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The role of corticotropin-releasing factor in anxiety disordersPietersen, Charmaine Y. 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: SEPARATION STUDY
Traumatic experiences during childhood can have a negative impact on behaviour
later in life. Kendier et al. (1992) found that the loss of a parent during childhood
increased the risk to develop major anxiety disorders and could also lead to
depressive-like behaviour (Furukawa et al., 1999).
Methods:
We subjected rat pups to maternal separation and determined the effects thereof on
adult behaviour. We removed rat pups from their mothers for 3 hours daily from
postnatal day 2 to 14. On day 60, the behaviours of the rats were tested using the
elevated plus-maze and the open field test. Controls were reared normally.
Behaviours: Amount of time spent and the number of entries into the arms of the
maze were noted on the elevated plus-maze, while the total time spent in each zone
(inner versus outer) and the number of zone crossings were noted for each rat on the
open field arena. The latency to move from the initial placement in the outer zone to
the inner zone as well as the number of quadrant crossings was also determined.
Defecation, freezing, rearing and grooming behaviours were also noted.
Neurotransmitter levels: Noradrenaline, serotonin and their metabolites were
evaluated in maternally separated rats and compared to controls. Their concentrations
at basal level, immediately after restraint stress and 15 minutes after restraint stress,
were also determined. A HPLC method was followed in these determinations. ACTH Determinations: All rats were subjected to restraint stress for a lO-minute
period. Trunk blood was collected for basal, as well as 15 and 60 minutes postrestraint
stress for ACTH determinations.
Results:
Behaviours: The amount of entries was significantly reduced in the separated
animals, indicating decreased locomotion. They spent significantly more time in the
closed maze arms. A significant increase in defecation frequency and rearing
behaviour was noted. These observations are typical of anxious behaviour. In the
open field test, the behavioural results were less convincing. Only a significant
increase in defecation frequency and a significant decrease in rearing behaviour in
separated animals, were observed.
Neurotransmitter levels: No significant differences were noted between separated
animals and controls with respect to basal monoamine levels. However,
noradrenaline levels were significantly decreased in the frontal cortex 15 minutes
after restraint stress and immediately after restraint stress in the hypothalamus and
hippocampus in separated animals. MHPG levels were significantly decreased in the
frontal cortex immediately after restraint stress. No significant differences were
found with respect to serotonin levels. However, significant increases were found in
5HIAA levels in the frontal cortex and hippocampus of separated rats, 15 minutes
after restraint stress.
The basal turnover ratios of serotonin (5HIAA/5HT) and noradrenaline (MHPGINA)
did not yield significant results. However, immediately after restraint stress, a
significant increase was found in serotonin turnover in the hypothalamus of separated rats when compared to controls. This turnover rate was also increased in separated
rats, 15 minutes after restraint stress in the frontal cortex and hypothalamus.
ACTH Determinations: Basal ACTH levels were significantly higher in separated
animals. At 15 minutes post-restraint stress, the levels were significantly lower than
controls, indicating a blunted stress response.
Our results therefore showed that maternal separation could lead to anxious
behaviours in adult life. These behavioural abnormalities were associated with
alterations in the central nervous and neuroendocrinological systems, particularly in
response to stressful situations.
CRF STUDY
The maternal separation study indicated that elevated CRF levels could possibly be
causally related to abnormalities observed in the anxious animals. We therefore
hypothesised that adverse development factors, such as maternal separation,
predisposes individuals to develop psychopathologies later in life and that this
process was driven by a presence of high CRF levels.
Methods:
Cannulas were implanted into the left lateral ventricles of normal rats, making use of
stereotaxic procedures. CRF (3 flg/fll) was injected into the ventricles daily for 5
days. Saline controls were handled similarly, but only injected with saline for the same time period. Both groups of animals were then compared to naïve controls.
Histology was performed to determine the correct placement of the cannulas.
Behaviours: The Elevated Plus-maze was employed to determine whether their
behaviours were anxious. The number of entries into the various arms of the maze as
well as the amount of time spent in the open and closed arms was accumulated.
Rearing, freezing, defecation and grooming were also noted.
ACTH Determinations: The ACTH levels ofCRF-injected, saline-injected and naïve
rats were determined 15 minutes after restraint stress.
Results:
Behaviours: A decrease in the number of entries into the closed arms of the maze
was noted in the CRF-injected rats when compared to naïve controls. No significant
differences were found between the groups with respect to the amount of time spent
in the various arms and the behaviours noted during the experiment.
ACTH Determinations: A decrease in ACTH levels was noted in CRF-injected rats
15 minutes after restraint stress when compared to naïve controls. Therefore,
although the CRF injections did not alter the behaviour of the rat, they did exhibit a
blunted stress response to the stressor.
Conclusion:
Our experiments led us to conclude that early adverse experiences, such as maternal
separation, can lead to the development of psychopathologies later in life. CRF, however, is not pivotal in the development of these abnormalities; rather it seems that
the neurochemical abnormalities (serotonin and noradrenaline) play a more important
role in the development of these mental disturbances. Finally, we hypothesise that
combination drug therapy that targets both the noradrenergic and serotonergic
neurotransmitter systems could be preferred above those aimed at rectifying the
individual neurotransmitter systems in the treatment of psychopathologies, such as
anxiety disorders. / AFRIKAANSE OPSOMMING: MOEDERLIKE SKEIDINGS STUDIE
Traumatiese gebeurtenisse wat gedurende kinderjare ervaar word, kan 'n negatiewe
impak op die gedrag van dieselfde individue hê, as hulle volwassenheid bereik het.
Kendier et al. (1992) het waargeneem dat die verlies van 'n ouer tydens die
kinderjare, die risiko om angssteumisse te ontwikkel, dramaties verhoog en kan ook
lei tot 'n depressiewe gemoedtoestand (Furukawa et al., 1999).
Metodes:
Ons het neonatale rotte aan moederlike skeiding blootgestel en die effekte daarvan op
gedrag tydens hul volwasse lewe beoordeel. Ons het daagliks die moeders vir 3 ure
van die kleintjies afweggeneem, vanafpostnatale dag 2 tot 14. Op dag 60, het ons die
gedrag van die diere op die "elevated plus-maze" en die" open field test" getoets.
Kontrole rotte het onder normale omstandighede opgegroei.
Gedrag parameters: Die hoeveelheid tyd en aantal kere wat die rotte in die
verskillende arms van die "elevated plus-maze" gespandeer het, was waargeneem. Die
totale tyd in die "open field" toets se binneste ofbuitenste sones, die hoeveelheid
kruisings tussen die twee sones, die tyd wat dit neem om beweging in die binneste
sone te inisiëer, sowel as die hoeveelheid kwadrante wat gekruis was, is genotuleer.
