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Improving methods for genotypic drug resistance testing in Mycobacterium tuberculosisMlamla, Zandile Cleopatra 03 1900 (has links)
Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: An important next step to Tuberculosis control relies on the translation of basic science and modern diagnostic techniques into primary health care clinics. These assays must be rapid, inexpensive, interpretation of results must be easy and they must be simple so that a healthcare worker with limited training can perform the tests under safe conditions. This study consists of four aims. The first aim was to develop a methodology to sterilize sputum specimens for rapid TB diagnosis and drug resistance testing. Candidate bactericides were identified from the literature, and tested for their bactericidal activity in Mycobacterium tuberculosis. We identified ultraseptin®aktiv as a powerful bactericidal agent which sterilizes sputum specimens for subsequent safe handling prior to light emitting diode microscopy and it also provides a DNA template for PCR-based tests. An algorithm has been proposed for the processing of specimens and rapid diagnosis of TB and drug resistant TB while patients wait for results.
Recently, the World Health Organization has endorsed the MTBDRplus test for diagnosis of TB and drug resistant TB. However genotypic tests may have more problems than anticipated. With the HIV pandemic, an increase of non-tuberculous mycobacteria has been reported. The sensitivity of genotypic tests in specimens with underlying non-tuberculous mycobacterial species therefore requires further evaluation. This study therefore also aimed at determining the reliability of the MTBDRplus assay for detection of drug resistant TB where non-tuberculous bacterial load is high. Clinically relevant non-tuberculous mycobacterium DNA and DNA from a multi-drug resistant TB isolate were obtained. Ratios of the different NTM with the MDR-TB DNA were made and subjected to the MTBDRplus assay. Known mix NTM and TB infected clinical isolates and sputum sediments were also evaluated for TB and drug resistance detection on the MTBDRplus assay. Under these conditions, this study provides evidence that the MTBDRplus test cannot reliably detect TB and drug resistance TB in specimens with underlying non-tuberculous mycobacteria.
Thirdly, to evaluate the sensitivity of the MTBDRplus assay for detecting drug resistance in hetero-resistant isolates, ratios were made using purified DNA from an MDR and pan-susceptible TB isolate. The MTBDRplus assay was then performed on the different ratios. We report that the MTBDRplus assay can efficiently detect wild type DNA in genes associated with resistance during the early evolution of drug resistance. However, in the later stage during treatment when both the wild type and mutants are present, the detection limit for the mutant DNA was 1:55. Due to these results, the MTBDRplus assay should still be further improved or other tests should be developed to address these limitations.
And finally to combat cross amplicon contamination during the final steps of genotypic detection with the MTBDRplus assay, a proof of concept for a patentable closed tube line probe device was proposed on the 4th aim. This device can be improved to enable automated drug resistance genotyping of multiple specimens.
The results of this study highlight the need for a sensitive inexpensive point of care drug resistance test that does not require intensive training. / AFRIKAANSE OPSOMMING: 'n Belangrike volgende stap om Tuberkulose te beheer is om basiese wetenskap resultate te gebruik sodat moderne diagnose tegnieke ontwikkel kan word wat in primêre gesondheidsorg klinieke toegepas kan word. Hierdie toetse moet vinnig, goedkoop, en die interpretasie van resultate moet maklik wees. Die toetse moet eenvoudig wees sodat 'n gesondheidswerker met beperkte opleiding die toetse onder veilige omstandighede kan uitvoer. Hierdie studie bestaan uit vier doelwitte, waarvan die eerste was om 'n metode te ontwikkel vir die sterilisasie van sputum monsters vir vinnige TB diagnose en die toesting van middelweerstandigheid. Kandidaat kiemdodende middels was geïdentifiseer vanaf die literatuur en die middels se kiekdodende aktiviteit was getoets op Mycobacterium tuberculosis. Ons het ultraseptin®aktiv geïdentifiseer as 'n kragtige kiemdodende middel wat bakteria in sputum monsters steriliseer vir veilige hantering voordat diagnose met 'n lig uitstralende diode mikroskopie gedoen kan word. Hierdie behandeling met ultraseptin®aktiv bied ook 'n DNA templaat vir PCR-gebaseerde toetse. 'n Algoritme is voorgestel vir die hantering van monsters en die vinnige diagnose van sensitiewe- en middel weerstandige Tuberkulose terwyl die pasiënte by die kliniek wag vir die resultate.
