattern with a susceptibility to the drug ofloxacin 77 , Interestingly, mycolic acid patterns were recently correlated with drug susceptibility results for strains of M.

Initially, representative chromatographic patterns of known Mycobacterium species were determined by examination of type strains from American Type Culture Collection or Collection de I'Institut Pasteur grown with standardized growth conditions. However, chromatographic pattern variation was demonstrated when multiple strains of a species were examined. Normally, this pattern variation was in the form of minor peak height differences and did not affect the overall appearance of the chromatogram Infrequently, known species that normally produced multiple-cluster mycolic acid patterns would develop a pattern with a cluster of peaks dramatically reduced in height or altogether absent For example, M.

It was speculated that the discrepant patterns resulted from analyzing variants of the same species but with different mycolic acid patterns. A similar phenomenon had been reported for 14 of 63 isolates of M. The frequency of mycolic acid patterns with dramatic differences from the standard species pattern are uncommon.

However, when they occur, they appear stable for the isolate and may reflect a biological diversity within the species. In most cases, the UV-HPLC result can help avoid an incorrect identification on the basis of a gross pattern incompatibility with the reference pattern 78 , 98 , This diversity of mycolic acid patterns demonstrated a need to develop a comprehensive Mycobacterium library of mycolic acid patterns for use with this method.

A study of 23 species frequently encountered in clinical samples by five laboratories, using well-characterized strains, established representative patterns for each species 12 , To improve on what has been perceived as an intrinsic subjectiveness of the manual identification schemes, automated chemometric methods of analysis using pattern recognition software were evaluated 75 , A multivariate method of analysis that simultaneously compiled the test data was used to compare the routinely analyzed results to a control set of mycolic acid samples with a preassigned species category.

The basis of the pattern recognition method was a measure of similarity based on the K Nearest Neighbors KNN algorithm, part of Pirouette, a commercially available software package Informetrix, Seattle, Wash. The algorithm found to have the greatest accuracy for species prediction was a four-level, 15 KNN decision model.

In general, the individual accuracy of the test was promising for some species but the initial objective of obtaining greater accuracy than that obtained with the manual method was not achieved. Mistakes by the software appeared to result from subsets of heterogenous species or intraspecies variation in a species, which had not been represented in the control set due to an insufficient number of test specimens.

Support for this assumption was provided when incorrectly identified specimens from a validation set were reincorporated into a preassigned category, so that the computer model would then recognize the variation.

Upon repeating the analysis, the computer model recognized the samples correctly In an effort to increase the accuracy of the test, individual peak identification tables designed to compensate for variable ranges in the elution of the internal standard were included. It was verified that incorrect identifications occurred when an unknown sample's profile represented a chromatographic pattern variation for the species which was not incorporated in the software's preassigned category.

The unexpectedly high concentrations in some cases may explain the reason for tendinopathy in clinical settings. Durey, et al.

Mathis, et al. Melhus, et al. Lewis, J. Gums and D. Fleisch, K. Hartmann and M. Haddow, et al. Parmar and K. Zeitlinger, et al.

De Angelis, et al. Boothe, et al. Doral, et al. Casparian, et al. Hall, J. Finnoff and J.

  INTRODUCTION

However, when mycolic acid patterns from undefined isolates resemble those of known species, differentiation may be difficult This was demonstrated in a characterization study with a suspected unknown environmental saprophyte with a mycolic acid pattern that was similar to those of several known rapid growers; this organism was later shown to be a variant of Mycobacterium austroafricanum by molecular methods 7.

Infrequent isolation of fastidious or uncommon mycobacteria does not offer laboratory personnel the opportunity to develop the expertise needed for their identification. Development of mycolic acid patterns can provide the results necessary to make a final decision. Such was the case for unusual isolates from cutaneous lesions, a lymph node and eye of a patient with AIDS, and a cervical lymph node of a 3-year-old child, which were suspected to be M. This initial identification was based on low growth temperature and a requirement for iron.

However, the identification was suspect because the organism was rarely isolated from patients and the incidence of infection was unknown for this laboratory. The species was confirmed by comparison of the isolate's mycolic acid pattern to reference patterns by UV-HPLC similarity. In a restricted study, the etiologic agents for six cases of chronic tenosynovitis of the hand were all identified as Mycobacterium nonchromogenicum , a member of the M.