Defekasie, botstilstande, steiering, en versorgingsgedragte was ook waargeneem
terwyl die rotte in die doolhowe was. Neurochemiese oordragstowwe: Die hippokampus, hipotalamus en frontale korteks
van moerderlik-geskeide rotte en kontroles, was uit hul brein gedissekteer om die
vlakke van noradrenalien, serotonien en hul metaboliete daarin te bepaal. Basale
vlakke sowel as hul konsentrasies onmiddelik na stres en 15 minute na stres, was
gedetermineer. 'n HPLC metode was gebruik vir hierdie bepalings.
ACTH bepalings: Rotte, moederlik-geskei en kontroles, was onderwerp aan
beperkingstres vir 'n tydsduur van 10 minute. Bloed was op die volgende
tydsintervalle gekollekteer vir die bepaling van ACTH vlakke, naamlik basaal, 15
minute en 60 minute na die einde van stresperiode.
Resultate:
Gedrag: Op die "elevated plus-maze" was moederlik-geskeide rotte minder
beweeglik omdat hul aanmerklik minder die arms van die doolhowe binne gegaan het.
Hulle het ook baie meer tyd in die geslote arms gespandeer. Verder het die
eksperimentele rotte meer defekasie bolusse uitgeskei en was die aantal steieringe
uitgevoer, ook aanmerklik verhoog. Hierdie patroon van gedrag is tipies die van
angstigheid.
Neurochemiese oordragstowwe: Daar was geen betekenisvolle verskil tussen die
basale neurotransmitter vlakke van moederlik-geskeide rotte en hul kontroles.
Daarenteen was die vlakke van noradrenalien in die frontale korteks dramaties
verhoog by die 15 minute tydsinterval na die stres, asook onmiddelik na die stres in
die hipotalamus en hippokampus. MHPG vlakke was egter aanmerklik verlaag in die
frontale korteks onmiddelik na die stres. Terwyl daar geen noemenswaardige verskil in serotonien vlakke waargeneem is nie, was die vlakke van 5HlAA betekenisvol
verhoog in die frontale korteks en hippokampus van moederlik-geskeide rotte, 15
minute na die beperkingstres. Geen verskil in die omsettingsverhoudinge van basale
serotonien (5HlAA/5HT) ofnoradrenalien (MHPGINA) vlakke is gevind nie. Daar
was egter 'n betekenisvolle verhoging in die serotonien omset in die hipotalamus van
moerdlik-geskeide rotte, onmiddelik na beperkingstres. Hierdie verskil het ook
voorgekom 15 minute na die stresperiode in die hipotalamus, sowel as in die frontale
korteks.
ACTH bepalings: Rotte wat onderwerp was aan moederlike skeiding het verhoogde
basale konsentrasies van ACTH getoon. Die ACTH vlakke was egter aanmerklik laer
15 minute na stres toe dit met kontrole groepe vergelyk is.
Ons resultate toon dus dat moerderlike-skeiding wel tot angstige gedrag tydens die
volwasse lewe kan lei. Hierdie afwyking in gedrag was geassosieër met
abnormaliteite in die sentrale senuwee sisteem sowel as die neuroendokrienologiese
sisteem van die dier, veralonder toestande van stres.
Na gelang van ons bevindinge in die moerderlike skeidingstudie, het dit geblyk dat
CRF 'n belangrike rol speel tot daarstelling van angstige gedrag. Daarom het ons in
die tweede deel van ons studie gaan kyk ofverhoogde vlakke van CRF in die brein
moontlik die gedrag van die rot kon verander. CRF STUDIE
Metodes:
Kannules was in die linker ventrikel van die breine van normale rotte geïmplanteer
deur gebruik te maak van stereotaktiese prosedures. CRF (3 Ilg/IlI) was daagliks vir 5
dae aan die rotte toegedien. Rotte wat presies dieselfde gehanteer was het 'n
fisiologiese soutoplossing ontvang. Hierdie rotte was met naïewe rotte vergelyk. Die
korrekte plasing van kannules was met histologiese metodes bevestig.
Gedrag: Die "elevated plus-maze" was gebruik om te bepaal of angstige gedragte
by behandelde rotte ontlok was. Die aantal kere wat 'n rot die verskillende arms van
die doolhofbinne gaan, sowel as die tyd wat die dier op elke arm deurbring was
genotuleer. Die aantal steierings, botstilstande, defekasies en versorgingsbewegings
was weereens waargeneem.
ACTH bepalings: Die vlakke van ACTH was bepaal in al die rotgroepe, 15 minute
nadat hulle aan 10 minute beperkingstres onderwerp was.
Resultate:
Gedrag: Rotte wat met CRF toegedien was, het op minder geleenthede die toe arms
van die "elevated plus-maze" binne gegaan toe hulle met die naïewe groep rotte
vergelyk was. Hierdie verskil was betekenisvol. Daar was geen ander
noemenswaardige verskille ten opsigte van die ander gedragsparameter nie. ACTH bepalings: Daar was 'n afname in die ACTH vlakke, 15 minute na die stres
toegedien was in rotte wat CRF ontvang het, in vergelyking tot die naïewe kontrole
groep.
Hierdie resultate dui daarop dat die toediening van CRF in die brein nie die rot se
gedrag, maar wel die dier se respons op stres, beïnvloed het.
Gevolgtrekking:
In die lig van die voorafgaande resultate verky, blyk dit dat moederlike-skeiding
tydens die vroeë kinderjare wel kan aanleiding gee tot angstige gedrag tydens
volwassenheid. Ons studies dui ook aan dat CRF nie die primêre bron van hierdie
gedrags afwykings is nie, maar dat abnormaliteite in die neurochemiese
oordragstowwe (serotonien en noradrenalien) eerder die bepalende faktore is. Ten
slotte, ons beveel aan dat geneesmiddels wat geskoei is om die serotonerge sowel as
die noradrenerge sisteme aan te spreek, voordeel moet geniet in die behandeling van
gedragstoomisse, soos angs.
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Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigensScriba, Thomas Jens 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an
estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is
overwhelmingly prevalent in Botswana and South Africa and to date no interventions
have been successful enough to curb the rapid spread of the virus. A number of
HIV-1 vaccine strategies are being developed, however the breadth and efficacy of
such candidate vaccines, many of which are based on the HIV-1 structural genes pol,
gag and env, have mostly been found to be inadequate.