Onlangs het die Wêreld Gesondheid Organisasie die genotipiese MTBDRplus toets vir die diagnose van Tuberkulose en middel-weerstandige Tuberkulose onderskryf. Hierdie toets word tans op groot skaal in Suid Afrika gebruik. Dit kan egter wees dat genotipiese toetse baie meer probleme kan he as wat aanvanklik verwag is. Die HIV pandemie gaan toenemend gepaard met n toename van nie-tuberkulose mycobacteria. Die sensitiwiteit van genotipiese toetse op monsters met onderliggende nie-tuberkulose mikobakteriese spesies vereis dus verdere evaluasie. Die doel van hierdie studie was ook om die betroubaarheid van die MTBDRplus-toets te bepaal vir die opsporing van middelweerstandige TB waar die nie-tuberkulose bakteriële lading hoog is. DNA van kliniese relevante nie-tuberkulose mikobakteria en multi-middelweerstige TB isolate was bekom. Verskillende verdunnigs van die spesifieke NTM DNA te same met die van MDR-TB DNA is gemaak en onderwerp aan die MTBDRplus toets. Bekende gemengde NTM- en TB geïnfekteerde kliniese isolate en sputum sedimente was ook geevalueer vir die opsporing van TB en middel weerstandigheid met die MTBDRplus toets. Hierdie studie verskaf bewyse dat die
MTBDRplus toets nie betroubaar is met die diagnose van sensitiewe- en middel weerstandige Tuberkulose in monsters met onderliggende nie-tuberkulose mycobacteria nie.
Verskillende verdunnings van gesuiwerde DNA van MDR en pan-sensitiewe TB isolate is gemaak om die sensitiwiteit van die MTBDRplus toets vir die opsporing van middelweerstandigheid te bepaal. Die MDRDRplus toets is gebruik met hierdie verdunnings. Resultate in hierdie studie toon dat die MTBDRplus toets effektief is met die identifisering van wilde-tipe DNA (dit beteken middel sensitief) in gene wat geassosieer word met middel weerstandigheid gedurende die vroeë ontwikkeling van weerstandigheid. Hier teenoor toon die resultate dat in die later stadium tydens behandeling, wanneer beide die wilde-tipe (sensitief) en mutante DNA (weerstandig) teenwoordig is, is die opsporingslimiet vir die mutante DNA maar 1:55. As gevolg van hierdie resultate raai ons aan dat die MTBDRplus toets nog verder verbeter moet word of dat ander toetse ontwikkel moet word om hierdie beperkinge aan te spreek.
Amplikon kruiskontaminasie kan n groot impak hê op die betroubaarheid van enige genotipiese diagnostiese toets. Die finale stappe van MTBDRplus toets behels die gebruik van 'n oop sisteem sodat kontaminasie maklik kan plaasvind. In die 4de doewit 'n konsep vir 'n patenteerbare geslotebuis toestel ontwikkel en die resultate het getoon dat kontaminasie suksesvol uitgeskakel kan word. Hierdie toestel kan verbeter na 'n outomatiese apparaat verbeter word sodat die module genotipering van verskeie monsters moontlik kan maak.
Die resultate van hierdie studie beklemtoon die noodsaaklikheid van 'n sensitiewe goedkoop “point of care” diagnostiese toets wat nie intensiewe opleiding benodig nie. / Medical Research Council of South Africa / University of Stellenbosch, Dept. of Molecular Biology and Human Genetics
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Immune regulation in children and adults in a community with a high incidence of tuberculosisAdams, Joanita Frances Ann 12 1900 (has links)
Thesis (MScMedSc) -- Stellenbosch University, 1998. / Bibliography / ENGLISH ABSTRACT: There is a progressive maturation of the immune system from infancy to adulthood. The
immature immune system in early life is characterised by impaired macrophage function
and antigen presentation as well as a higher naIve to memory T cell ratio with subsequent
diminished IFN-y production. Children with tuberculosis often present with
lymphadenopathy, the complications thereof or with systemic spread of the organisms.