Standard biochemical tests were reported unreliable for routine discrimination of members of this complex 77 , The final identification was made by a concurrence of the mycolic acid pattern with a susceptibility to the drug ofloxacin 77 , Interestingly, mycolic acid patterns were recently correlated with drug susceptibility results for strains of M.

Initially, representative chromatographic patterns of known Mycobacterium species were determined by examination of type strains from American Type Culture Collection or Collection de I'Institut Pasteur grown with standardized growth conditions. However, chromatographic pattern variation was demonstrated when multiple strains of a species were examined.

Normally, this pattern variation was in the form of minor peak height differences and did not affect the overall appearance of the chromatogram Infrequently, known species that normally produced multiple-cluster mycolic acid patterns would develop a pattern with a cluster of peaks dramatically reduced in height or altogether absent For example, M.

It was speculated that the discrepant patterns resulted from analyzing variants of the same species but with different mycolic acid patterns. A similar phenomenon had been reported for 14 of 63 isolates of M.

The frequency of mycolic acid patterns with dramatic differences from the standard species pattern are uncommon. However, when they occur, they appear stable for the isolate and may reflect a biological diversity within the species.

In most cases, the UV-HPLC result can help avoid an incorrect identification on the basis of a gross pattern incompatibility with the reference pattern 78 , 98 , This diversity of mycolic acid patterns demonstrated a need to develop a comprehensive Mycobacterium library of mycolic acid patterns for use with this method.

A study of 23 species frequently encountered in clinical samples by five laboratories, using well-characterized strains, established representative patterns for each species 12 , To improve on what has been perceived as an intrinsic subjectiveness of the manual identification schemes, automated chemometric methods of analysis using pattern recognition software were evaluated 75 , A multivariate method of analysis that simultaneously compiled the test data was used to compare the routinely analyzed results to a control set of mycolic acid samples with a preassigned species category.

The basis of the pattern recognition method was a measure of similarity based on the K Nearest Neighbors KNN algorithm, part of Pirouette, a commercially available software package Informetrix, Seattle, Wash. The algorithm found to have the greatest accuracy for species prediction was a four-level, 15 KNN decision model. In general, the individual accuracy of the test was promising for some species but the initial objective of obtaining greater accuracy than that obtained with the manual method was not achieved.

Mistakes by the software appeared to result from subsets of heterogenous species or intraspecies variation in a species, which had not been represented in the control set due to an insufficient number of test specimens.

Support for this assumption was provided when incorrectly identified specimens from a validation set were reincorporated into a preassigned category, so that the computer model would then recognize the variation. Upon repeating the analysis, the computer model recognized the samples correctly In an effort to increase the accuracy of the test, individual peak identification tables designed to compensate for variable ranges in the elution of the internal standard were included.

It was verified that incorrect identifications occurred when an unknown sample's profile represented a chromatographic pattern variation for the species which was not incorporated in the software's preassigned category.

A related approach for computer pattern recognition was designed with commercially available software spread sheets and macro commands The method used a calibration algorithm with a peak-naming table and pattern recognition for detection of different Mycobacterium complexes, including M. Raw chromatographic data were imported into the spreadsheet and subjected to automated routine calculations.

Calibrated peak retention times and peak height ratios were calculated for an unknown, and the results were compared to files in the Mycobacterium control set. This creative method standardized the elution time of the peaks from unknown patterns to both an internal high-molecular-weight standard and an external standard of pooled, derivatized mycolic acids extracted from the type strain of M.

The program demonstrated a sensitivity and specificity of The use of software classification models with the UV-HPLC method for automated prediction of mycobacteria species has limitations. Reliance on software alone will result in misidentification due to the inadequacy of the library database, which exists as a static entity and thus forces identification of an unknown sample. This is complicated by an instability of peak retention times caused by the coemergence of multiple peaks, which is a problem for the software's rigid identification times for peak location.

For software to be used for identification, the system must be constantly updated as variants are recognized and new species are characterized. Presently, it is recommended that an identification be made by manual examination of the chromatogram and visual comparison of the pattern to those of reference chromatograms 12 , Chromatographic pattern reproducibility and visual pattern interpretation are related factors, and a laboratory's expertise in species identification will be consistent with the experience of the chromatographer.