The HIV-1 accessory genes are attractive components of HIV vaccines due to their
role in viral pathogenesis, early expression and the high ratio of conserved CTl
epitopes. Yet, because of undesirable properties questions regarding their safety as
vaccine components are raised. In this study candidate tat, rev and nefmutants were
assessed for efficient expression and inactivation of undesirable functionality.
/
Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat,
Rev and Nef proteins were constructed. The coding sequences of the genes were
codon-optimised for optimum protein expression and these synthetic genes were
constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins
were mutated at previously described sites by PCR-based site-directed mutagenesis
to render them inactive for their respective functions. Corresponding wild-type Tat,
Rev and Nef constructs were also made from viral isolates that were least dissimilar
to the respective consensus amino acid sequences. tn vitro expression of the
different constructs were assessed in 293 cells by Western blotting with polyclonal
mouse sera, which were generated by DNA immunisation with one of the Tat, Rev
and Nef constructs. The transactivation activity of Tat variants and Rev-mediated
nuclear export activity of RRE-containing transcripts were studied in cotransfection
experiments using reporter-gene-based assays while Nef functionality was assessed
in a cotransfection assay with subsequent flow cytometric analysis of surface CD4
and MHC-I expression on 293 cells.
Sequence analysis of the South African HIV-1 subtype C consensus sequences of
Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence
that was drawn up from a large number of viruses from southern Africa. These
consensus sequences were also closer to individual viral isolate sequences than any
individual sequences were, indicating that the use of a consensus sequence may
serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not
significantly higher than the wild-type constructs, however, the codon-optimised rev
mutant exhibited markedly higher expression than the wild-type rev construct.
Immunoreactivity of the protein with the mouse sera demonstrates expression and
immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of
the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent
CAT expression in 293T and Hela cells. Yet, this activity was significantly
impaired using the single mutation, C3?, or the double mutation, C22C3? Compared
to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was
completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised
consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4
downregulation was completely abrogated in one of the mutants, while both Nef
mutants were entirely deficient for MHC-I downregulation.
These data demonstrate the high expression levels and impaired functionality of
sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens
that may be used as single-standing vaccine components or form part of a multicomponent
HIV-1 vaccine. / AFRIKAANSE OPSOMMING: Sedert die eerste gevalle van MIV in die vroeë 1980's beskryf is het die virus
wêreldwyd versprei en 'n beraamde 28.1 miljoen mense in sub-Sahara Afrika was
teen die einde van 2001 geïnfekteer. MIV-1 subtipe C kom verreweg die meeste voor
in Botswana en Suid-Afrika en tans is daar geen suksesvolle tussenkoms wat die
vinnige verspreiding van die virus kan stuit nie. 'n Aantal MIV-1 subtipe C
entstofstrategieë word tans ontwikkel maar die spektrum en effektiwiteit van sulke
entstowwe, waarvan baie op die MIV strukturele gene gag, pol en env gebaseer is, is
tans onvoldoende.
Die MIV-1 bykomstige gene is aantreklike entstofkomponente omdat hulle vroeg
uitgedruk word, 'n belangrike rol in virale patogenese speel en omdat hulle 'n hoë
verhouding van gekonserveerde sitotoksiese T-limfosiet (STL) epitope tot grootte
besit. Vanweë hierdie gene se verskeie ongewenste eienskappe word vrae ten
opsigte van hul veilige insluiting in enstofstrategieë geopper. Hierdie studie omskryf
die evaluasie van kandidaat tat, reven nef mutante vir doeltreffende
proteïenuitdrukking en funksionele onaktiwiteit.
Plasmiedkonstrukte wat vir die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Rev
en Nef proteïene kodeer is saamgestel. Die koderingsvolgordes van die gene is
geoptimiseer vir optimale uitdrukking en die sintetiese gene is van oorvleuelende 50-
mer oligonukleotiede vervaardig. Deur van PKR-gebaseerde site-directed
mutagenese gebruik te maak is hierdie proteïene gemuteer op posisies wat voorheen
geïdentifiseer is. Ooreenstemmende wilde-tipe Tat, Reven Nef konstrukte is gemaak
vanaf virale isolate waarvan die aminosuurvolgordes die meeste ooreenstem met dié
van die konsensusvolgorde. In vitro uitdrukking van die konstrukte in 293 selle is met
behulp van immunoklad met poliklonale muissera bepaal. Die serum is gegenereer
deur DNS immunisasie van muise met een elk van die Tat, Reven Nef konstrukte.
Die transaktiverings-aktiwiteit van Tat variante en Rev bemiddelde uitvoer van RREbesittende
transkripte uit die nukleus is in verklikkergeen kotransfeksie-eksperimente
bestudeer. Nef se funksionaliteit is deur kotransfeksie en die daaropvolgende
vloeisitometriese analise van 293 selle se oppervlak-CD4 en MHC-I uitdrukking
bestudeer.
Nukleotiedvolgorde-analise van die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat,
Reven Nef proteiëne toon 'n hoë vlak van ooreenkoms met 'n konsensusvolgorde
wat afgelei is vanaf 'n groot aantal suider-Afrikaanse virusse. Hierdie konsensusvolgordes is ook meer soortgelyk aan individuele virale isolate as enige
individuele volgordes. Vanuit hierdie data kan afgelei word dat die gebruik van so 'n
konsensusvolgorde die genetiese diversiteit tussen 'n entstof en sirkuierende virusse
kan verminder.
Uitdrukkingsvlakke van die volgorde-geoptimiseerde tat en nef geenkonstrukte is nie
merkbaar hoër as die van die wilde-tipe konstrukte nie. In teenstelling het die
volgorde-geoptimiseerde rev mutant merkbaar hoër uitdrukkingsvlakke as die wildetipe
getoon. Immunoreaktiwiteit van die proteïene met die muissera demonstreer dat
die Tat, Reven Nef proteïene uitgedruk word en immunogenies in muise is. 'n
Enkele C22 mutasie in Tat is nie genoeg om lTR-afhanklike CAT uitdrukking in 293T
en Hela selle te inaktiveer nie. In teenstelling is hierdie aktiwiteit geïnhibeer vir Tat
proteïene met die enkel mutasie C37 en die dubbel mutasie C22C37. In vergelyking
met die funksionele aktiwiteit van die wilde-tipe Rev is dié van die Rev mutant
M5M10 heeltemal geïnhibeer. Die wilde-tipe en geoptimiseerde, konsensus Nef
proteïene het seloppervlak-CD4 en -MHC-I uitdrukking verlaag, maar hierdie effek
van afregulering van CD4 uitdrukking was heeltemaal opgehef in een Nef mutant en
van MHC-I uitdrukking in beide Nef mutante.