Adults generally manifest with pronounced systemic effects (such as weight loss and high
fever) and immunopathology (such as cavitation and fibrosis). We hypothesised that the
immunopathology in adults may be due to enhanced cytokine production in comparison
to children. The first aim of this study was therefore to measure cytokine responses in
healthy children and adults. Cytokine responses in patients with tuberculosis will be
examined in future studies. Peripheral blood mononuclear cells (PBMC) were isolated
from whole blood obtained from 9 healthy children and 9 healthy adults. The cells were
cultured in serum-free medium, unstimulated or polyclonally stimulated with
Phytohaemagglutinin (PHA). Supernatants were harvested after which IFN-y, IL-2,
TNF-a., IL-4 and IL-IO production was determined by means of ELISA analysis. Ri'J"A
was ~ubsequently extracted from the cells followed by RT-PCR analysis for the semiquantitative
determination of mRNA levels of these cytokines. PBMC isolated from
healthy children produced significantly less IFN-y protein than adults. Futhermore, IFN-y
production in the adults seemed to be trimodally distributed. No significant differences
could be found in the production of IL-2, TNF-a, IL-4 and IL-] O. Although children
produced low levels of IFN-y protein, their IFN-y, TNF-a, IL-2, IL-4 and IL-IO mRNA
levels were comparable to that of adults.
Tuberculosis is a major cause of mortality and morbidity, particularly in the third world.
Ravensmead and Uitsig, two adjacent suburbs in the Western Cape, have a tuberculosis
incidence of> I 000/100000 population. Also, up to 90 % of the children in the Western
Cape have been reported to be infested by intestinal parasites such as Ascaris
lumbricoides and Trichurius trichl/ria. Infection with M tuberculosis indut:es a Th 1
Stellenbosch University http://scholar.sun.ac.za
iv
In:.,c response, while intestinal parasites elicit a Th2 immune response. Th2
dominance induced by intestinal parasite infestations could predispose individuals to an
enhanced susceptibility to M. tuberculosis. The second aim of this study was to
investigate serum IgE levels, surrogate markers for Th2 activation, in the community.
The serum 19B levels were subsequently correlated to the tuberculosis incidence per
enumerator sub-district (ESD), crowding, female literacy and socio-economic levels. Similarly, the tuberculosis incidence per ESD was correlated with the above mentioned
parameters. A significant positive correlation was found between tuberculosis incidence
and the serum 19E levels in the community. However, further studies are needed to
determine if intestinal parasites are the main cause of the high 19B levels in the
community and to dCh111ine if parasite loads or Th2 dominance are causally linked to the
incidence of tuberculosis. Correlation between serum 19E levels and tuberculosis
incidence with the other parameters were significant, except in the case of crowding.
The third aim of this study was to measure serum IgE and specific 19E levels against
Ascaris and common allergens on presentation of tuberculosis and again after completion
of successful treatment. Significant declines in serum 19B and Ascaris specific 19B levels
were observed after completion of tuberculosis treatment. This down regulation of IgE
levels may be due to an up regulation of ThI responses in patients following successful
treatment for tuberculosis. / AFRIKAANSE OPSOMMING: Die immuunsisteem matureer toenemend vanaf kinderjare tot en met volwassewording.
Die onvolwasse immuunsisteem van jong kinders word gekenmerk deur verswakte
makrofaag-funksionering en antigeenpresentering, sowe) as 'n verhoogde naiwe tot
geheue T-sel verhouding met gevolglikc verminderde IFN-y produksie. Kinders met
tuberkulose presenteer gewoonlik met Iimfadenopatie, komplikasies daarvan of met
gedissemineerde siekte. Volwassenes presenteer met sistemiese gevolge (soos
gewigsverlies en hoe koors) en immunopatologie (soos kavitasie en fibrose). Ons
hipotese is dat die immunopatologie in volwassenes die gevolg is van 'n verhoogde
sitokienproduksie in vergelyking met kinders. Die eerste doelwit van die studie was om
sitokienproduksie in gesonde kinders en volwassenes te meet. Sitokienproduksie in
tuberkulose pasiente sal in 'n opvolgstudie bepaal word. Perifere bloed mononukleere
selle was geisoleer vanuit heel bloed verkry vanaf 9 gesonde kinders en 9 gesonde
volwassenes. Die selle was gekweek, ongestimuleer of gestimuleer met
Phytohaemagglutinien (PHA). Supernatante was geoes vir die bepaling van IFN-y, IL-2,
IL-4, IL-I0 en TNF-a. produksie, deur gebruik te maak van ELISA analise. RNA was
gevolglik vanaf die selle ge-ekstraheer vir die tru-transkriptase polimeerketting reaksie
analise, waartydens sitokien mRNA vlakke op 'n semi-kwantitatiewe wyse bepaal was.