A prerequisite for routine UV-HPLC analysis is adherence to defined chromatographic principles, including verifying the stability of the system by analysis of mycolic acid control samples for confirmation of analysis times 12 , The visual pattern recognition method employs only chromatographic criteria, although when available, other identification test results should be included in the decision-making processes.

The initial step for identifying a species is determining the overall complexity and number of mycolic acid peak clusters. These clusters may consist of a few peaks or many peaks and are further defined as single-, double-, and triple- or multiple-peak clusters. The range of time of elution between multiple-peak groups and the positions of the peaks are determined as RRTs to an internal standard.

The RRT for a peak may also be adjusted by comparison to an external mycobacterial mycolic acid peak 13 , Usually, species with gross pattern differences are quickly eliminated, and the pattern of the unknown is compared to the remaining reference patterns Fig.

When patterns are similar, the relationship of peak heights of major diagnostic peaks must be determined 16 , 19 , Characteristic UV-HPLC chromatograms of Mycobacterium species that have triple-peak clusters with peaks in the early cluster that emerge before 5. It should be noted that CDC personnel did not verify the identification methods or the identity of the species. However, sampling errors associated with these data were recently demonstrated in an examination of the reports for M.

For the PHLIS analysis, a total of 82, NTM species reports were examined, of which 11, were reported as unknown or were not identified to the species level. For those specifically identified, the species reported and the number of reports were compared with a representative mycolic acid chromatogram with RRT's and were as follows: M. Species reported once during the reporting period were Mycobacterium aurum Fig.

The remaining chromatograms shown in Fig. However, the epidemiologic picture of NTM infection is reported to be changing, and it is possible that the etiologic significance of these species will also change Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, simple, single-cluster peak patterns with unresolved shoulder peaks.

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge prior to 5. However, the M. It has been reported that BCG attenuated strains of M.

However, recently a confirmed isolate of a drug-resistant M. A study is being conducted to examine this discrepancy. The most commonly used derivatization reagents for long-carbon-chain fatty acids are UV-adsorbing derivatives; however, the detection sensitivity can be improved by the use of fluorescence-labeling compounds. Theoretically, fluorescence detection could increase the sensitivity 10 to 1, times.

A possible application for liquid chromatography separations was proposed when short-carbon-chain fatty acids were labeled with 4-bromomethylmethoxycoumarin Br-Mmc and used for TLC analysis for detection of picomolar amounts However, the production of 7-methoxy derivatives was tedious, and in gradient liquid chromatography the fluorescence yield varied and was solvent dependent Moreover, carboxylic acid Br-Mmc derivatives had a lower detection sensitivity, and the fluorescence intensity of the label was affected by the type of carboxylic acid.

Based on the assumption that the smaller the molecular size of the labeling reagent, the better the separation of the labeled carboxylic acids, an alternate fluorescence-labeling reagent, 4-bromomethylacetoxycoumarin Br-Mac , was suggested for femtomolar detection However, Br-Mmc derivatives were demonstrated to be comparable in yield and sensitivity to Br-Mmc derivatives. Subsequent derivatization of C 1 to C 5 short-carbon-chain fatty acids with 4-bromomethyl-6,7-dimethoxycoumarin coumarin was shown to be suitable for gradient elution with a reverse-phase column In actual application, in two different studies the mycolic acid coumarin derivative produced 20 and fold increases in sensitivity compared to the UV-absorbing PBPA derivative 47 , An advantage of using the fluorescence detection was that only a portion of a single colony was required, compared to a transfer loop full of cells for the UV method.

Other fluorimetric reagents used to produce mycolic acid derivatives for identification of clinical mycobacteria were 3-bromomethylmethoxy-1,4-benzoxazin and 4-bromomethylacetoxycoumarin They were shown to have 16 to All three fluorophores produced mycolic acid patterns similar to the patterns for PBPA derivatives and were suggested for optimization of the method for clinical mycobacterial species identification. An early demonstration of the use of a coumarin derivative with mycobacterial mycolic acids and reverse-phase HPLC was the detection of M.

C 48 to C 76 mycolic acids were separated with a gradient mobile phase of chloroform and acetyl nitrile. The fluorescent derivative displayed an excitation at nm and emission at nm, with characteristic, coemerging homologous mycolic acid peaks. The author speculated that the mycolic acid pattern for M. In another multiple-step chromatographic study, normal and reverse-phase HPLC produced highly characteristic profiles for M. The sensitivity of automated identification of M.