Hierdie data demonstreer die hoë uitdrukkingsvlakke en geïnhibeerde funksionaliteit
van volgorde-gemodifiseerde MIV-1 subtipe C konsensus Tat, Reven Nef DNS
immunogene wat as enkelstaande enstof kan optree of deel kan uitmaak van 'n
multi-komponent MIV-1 entstof.
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Phenotypic factors influencing Mycobacterium tuberculosis phenotypeMoses, Lorraine 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT:
Please see fulltext for abstract. / AFRIKAANSE OPSOMMING:
Sien asb volteks vir opsomming.
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Analysis of hereditary haemochromatosis and clinical correlations in the elderlyBouwens, C. S. H. 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Hereditary haemochromatosis (HH) is an autosomal recessive iron storage disease where
the accumulation of iron in parenchymal organs may lead to diabetes, heart failure, liver
cirrhosis, arthropathy, weakness and a variety of other ailments if preventive measures are
not taken. HH is often not considered as a cause of these conditions, particularly not in the
elderly where the background frequencies of type II diabetes, osteoarthritis and heart
failure are generally high. Heterozygosity for C282Y, the HFE-mutation causing HH in
approximately 80% of affected individuals worldwide, has been linked to a raised
incidence of malignancies of the colon and rectum, stomach and the haematological
system. One of the highest carrier-frequencies (116) in the world for this mutation has been
reported in the South-African Afrikaner population, resulting in C282Y-homozygosity in
approximately 1 in every 115 people in this group.
A sample of 197 elderly Afrikaner volunteers was recruited for genotype/phenotype
association studies. Their clinical presentation was denoted, biochemical iron-status
determined and HFE genotyping performed. Either an increase or decrease in survival, or
both, were proposed, depending on possible gender effects. HH has been positively
associated with various cancer types, but may also protect against iron-deficiency anaemia
which is by far the most frequent cause of anaemia in the older person.
This study has led to the following findings:
1. The carrier frequency of mutation C282Y was found to be 1/8 in the elderly population
(similar in males and females), which is slightly lower than the 1/6 reported in younger
adults from the same population. Only one C282Y homozygote and two C282YIH63D
compound heterozygotes were detected, all of them female.
2. The prevalence of diabetes, heart disease, arthropathy or a combination of these
conditions did not differ significantly in C282Y heterozygotes and the mutationnegative
group.
3. Among 24 C282Y heterozygotes only one individual with rectal carcmoma was
detected compared with two cases with rectal- and seven with colonic malignancies in
153 mutation-negative individuals. The single female C282Y homozygote identified
suffered from both rectal and colon carcinoma and died approximately 6 months ago as
a consequence of her colon malignancy.
4. Serum ferritin appears to be a highly unreliable parameter of iron status, particularly in
the elderly where a variety of factors that may influence the levels are often present in
elderly individuals. This may be due to ageing alone or as a result of multiple comorbidities.
5. Serum ferritin levels were lower than expected in elderly subjects with mutation
C282Y and compound heterozygotes with both C282Y and H63D, which may be
related to a variable penetrance of the HFE gene mutations. It is possible that variation
in other genes exist that confer protection against iron-loading by gene-gene
interaction. The probability that environmental factors (e.g. a low iron diet) are more
important in this respect cannot be excluded, although this is considered less likely in
the light of the fact that the same trend was observed in all mutation-positive elderly individuals. It is therefore highly likely that C282Y -positive subjects with significant
iron loading have died before reaching their seventies, particularly since none of the
males included in this study were homozygous or compound heterozygous for the
mutations analysed.
In conclusion, possession of a mutant HFE gene does not appear to confer a survival
advantage in old age, neither does it seem that mutation carriers with significant ironloading
are overlooked by the medical fraternity. Further investigations are warranted to
shed more light on the contributions of gene-gene and gene-environment interaction in the
clinical manifestation of Hll, and how these processes can be manipulated to prevent the
symptoms of this largely underdiagnosed disease. / AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is 'n outosomaal resessiewe yster-oorladingssiekte waar
akkumulasie van yster in parenkimale organe kan lei tot suikersiekte, hartversaking, lewer
sirrose, artropatie, moegheid en 'n verskeidenheid van ander probleme indien
voorkomende maatreëls nie getref word nie. OH word gewoonlik nie oorweeg as
moontlike oorsaak vir hierdie toestande nie, veral nie in ouer mense nie waar die
agtergrond-frekwensie van tipe II diabetes, osteoartritis en hartversaking in elk geval hoog
is. Heterosigositeit vir die HFE mutasie C282Y, wat OH veroorsaak in ongeveer 80% van
geaffekteerde gevalle wêreldwyd, is geassosieer met 'n verhoogde voorkoms van kanker
van die kolon, rektum, maag en ook die hematologiese sisteem. Van die hoogste draer
frekwensies ter wêreld vir hierdie mutasie (1/6) is gevind in die Afrikaner populasie van
Suid-Afrika, wat daarop dui dat 1 uit elke 115 mense in die groep homosigoties vir die
C282Y mutasie kan wees.
Eenhonderd sewe-en-negentig bejaarde Afrikaner vrywilligers het aan die studie
deelgeneem wat daarop gemik was om genotipe/fenotipe korrelasies uit te voer. Die kliniese
beeld van elke individu is gedokumenteer, die yster status biochemies bepaal en HFE
genotipering uitgevoer. Die a priori veronderstelling was dat oorlewing sou toeneem of
afneem, of beide, afhangende van die geslag van die individu. Daar is voorheen 'n verband
gevind tussen OH en die ontwikkeling van bogenoemde maligniteite, maar aan die ander
kant kan dit moontlik ook beskerm teen anemie as gevolg van yster gebrek, wat juis die
mees algemene oorsaak van anemie in die ouer persoon is. Hierdie studie het tot die volgende bevindings gelei:
1. Die draer frekwensie van mutasie C282Y was 1/8 in die bejaardes (dieselfde in mans
en vrouens), wat effens laer is as die 1/6 wat gerappoteer is in jonger volwassenes.
Slegs een C282Y homosigoot en twee C282YIH63D saamgestelde heterosigote is
opgespoor, en al drie was vroulik.
2. Die voorkoms van suikersiekte, hartsiekte, gewrigspyne of 'n kombinasie van hierdie
aandoenings het nie betekenisvol verskil tussen die C282Y heterosigote en die mutasienegatiewe
groep nie.
3. Daar was slegs een persoon met rektum karsinoom in die groep van 24 bejaarde C282Y
heterosigote, terwyl daar twee gevalle met rektum kanker en sewe gevalle met kolon
kanker gevind is onder die 153 mutasie-negatiewe individue. Die enkele vroulike
C282Y homosigoot wat opgespoor is het beide rektum- en kolonkanker gehad en is
ongeveer 6 maande vóór voltooing van die tesis oorlede aan haar kolon karsinoom.
