A new liquid chromatographic method for the identification of tuberculosis and other mycobacterium species

by Schillack, Volker Reinhard.

An important class of fatty acids present in the cell wall of the mycobacterium organisms is the group of high-molecular-weight, long chain C60 – C90, ?-branched, ?-hydroxylated, keto, methoxy mycolic acids that were already identified in 1950 by J. Asselineau. Mycolic acids were detected in both reference strains and clinical isolates of mycobacteria by using high performance liquid chromatography together with fluorescence detection (HPLC-FL), to identify the different mycolic acid profiles within the cell wall of different mycobacterium species. Analysis of cellular “mycolic acids” by HPLC indicated a distinct pattern, which is known as the “fingerprint” of that mycobacterium species. This is achieved by profiling the mycolic acid 6,7dimethoxycoumarin esters. Two internal standards with different molecular masses (C60 – C90) are used to determine the relative retention index (RRI), of the different mycolic acid profiles within the cell wall of the mycobacterium. Peak ratios between two or more prominent peaks are also used to identify different species (eg. Mycobacterium kansasii and Mycobacterium asiaticum). The identification of different mycobacteria by conventional tests is often difficult, costly and time consuming. Without the aid of HPLC/FL, it is almost impossible to correctly identify atypical mycobacterium species. Sophisticated techniques, like genetic sequencing and HPLC, by now seem indispensable for differentiating unusual and new mycobacteria from the well established ones. The HPLC technique and the distinct patterns of mycobacteria can be used not just as a research tool but also as a diagnostic tool in the clinical environment. This technique is currently used to identify most of the known mycobacterium species according to their mycolic acid profiles. This technique is able to identify certain multiple mycobacterial infection in patients by comparing the different mycolic acid profiles within the cell wall of the 19 mycobacterium. Unknown mycobacterium species are not uncommon and are easily recognized with this technique. The HPLC/FL technique together with an optimised culturing process, has been proven to be a powerful tool in the clinical diagnostic field. 20 Opsomming ‘n Belangrike groep vetsure teenwoordig in die selwand van mikobakterium organismes, is die groep hoë - molekulêre-massa, langketting (C60 – C90), ?- vertakte, ?-gehidroksileerde keto, metoksie, mikoliese sure wat alreeds in 1950 deur J.Asselineau geïdentifiseer is. Mikoliese sure in beide kliniese en verwysings - mikobakterium isolate is ontleed deur gebruik te maak van hoëdrukvloeistof chromatografie met ‘n fluoressensie - detektor (HDVC / FL), om die verskillende mikoliese sure in die selwand van die mikobakterium te identifiseer. Ontleding van selwand - mikoliese sure met behulp van HDVC lewer ‘n definitiewe vingerafdruk van die betrokke mikobakterium - spesie. Dit is bereik deur profiele van die 6,7dimetoksie-kumarien esterderivate van mikoliese sure op te neem. Twee interne standaarde met verskillende molekulêre massas (C60 – C90) is gebruik om die relatiewe retensie indeks (RRI) van die verskillende mikoliese – suur - profiele binne die selwand van ‘n mikobakterium te bepaal. Piekverhoudings tussen twee of meer prominente pieke is ook gebruik vir die identifikasie van verskillende spesies (bv. Mikobakterium kansasii en Mikobakterium asiaticum). Die identifikasie van ‘n mikobakterium deur konvensionele toetse is dikwels moeilik, duur en tydrowend. Sonder die hulp van HPVC-FL, is dit prakties onmoontlik om atipiese mikobakterium - spesies korrek te identifiseer. Gevorderde tegnieke, soos nukleotied – volgorde - bepaling en HPVC blyk onmisbaar te wees in die onderskeiding van ongewone en nuwe mikobakteria van die goed gevestigde spesies. Die HPVC - tegniek en die bekende patroon mikoliese sure wat mikobakteria lewer, verskaf saam ‘n goeie diagnostiese en navorsingsmetode vir die gebruik in die kliniese omgewing. Die tegniek word tans gebruik vir identifikasie van meeste mikobakterium-spesies aan hand van die mikoliese – suur - profiele. Die 21 tegniek is ook in staat om sekere multi – mikobakterium - infeksies in pasiënte te identifiseer aan hand van die verskillende mikoliese – suur - profiele in die selwand van die mikobakterium. Die HPVC/FL tegniek, tesame met ‘n geoptimiseerde kweekproses, het homself bewys as ‘n kragtige tegniek in die klinies - diagnostiese veld. 22