Perifere bloed mononukleere selle geisoleer vanaf die kinders het minder IFN-y
geproduseer as die van volwassenes. Hierdie verminderde produksie was hoogs
betekenisvol. Dit wou voorkom asof die IFN-y produksie deur volwassenes trimodaal
versprei was. Geen betekenisvolle verskille tussen kinders en volwassenes kon gevind
word in die produksie van IL-2, IL-4, IL-IO en TNF-a nie. Alhoewel kinders minder
IFN-y proteien geproduseer het, het hulle IFN-y, IL-2, IL-4, IL-JO en TNF-a mRNA
produksie met vlakke van volwassenes ooreengestem.
Tuberkulose speel 'n groat rol in morbiditeit en mortaliteit in veral die derde wereld.
Ravensmead en Uitsig, twee aangrensende voorstede in die Wes-Kaap, het 'n tuberkulose
voorkomssyfer van> 1 000/1 00000 populasie. Verder, is tot 90 % van die kinders in die
Stellenbosch University http://scholar.sun.ac.za
VI
Wes-Kaap gei'nfesteer met intestinale parasiete soos Ascaris Ilimbricoides en Trichllrills
trichllria. M. tuberculosis infeksie induseer 'n Thl immuunrespons, terwyl intestinale
parasiete 'n Th2 immuunrespons uitlok. 'n Dominante Th2 respons mag moontlik
individue predisponeer tot 'n verhoogde vatbaarheid vir M. tuberculosis. Gevolglik was
die tweede doelwit van die studie om serum IgE vlakke as surrogaat merkers vir Th2
aktivering in die gemeenskap bestudeer. Die serum IgE vlakke was gevolglik gekorreleer
met die tuberkulose voorkoms per opnemerssensusgebied (OSG), saamdringing, vroulike
geletterdheid en sosio-ekonomiese vlakke. Die tuberkulose voorkoms per OSG, is op
dieselfde wyse gekorreleer met die bogenoemde parameters. 'n Betekenisvolle positiewe
korrelasie is gevind tussen tuberkulose voorkoms en serum IgE vlakke in die
gemeenskap. Verdere stuciies is egter nodig om te bepaal of intestinale parasiete weI die
oorsaak van die hoe IgE vlakke in die gemeenskap is en of parasiet ladings of Th2
dominansie oorsaaklik verbind kan word aan die tuberkulose voorkoms.
Die derde doelwit van die studie was om serum 19E en spesifieke IgE vlakke teen Ascaris
en algemene allergene te meet met presentering van tuberkulose en weer na voltooing
van suksesvolle behandeling. 'n Betekenisvolle afname in serum 19E en Ascaris
spesifieke 19E vlakke is waargeneem na vohooing van tuberkulose behandeling. Die
afregulering van 19E vlakke kan moontlik toegeskryf word aan die opregulering van Th1
response in pasi"ente na voltooing van suksesvolle behandeling van tuberkulose.
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Investigation into genotypic diagnostics for mycobacterium tuberculosisHoek, Kim Gilberte Pauline 12 1900 (has links)
Thesis (PhD )--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB)
cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and
extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid
diagnostic methods are developed. Molecular-based assays have the potential for the rapid
species-specific diagnosis of TB and associated drug-resistances directly from clinical
specimens. We investigated whether high resolution melting analysis (HRM) could enable
the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and
reagents are relatively inexpensive and the methodology can easily be implemented in high incidence,
low income regions.
Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis
from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the
region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other
members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%,
respectively) when compared to the reference standard (positive cytology and/or positive
culture). Despite this limitation our method was able to provide a rapid diagnosis in more than
half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We
therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM
and cytology results indicative of mycobacterial disease.
We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which
allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the
rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method
was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene
with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA
sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture
based method. We identified a strong association between EMB resistance (and pyrazinamide
resistance) and MDR-TB and subsequently advised modifications to the current (2008) South
African National TB Control Programme draft policy guidelines.
Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to
a closed-tube one-step method using the detection of inhA promoter mutations conferring
isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA
promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of
particular interest as they confer low level INH resistance and cross-resistance to ethionamide
(Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and
Eastern Cape Provinces of South Africa, data generated by the recently implemented
GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a
patient depending on their INH mutation profile. Our proposed treatment algorithm may be
particularly useful in XDR-TB cases, for which only few active drugs remain available.
Since current diagnostic methods all carry advantages and disadvantages, a combination of
phenotypic and genotypic-based methodologies may be the best scenario while awaiting
superior methods. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters
weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die
volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat
vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig
spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie
studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die
weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense
en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit
uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste.
Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies,
het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die
gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar
afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was
(51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard
(positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige
diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit
(94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die
resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel.
‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur
mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%,
onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum
rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode
kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid
teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit
en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling.
Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate
kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en
weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge
aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel.
Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas
tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies
wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n
sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies
veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide
(Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en
Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets
moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van
die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte
vir wie daar weinig aktiewe antibiotika beskikbaar is.
Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese
en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.
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Imunohromatografski test u diferencijalnoj laboratorijskoj dijagnostici tuberkuloze pluća / Immunochromatographic test in differential laboratory diagnostic of tuberculosisSavković Tijana 01 April 2016 (has links)
<p>UVOD: Tuberkuloza je odavno poznata bolest koja i danas u 21. veku još uvek predstavlja veliki javnozdravstveni problem, uprkos primeni moćnih antituberkuloznih lekova. Trećina svetske populacije inficirana je bacilom tuberkuloze. Svake godine oboli oko osam miliona, a umre oko dva miliona ljudi, zbog čega je tuberkuloza i dalje infektivno oboljenje sa najvećom stopom smrtnosti. Kasna dijagnoza, multirezistentna tuberkuloza i udruženost sa HIV infekcijom predstavljaju jednu od najvećih prepreka za efikasnu kontrolu ove bolesti u svetu. Rano otkrivanje se oslanja na kvalitetnu bakteriološku dijagnostiku koja je kamen temeljac svakog nacionalnog programa za kontrolu tuberkuloze. Brza i tačna mikrobiološka dijagnostika predstavlja osnovu programa kontrole tuberkuloze i zbog toga je uvođenje novih i brzih laboratorijskih testova od veoma velikog značaja. Razvijen je novi komercijalno dostupni imunohromatografski test koji se zasniva na detekciji antigena MPT64 glavnog sekretovanog proteina M. tuberculosis. Test je brz i pouzdan u identifikaciji izolovanih sojeva M. tuberculosis i jeftiniji je od konvencionalnih biohemijskih i molekularnih testova. CILJ: Ciljevi istraživanja su bili da se evaluiraju karakteristike novog brzog imunohromatografskog testa u identifikaciji mikobakterija izolovanih iz respiratornih uzoraka bolesnika sa tuberkulozom pluća i referentnih sojeva klinički značajnih vrsta netuberkuloznih mikobakterija (NTM). MATERIJAL I METODE: Istraživanje je sprovedeno u periodu od 1.1.2010. do 31.12.2013. i obuhvatilo je 43563 respiratornih uzoraka dobijenih od bolesnika hospitalizovanih u Institutu za plućne bolesti Vojvodine. Iz obrađenih respiratornih uzoraka izolovano je 3469 izolata mikobakterija. Identifikacija do nivoa vrste urađena je primenom standardnih biohemijskih testova, molekularnog testa (GenoType® Mycobacterium) i imunohromatografskog testa (BDMGIT Tbc). U istraživanje je uključeno 100 sojeva Gram pozitivnih i Gram negativnih bakterija (n = 19 vrsta) izolovanih iz respiratornih kliničkih uzoraka. Identifikacija do nivoa vrste je potvrđena komercijalnim identifikacionim sistemima. REZULTATI: U toku četvorogodišnjeg istraživanja izolovano je 3469 izolata mikobakterija iz respiratornih uzoraka. U ispitivanom periodu ne postoji opadajući trend izolacije mikobakterija što potvrđuje i koeficijent korelacije (r = 0,31). Svi izolati mikobakterija su identifikovani konvencionalnim biohemijskim ispitivanjima koja pokazuju da je 89% od svih izolata identifikovano kao Mycobacterium tuberculosis (M. tuberculosis), a 11% izolata kao NTM. Mycobacterium xenopi je bila najzastupljenija NTM vrsta identifikovana kod 55,3% izolata. Nakon biohemijske identifikacije kod 300 izolata M. tuberculosis i 100 izolata NTM, identifikacija je potvrđena komercijalno dostupnim molekularnim i imunohromatografskim testom. Na osnovu rezultata testiranja mikobakterija imunohromatografskim testom, senzitivnost, specifičnost, pozitivne i negativne prediktivne vrednosti bile su: 99,7%, 100%, 100% i 99%. U poređenju