4. Dit wil voorkom asof serum ferritien veral in bejaardes 'n hoogs onbetroubare maatstaf
is vir yster status, aangesien dit deur 'n verskeidenheid faktore beïnvloed word wat
dikwels in bejaardes aanwesig is as gevolg van veroudering of veelvuldige komorbiditeite.
5. Die serum ferritien vlakke was laer as verwag in sowel die bejaarde C282Y-homosigoot
as in die twee saamgestelde heterosigote met mutasies C282Y en H63D,
wat moonlik die gevolg is van die wisselende graad van penetrasie van HFE mutasies.
Dit is moontlik dat variasie in ander gene beskerming bied teen yster-oorlading deur
middel van geen-geen interaksie. Die moontlikheid dat omgewingsfaktore (soos 'n lae-yster
dieet) 'n belangrike rol speel in hierdie verband kan nie uitgesluit word nie,
hoewel dit minder waarskynlik lyk te wees in die lig van die feit dat dieselfde neiging waargeneem is in alle mutasie-positiewe bejaardes. Die kans is dus redelik groot dat
individue met die C282Y mutasie en betekenisvolle yster oorlading oorlede is voordat
hulle die sewentiger jare kon bereik, veral omdat geeneen van die mans wat ingesluit is
in die studie homosigoot of 'n saamgestelde heterosigoot was vir die mutasies wat
geanaliseer is nie.
Opsommend wil dit voorkom asof die teenwoordigheid van 'n mutante HFE geen nie 'n
beter oorlewingskans bied op ouer leeftyd nie, en dit blyk ook dat mutasie draers met
betekenisvolle ysteroorlading nie deur dokters misgekyk word nie. Verdere navorsing is
nodig om meer lig te werp op die bydrae van geen-geen- en geen-omgewing interaksie in
die kliniese manifestasie van OH, en ook hoe hierdie prosesse gemanipuleer kan word om
die simptome van hierdie onder -gediagnoseerde siekte te voorkom.
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The MED-PED project : presymptomatic diagnosis in families with disease- related LDL receptor gene mutationsVergotine, Joseph Vincent 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Familial hypercholesterolaemia (FH) contributes significantly to the high death rate
from cardiovascular disease worldwide. FH is a common autosomal co-dominant
disease characterised by raised cholesterol levels and premature coronary heart
disease (CHD). Whilst these features usually are very prominent in homozygotes the
clinical diagnosis of heterozygotes is complicated by variable phenotypic expression.
Specific founder genes in the low-density lipoprotein receptor (LDLR) gene have
increased the prevalence of FH in South African Afrikaners, Indians, Jews and
Coloureds, and screening for these known mutations allows unequivocal diagnosis
of FH-affected individuals. The systematic molecular analysis of FH resulted in the
identification of at least ten founder-type LDLR gene mutations among the 56
different gene defects described to date in the diverse South African population.
DNA screening of 792 at-risk family members for the FH-related mutations identified
in 379 index cases, allowed accurate disease diagnosis in an additional 340
relatives and exclusion of the relevant mutation in 452 individuals. This effort forms
part of the MED PED FH initiative, a collaborative project to "Make Early Diagnosis
and Prevent Early Deaths in MEDical PEDigrees with FH".
Evaluation of clinical criteria versus DNA diagnosis of three founder-related
mutations (D154N, D206E and V408M) in the South African population
demonstrated that the sensitivity and specificity of diagnoses, based on total
cholesterol values measured in family members of index cases recruited for this
study, were 88% and 77%, respectively. A population-directed DNA diagnosis of FH is therefore justified in South Africa on a routine basis, since expression of the
defective gene measured in biochemical tests does not allow accurate diagnosis of
FH in all cases.
The application of mutation detection was illustrated by prenatal diagnosis of FH
performed for a couple who are both heterozygous for the most common Afrikaner
mutation, D206E. The mutation was absent in the foetus and a
normocholesterolaemic infant was born. Prenatal diagnosis of FH, aimed at the
detection of homozygous cases, is particularly applicable in populations and families
with molecularly defined LDLR gene mutations.
The MED-PED approach resulted in accurate diagnosis and subsequent treatment
of FH in more patients, and referral to lipid clinics where they could receive the
intensive care their condition justifies. Molecularly diagnosed FH patients will be the
first to benefit from future treatment approaches based on mutation type. / AFRIKAANSE OPSOMMING: Familiële hiprcholesterolemie dra grootliks by tot die wêreldwye hoë sterftesyfer van
kardiovaskulêre siekte. FH is 'n algemene outosomale ko-dominante siekte wat
gekenmerk word deur verhoogde cholesterolvlakke en vroeë koronêre hartsiekte.
Terwyl hierdie kenmerke prominent is in homosigote, word die kliniese diagnose van
heterosigote bemoeilik deur variasie in fenotipiese uitdrukking. Spesifieke
stigtergene in die lae-digtheids lipoproteien reseptor (LDLR) geen het die
voorkomssyfer van FH verhoog in Suid Afrikaanse Afrikaners, Indiërs, Jode en
Kleurlinge. Sifting vir hierdie bekende mutasies maak akkurate diagnose van FH geaffekteerde
individue moontlik. Die sistematiese molekulêre analise van FH het
aangetoon dat ten minste tien van die 56 verskillende geen defekte wat tot dusver
beskryf is in die Suid-Afrikaanse populasie stigtertipe LDLR geen mutasies is. DNA
sifting van 792 familielede vir die FH-verwante mutasie in 379 indeksgevalle
geïdentifiseer is, het akkurate diagnose moontlik gemaak in 340 addisionele
familielede, en uitsluiting daarvan in 452 individue. Hierdie poging vorm deel van die
MED-PED FH ("Make Early Diagnosis and Prevent Early Deaths in MEDical
PEDigrees with FH) inisiatief.
Evaluering van kliniese kriteria teenoor DNA diagnose van drie stigter verwante
mutasies (D154N, D206E en V408M) in die Suid Afrikaanse populasie het getoon
dat die sensitiwiteit en spesifisiteit van die diagnose, wat gebasseer is op totale
cholesterol waardes in familielede van indeksgevalle, onderskeidelik 88% en 77% was. 'n Populasie gerigte DNA diagnose van FH is dus geregverdig in Suid-Afrika op
"n roetine basis, omdat die defektiewe geen nie altyd in biochemiese toetse
uitgedruk word nie.