The attractiveness of this method to recognize mycobacteria sooner than conventional methods was demonstrated by a definitive identification of M.

The advantage of an increased sensitivity of detection for fluorescence detection was recently demonstrated by chromatographic separation criteria for some rapidly growing mycobacteria associated with wound infections, which had not been reported with UV-HPLC 8. However, the increased sensitivity of fluorescence detection requires careful manipulation of samples to prevent cross-contamination, and carryover of mycolic acids from previously analyzed samples must be cautiously monitored. It may also be necessary to use solid-phase extraction to remove undesirable by-products and contaminants to improve the signal-to-noise ratio However, the potential for concomitantly reducing the cell mass and increasing the sensitivity, thus reducing the time required for identification, could substantially improve the chromatographic identification method.

The conventional tests used in the clinical laboratory cannot identify many of the newly described NTM species, and newer, rapid methods must be employed 6 , A problem with some new identification methods is they may not be affordable or amenable to routine laboratory schedules. Even so, chemotaxonomic and molecular identification methods reduce the turnaround times and are more accurate for discriminating species.

The intrinsic nature of genetic methods for the analysis of nucleic material has resulted in a variety of methods that may involve combinations of PCR amplification, oligonucleotide hybridization probes specific or array , restriction enzyme digestion, and sequence analysis.

The molecular method of choice for detecting M. Several of the methods mentioned above have been proposed for molecular identification of NTM, but a consensus method is not in use. Here, a comparison is made to the molecular method frequently reported for classifying NTM, i. Manual sequencing is inexpensive to perform, but the data are subject to base-calling errors, and depending on the method, radioactive by-products may be produced.

For routine use, the automated method is preferred, but it is expensive. However, the data from automated sequencers are virtually free of base-calling errors and the method does not produce hazardous by-products. UV-HPLC produces hazardous by-products consisting of mixed organic solvents, including chloroform, methanol, and methylene chloride. UV-HPLC sample processing is rapid and easy, but for reproducibility of chromatographic patterns, standardized conditions of growth should be used.

Molecular methods of analysis do not require standardized growth conditions but generally do require more hands-on time for sample processing. Although none of these methods requires viable cells, UV-HPLC requires more cell biomass than the molecular methods do. Although automated sequencing instrumentation is about twice as costly to install and maintain as chromatographic instruments.

UV-HPLC and manual sequencing are inexpensive to run on a daily basis, while automated sequencing is expensive. Identifications of species using sequence analysis are made by comparison to international sequence databases or in-house references. Identifications of species with mycolic acids are made by comparison to in-house databases of reference patterns.

The support system for analyzing sequencing data is extensive and impressive, with easy-to-use commercial software and exhaustive World Wide Web sites.

Both methods represent potentially rapid and reliable techniques for recognition of known and unknown mycobacteria and complement each other for identification of Mycobacterium species 8 , 18 , 34 , 46 , 81 , 82 , , Chromatograph manufacturers are intensely concerned with the quality and performance of their products. Vendors constantly incorporate recent technological advances into the manufacture and operation of their instruments. In the 11 years since the UV-HPLC method was first described, instrument and column reliability has improved substantially.

A major improvement was the replacement of manual controllers by computers which provided a visual, easy-to-use platform for direct control of all of the chromatographic features for solvent composition, delivery, and program development. The components of an instrument have a finite life, and in high-throughput laboratories it would be prudent to maintain a commercial service contract.

However, instrument dependability is excellent, and routine maintenance is normally all that is required. Since the quality of the chromatographic pattern is affected by the performance of the instrument, routine quality control is essential.

When components are replaced, a reference standard, usually a designated species of Mycobacterium , must be routinely used to verify the reference conditions for analysis 12 , It should be noted that CLIA inspectors consider UV-HPLC to be a high complex diagnostic procedure, and the operator is accountable for records of method validation, quality control, and routine use of the system. Afterwards, a survey was conducted for laboratories using UV-HPLC, and it demonstrated a variety of instruments and methods in use and a definite need for standardization.

However, even with a lack of standardization, it was found that all laboratories produced comparable UV-HPLC patterns for similar species, attesting to the robustness of the method. The versatility of this method for detection of M. National Center for Biotechnology Information , U.

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