imunohromatografskog testa sa konvencionalnim biohemijskim ispitivanjima nije nađena statistički značajna razlika (p> 0,5). Kappa vrednost testa je iznosila 0,993, a interval poverenja CI =0,98 – 1,00. U poređenju imunohromatografskog sa molekularnim testom vrednost kappa je iznosila 0,993, a interval poverenja CI = 0,98 – 1,00. Slaganje rezultata je potvrđeno i McNemar testom sa vrednošću 0,99. Utvrđena je stabilnost sekretovanog antigena MPT64 i posle 5 godina od prvog testiranja. ZAKLJUČAK: Visoka senzitivnost i specifičnost imunohromatografskog testa omogućuju tačnu i preciznu identifikaciju M. tuberculosis kao i pouzdanu diferencijaciju M.tuberculosis od NTM – a. Imunohromatografski test može da predstavlja zamenu za konvencionalne biohemijske i molekularne testove u identifikaciji M. tuberculosis. Jeftiniji je, jednostavniji za izvođenje i brže se dobijaju rezultati čime seskraćuje vreme za postavljanje dijagnoze.</p> / <p>INTRODUCTION: Tuberculosis (TB) has been known as a disease for a long time, but nevertheless it represents a major public health issue even nowadays in the 21st century, despite potent antituberculous drugs applied. One third of the world population is infected by the TB bacillus. About eight million people get infected and two million die of tuberculosis in a year, so tuberculosis is still an infectious disease with the greatest mortality rate. Late diagnosis, multiresistant tuberculosis and concomitant HIV infection interfere mostly with an efficient control of the disease all over the world. Early TB detection largely depends on the high-quality bacteriological diagnostics, which is the corner stone of each national TB control programme. A fast and accurate microbiological TB diagnosis plays a crucial role in any TB control programme. It is therefore very important to introduce new and fast laboratory tests. A novel commercially available immunochromatographic test has been designed, based on the MPT64 antigen of the major M. tuberculosis – secreted protein. This is a rapid and reliable test to identify the isolated strains of M. tuberculosis, which is not expensive as conventional biochemical and molecular tests. OBJECTIVE: The objective of the investigation was to evaluate the new immunochromatographic rapid test to identify mycobacteria isolated from respiratory samples from pulmonary TB patients, and referential strains of clinically relevant species of nontuberculous mycobacteria (NTM). MATERIAL AND METHODS: The research was carried out in the period from 1st January, 2010 to 31st December, 2013. It included 43 563 respiratory samples obtained from the patients hospitalized in the Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica (Serbia). There were 3 469 mycobacterial isolates obtained from the processed respiratory samples. The species – level identification was performed by standard biochemical tests, the molecular test (GenoType®Mycobacterium), and the immunochromatographic test (BD MGIT Tbc). The study included one hundred (100) of Gram positive and Gram negative bacteria (n = 19 species) isolated from respiratory clinical samples. The species – level identification was confirmed by commercial identification systems. RESULTS: During the four – year investigation, 3 469 mycobacterial isolates were obtained from respiratory samples. No declining tendency of mycobacterial isolation was registered in the examined period, as confirmed by the correlation coefficient (r = 0.31). All mycobacterial isolates were identified by conventional biochemical tests showing that 89% of all isolates were identified as M. tuberculosis, and 11% of the isolates as NTM. Mycobacterium xenopi was the most common NTM species identified in 55.3% of the isolates. Following the biochemical identification in 300 M. tuberculosis isolates and 100 NTM isolates, the identification was confirmed by commercially available molecular and immunochromatographic tests. Based on immunochromatographic testing of mycobacteria, the sensitivity, specificity, positive and negative predictive values of the test were 99.7%, 100%, 100% and 99% respectively. There is no statistically significant difference (p> 0.5) when comparing features of immunochromatographic test with conventional biochemical assay. The kappa test value was 0.993, and the confidence interval CI = 0.98 – 1.00. Comparing the immunochromatographic with the molecular test, the kappa value was 0.993, and the confidence interval CI = 0.98 – 1.00. The congruence of the tests findings was also confirmed by the McNemar test, estimated to 0.99. The stability of the secreted MPT64 antigen was registered even five years after the first testing episode. CONCLUSION: The high sensitivity and specificity of the imunochromatographic test enable an accurate and precise identification of M. tuberculosis, as well as a reliable differentiation of M. tuberculosis from NTM. The immunochromatographic test may substitute conventional biochemical and molecular tests to identify M. tuberculosis. It is easier to perform and provides faster test results, thus reducing the time of establishing the diagnosis.</p>
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