Die waarde van mutasie opsporing is geillustreer deur 'n voorgeboortelike diagnose
van FH wat aangevra is vir ouers wat beide heterosigoties is vir die mees algemene
Afrikaner mutasie, D206E. Die mutasie was afwesig in die fetus en 'n
normocholesterolemiese baba is gebore. Voorgeboortelike diagnose van FH, wat
gemik is op die opsporing van homosigotiese gevalle, is veral van toepassing in
populasies en families met bekende LDLR geen mutasies.
Die MED-PED benadering het gelei tot akkurate diagnose en daaropvolgende
behandeling van FH in meer pasiënte, en verwysings na lipiedklinieke waar hulle
intensiewe aandag kan geniet. Molekulêre gediagnoseerde FH pasiënte sal die
eerste wees om baat te vind by toekomstige behandeling wat moontlik gebasseer
sal word op mutasie status.
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The relevance of apoptosis in the pathogenesis of human immunodeficiency virus-1 diseaseCotton, Mark Fredric 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: A simple and rapid scatter-based flow cytometric assay was developed to detect
apoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay was
suitable for children.
Apoptotic PBMCs were confirmed by morphologic assessment in clinical samples
ex vivo and after overnight culture. The scatter-based assay was validated in a number of
ways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induce
apoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sorted
into apoptotic and viable populations by scatter characteristics (diminished forward and
increased side scatter). Morphology was assessed by fluorescence microscopy. The
majority of cells with apoptotic scatter characteristics had apoptotic morphology
(chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region had
normal morphology. CD4+ T cell apoptosis measured by scatter was then correlated with
the TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter and
annexin V uptake were also shown to correlate. In the latter experiments, PBMC
morphology and cell death by trypan blue uptake were studied simultaneously and
confirmed the two flow cytometric assays.
Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infected
adults analyzed after overnight culture. Since cell death may be an artifact of in
vitro culture, and because there is little information on apoptosis in paediatric HIV disease,
I undertook a cross-sectional analysis in PBMCs analyzed immediately ex vivo from HIV infected
children and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stained
for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized
flow cytometric assay by diminished forward and increased side scatter.
For the scatter assay, PBMCs had been labeled initially by an indirect method
involving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30
minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, the
intermediary incubation step was removed and PBMCs were incubated with PE-conjugated
CD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appeared
enhanced in the indirect method. The significant differences were abolished after
subtraction of data from simultaneously studied time-matched controls.
CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected study
subjects than in simultaneously studied seronegative controls. PBMCs were assayed
immediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb as
well as spontaneously. There was a direct correlation between CD4+ and CD8+ T cell
apoptosis and CD4+ T cell depletion. A significant correlation was also shown between
apoptosis immediately ex vivo and after overnight culture.
I then studied apoptosis in a South African population comprising 18
symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis were
significantly higher in symptomatic HIV-1-infected children than in seroreverters and
seronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cell
percentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cell
apoptosis in patients recovering from intercurrent disease in comparison to those who
were either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, CO
and Tygerberg Children’s Hospital, South Africa. I selected only patients with moderate
or severe HIV infection from both centers. South African patients were significantly
younger, more malnourished, had higher gamma globulin levels and were less likely to
receive ART. CD8+ T cell apoptosis was higher in North American patients suggesting a
possible impairment in CD8+ activity in the South African study subjects. / AFRIKAANSE OPSOMMING: ‘n Eenvoudige en vinnige vloei sitometriese toets is ontwikkel om apoptose aan te
toon vanuit ‘n gemengde populasie selle. Dit moes geskik wees vir kinders van wie net
klein volumes bloed getrek kan word.
Die teenwoordigheid van apoptotiese perifere bloed mononuklere selle (PBMS)
was vasgestel deur morfologiese beoordeling in kliniese monsters ex vivo en na oornag
kultuur. Die ondersoek is gebasseer op die verstrooings patroon van bestraalde PBMS
wat apoptose induseer. PBMS is gemerk met a CD4 MAb. CD4+ selle is gesorteer in
apoptotiese en lewensvatbare populasies deur verstrooings karakteristieke. Morfologie is
beoordeel deur fluoreserende mikroskopie. Die meerderheid van selle met apoptotiese
verstrooings karakteristieke (verminderde voorwaartse en verhoogde sywaartse
verstrooings patroon) het apoptotiese karakteristieke gehad (80.6%). Twee-en-negentig
persent van selle van die lewensvatbare area het normale morfologie gehad. Verstrooings
patroon is ook gekorreleer met die TdT meting vir DNA fragmentasie in kliniese
monsters van MIV-geinfekteerde kinders. Daarna is Annexin V gekorreleer met verstrooings patroon, apoptotiese morfologie en trypan blou opname in selle wat
blootgestel is na verskillende konsentrasies van beauvericin.
Apoptose van CD4+ en CD8+ T-selle is bewys in PBMS van MIV-geinfekteerde
volwassenes na oornag kultuur. Omdat sel dood ‘n artefak van in vitro kultuur kan wees,
en omdat daar min inligting is oor apoptose in paediatriese MIV siekte, het ek onderneem
om ‘n deursnee analiese te doen in PBMS wat onmiddelik ex vivo geanaliseer is vanaf
MlV-geinfekteerde kinders en volwassenes. Die pasiente is bestudeer in Denver,
Colorado, VS A.
PBMS van 22 kinders, 4 adolessente en 9 volwassenes en seronegatiewe
ouderdoms-gepasde kontroles is gekleur vir CD4+ en CD8+ oppervlaksmerkers.
Apoptotiese selle is vloeisitometries aangedui deur verandering in verstrooings patroon.
Vir die doeleindes van die verstrooings assay is die PBMS aanvanklik deur ‘n
indirekte metode gemerk, wat ‘n intermediere inkubasie in die teenwoordigheid van
biogetinileerde MAbs by 37°C vir 30 minute voor dit geinkubeer is met streptavidin-
FITC by 4°C vir 20 minute behels. Daarna is die intermediere inkubasie stap verwyder
en PBMC is geinkubeer met PE - gekonjugeerde CD4+ and CD8+ MAbs. Beide die
CD4+ en CD8+ T-sel apoptose het verhoog voorgekom met die indirekte metode. Die
betekenisvolle verskille het verdwyn na data van gelyktydige tyd - gepaarde kontroles
afgetrek is.
CD4+ en CD8+ T-sel apoptose was betekenisvol hoër in MIV-geinfekteerde
studie gevalle as in gelyktydig bestudeerde seronegatiewe kontroles. PBMS assays is
gedoen onmiddelik ex vivo en na oornag inkubasie na stimulasie deur ‘n anti-TCR MAb,
sowel as spontaan. Daar was ‘n direkte korrelasie tussen CD4+ en CD8+ T sel apoptosis en CD4+ T sel vermindering. ‘n Beduidende korrelasie is ook getoon tussen apoptose
onmiddelik ex vivo en na oornag kultuur.
Daaropvolgend het ek apoptose in ‘n Suid Afrikaanse populasie van 18
simptomatiese kinders en 4 serologies terukerende gevalle bestudeer. CD4+ en CD8+ T
sel apoptose was aansienlik hoër in siptomatiese MIV - 1-geinfekteerde kinders as in die
serologies terukerende gevalle en seronegatiewe kontroles. CD4+ T sel apoptose het
gekorrelleer met vermindering van CD4+ T sel persentasie. Ek het ook opgemerk dat daar
‘n tendens bestaan het tot verhoogde CD4+ T sel apoptose in pasiente wat besig was om
te herstel van bykomende siektes.
Ek het CD4+ en CD8+ T sel apoptose in kohorte van Denver, Colorado en
Tygerberg, Suid Afrika vergelyk. Suid Afrikaanse pasiente was jonger en meer
wangevoed as hul Noord Amerikaanse ewekniee. Suid Afrikaanse kinders het ook meer
gevorderde siekte gehad. Wanneer pasiente gepas is vir die graad van ernstigheid van
siekte en slegs die minder ernstige (B) en ernstige siekte (C) vergelyk is, was CD8+ T sel
apoptose beduidend hoër in Noord Amerikaanse pasiente. Hierdie waarneming
ondersteun die hipotese dat CD 8+ T sel aktiwiteit moontlik onderdruk mag wees in
simptomatiese Suid Afrikaanse MIV-1-geinfekteerde kinders.
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Mycobacterium tuberculosis : genetic and phenotypic comparisonSampson, Samantha Leigh 03 1900 (has links)
Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in
the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the
phenotypic and molecular epidemiological implications thereof.
The study was initiated by investigation of the insertion sequence IS6110, the primary
DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable
element is present in variable copy number and chromosomal location in clinical isolates of
M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this
study, little was known about this element in terms of the genetic identity of its surrounding
regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity.
These shortcomings were therefore addressed by a number of approaches.
Firstly, to establish their genetic identity and chromosomal distribution, IS6110
insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data
was examined in conjunction with available genome sequence data. The results demonstrated
that the majority of insertions occurred within coding regions. Furthermore, the element was
shown to have a non-random chromosomal distribution, and a number of preferential
integration sites were identified. Secondly, the stability of chromosomal domains flanking
IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes
against clinical isolates. This allowed the identification of extensive genetic variation
associated with these chromosomal domains, arising from IS6110 transpositions, deletions
and point mutations. These events were expressed in terms of a phylogenetic tree which
demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the
hypothesis that IS6110-mediated deletions occur via homologous recombination between
adjacent elements, deletion junctions were mapped and sequenced in clinical isolates
representing predecessor and descendant strains. While these results support the involvement
of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the
existence of unidentified intermediates.
The investigation of IS6110 flanking regions identified the disruption of a number of
members of the PPE gene family, leading to the second main area of investigation. The PPE
gene family was newly identified as a result of the M. tuberculosis genome sequencing
project, and its products are speculated to be of antigenic importance. However, at the
commencement of this study very little data was available regarding the biological role of
PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated.
Firstly, phylogenetic relationships between members of the PPE family were
elucidated, which suggested an evolutionary progression, and highlighted the possibility that
there may be functional subdivisions within the gene family. Secondly, the extent and
mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR
and sequence analysis. This approach revealed extensive variation associated the gene family,
although different members of the family exhibit different levels of variation. Of special
interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members
of the gene family demonstrate variation in the numbers of these tandem repeats. A third
avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT-
, peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ
hybridization demonstrated the expression of PPE genes in human tissue samples.
Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within
the human granuloma. The final approach reported here focused on the subcellular
localization of one member of the PPE family, Rv1917c. A combination of cell fractionation
and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated,
surface exposed molecule.
In summary, the results obtained have potential implications for the interpretation of
molecular epidemiological data, support the role of IS6110 as an agent of genome evolution,
and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the
PPE family demonstrated that this gene family contributes to genetic variation, is expressed in
vitro and in vivo and that at least one protein encoded by the gene family is cell wall
associated. Together, the results obtained support the hypothesis that selected members of the
PPE gene family may encode products involved in antigenic variation. / AFRIKAANSE OPSOMMING: Dié studie maak gebruik van die Mycobacterium tuberculosis H37Rv genoom
volgorde data in die konteks van M. tuberculosis kliniese isolate, om genetiese variasie toe te
lig en die fenotipiese en molekulêre epidemiologiese implikasies daarvan te ondersoek.
Die studie het 'n aanvang geneem deur die ondersoek van die inset-volgorde /S6110,
wat die primêre DNS vingerafdruk pylfragment vir die molekulêre epidemiologie van
tuberkulose is. Hierdie transponerende element is in wisselende kopiegetal en chromosomale
posisies teenwoordig in kliniese isolate van M. tuberculosis stamme, en gee so oorsprong aan
omvangryke genetiese afwisseling. Met die aanvang van hierdie studie was min bekend
omtrent hierdie element betreffende die genetiese identiteit van die areas wat die insetsels
omring, die chromosomale distribusie van insetsels, asook die meganismes wat bydra tot
genetiese afwisseling. Hierdie gebreke is dus deur 'n aantal benaderings aangespreek.
Eerstens is IS6110 insettingsetels van kliniese M. tuberculosis isolate gekloneer en hul
nukleotiedvolgorde bepaal om sodoende hul genetiese identiteit en chromosomale
verspreiding vas te stel. Hierdie data is in oorleg met beskikbare genomiese volgorde data
geanaliseer. Die resultate het gewys dat die meerderheid van insetsels binne koderende
gebiede plaasgevind het. Verder is gewys dat hierdie element nie na willekeur deur die
chromosoom versprei is nie, en 'n aantal gebiede waar insetting by voorkeur plaasvind, is
geïdentifiseer. Tweedens is die stabiliteit van die chromosomale gebiede wat IS6110
elemente flankeer ondersoek deur die insettingsetel klone as pylfragmente te gebruik in
hibridisasie van kliniese isolate. Dit het die identifisering van omvangryke genetiese
afwisseling binne hierdie chromosomale gebiede, wat ontstaan deur IS611 0 transposisies,
delesies en puntmutasies, tot gevolg gehad. Hierdie afwisselings is uitgedruk as 'n
filogenetiese boom waarin die voortdurende genomiese evolusie wat geassosieer word met
IS6110 gewys word. Derdens, om die teorie dat IS6110-gedrewe delesies deur middel van
homoloë rekombinasie tussen naasliggende elemente plaasvind te ondersoek, is die grense
van delesies gekarteer en die nukleotiedvolgorde daarvan bepaal in kliniese isolate wat
voorganger- en afstammelingstamme verteenwoordig. Alhoewel die resultate die
betrokkenheid van IS6110 as 'n bemiddelaar van genetiese delesie ondersteun, stel dit ook die
bestaan van of alternatiewe meganismes of van onbekende intermediêre vorme voor.
Ondersoek van die IS6110-flankerende gebiede het gelei tot die ontdekking van
ontwrigting van 'n aantal gene wat behoort tot die PPE geenfamilie, en het so gelei tot die
tweede hoof ondersoek tema. Die PPE geenfamilie is ontdek as gevolg van die M. tuberculosis genoomprojek, en dit word gespekuleer dat die produkte van hierdie gene van
antigeniese belang mag wees. Daar was egter met die aanvang van hierdie studie baie min
data beskikbaar omtrent die biologiese rol van die PPE proteïene. Om die fenotipiese
implikasies van ontwrigting van PPE gene te ondersoek is daar dus ondersoek ingestel na
verskeie aspekte van hierdie geenfamilie.
Eerstens is filogenetiese verwantskappe tussen lede van die PPE familie bepaal, wat
gedui het op 'n evolusionêre progressie en wat ook aangedui het dat daar moontlik
funksionele onderverdelings binne hierdie geenfamilie mag bestaan. Tweedens is die omvang
en meganismes van PPE geenvariasie geanaliseer deur 'n kombinasie van hibridisasie, PKR
en nukleotiedvolgorde analise. Hierdie benadering het omvangryke afwisseling binne hierdie
geenfamilie getoon, alhoewel verskillende lede van die familie verskillende vlakke van
afwisseling demonstreer. Wat veral interessant was, was die ontdekking dat lang tandem
herhalingsvolgordes (~69 bp) wat in 3 lede van hierdie geenfamilie voorkom, variasie toon in
die getalle van hierdie tandem herhalingsvolgordes. 'n Derde been van ondersoek het gefokus
op in vitro en in vivo PPE geen uitdrukkingsprofiele. RT-PKR is gebruik om te toon dat PPE
gene in vitro uitgedruk word, terwyl RNA:RNA in situ hibridisasie getoon het dat PPE gene
ook in menslike weefsel uitgedruk word. Interessant genoeg dui in situ hibridisasie daarop
dat daar wisselende PPE geen uitdrukking binne die menslike granuloom voorkom. Die
laaste benadering wat hier gerapporteer word fokus op die sub-sellulêre lokalisering van een
lid van die PPE familie, Rv1917c. 'n Kombinasie van selfraksionering en heel-sel
antiliggaam-bindingseksperimente dui daarop dat Rv1917c 'n selwand-geassosieerde
molekuul is wat aan die oppervlak blootgestel word.
Ter opsomming het die resultate wat bereik IS potensiële implikasies vir die
interpretasie van molekulêr-epidemiologiese data, dit ondersteun die rol van IS6110 as 'n
bemiddelaar van genoom evolusie en beklemtoon die potensiaal vir IS6110 om 'n invloed te
hê op die fenotipe van die stam. Ondersoek van die PPE familie het getoon dat hierdie
geenfamilie bydra tot genetiese afwisseling, dat dit uitgedruk word beide in vitro en in vivo en
dat ten minste een lid van hierdie geenfamilie geassossieer word met die selwand. Tesame
ondersteun hierdie resultate die teorie dat geselekteerde lede van die PPE geenfamilie wel
produkte enkodeer wat betrokke is by antigeniese variasie.
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A multi-disciplinary approach towards elucidating the genetics of multiple sclerosisDe Villiers, J. N. P. 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system.
Current knowledge suggests that MS is associated with autoimmunity and that infectious
agents and hereditary factors may be involved. The demonstration of a higher
recurrence risk of MS in families (4-5%) compared with the general population (0.1%)
provides strong evidence for a genetic basis. Extensive analyses of the entire human
genome to identify new genes that may underlie MS have indicated that several genes
may contribute to disease susceptibility, but these remain largely unidentified.
In this study candidate genes involved in iron metabolism and immunology have been
analysed for the first time within the context of both autoimmune and infectious disease
susceptibility, in order to investigate the role of genetic and viral factors implicated in the
pathogenesis of MS.
The Z-DNA forming repeat polymorphism in the promoter region of the solute carrier
family 11 (proton-coupled divalent ion transporters) member 1 (SLC11A1) gene was
found to be significantly associated with MS (P<0.01) in the genetically homogeneous
Afrikaner population of South Africa, but not in the German and French populations
using a case-control study and transmission linkage disequilibrium approach,
respectively. However, significant differences were observed in genotype distribution
between German MS patients with a primary- and secondary progressive disease
course (P<0.05), and between the German patients with relapsing remitting and primary
progressive MS (P<0.05). These findings provide further evidence that the SLC11A1
gene is associated with MS, most likely due to its role in iron homeostasis.
In order to investigate the influence of viruses in the apparent multi-step aetiology of MS,
serum and peripheral blood mononuclear cells (PBMCs) of MS patients, close relatives
and unrelated controls were screened for the presence of MS-associated retrovirus
(MSRV) and two herpes virus (HHV-6 and EBV) sequences. Detection of the pol gene
expression of MSRV in the serum RNA of 69% of South African MS patients and in 70% of their unaffected close relatives, whilst absent in the serum of 39 unrelated healthy
control individuals (P<0.001), indicated that virus infections affect the population risk but
not the familial risk in MS. HHV-6 sequences were also present at a significantly lower
frequency (P<0.04) in the PBMCs of unrelated controls (5%) compared to MS patients
(22.5%).
A point mutation (77C^G) in the gene encoding protein-tyrosine phosphatase, receptortype
C (PTPRC), which is essential for activation of T and B cells, was found to be
associated with MS in the German population. Analysis of the Afrikaner and German
study populations included in our study did not indicate a causative role for the PTPRC
gene in MS. However, it seems likely that this mutation may contribute to disease
expression, since in one of the South African families with two MS affected sibs, the
most severely affected sister was heterozygous for the 77C-»G mutation. The PTPRC
mutation may therefore be of significance in disease prognosis.
The multidisciplinary study approach has led to a stepwise accumulation of scientific
information, which forever changed our understanding of the disease process underlying
MS. / AFRIKAANSE OPSOMMING: Veelvoudige sklerose (VS) is ‘n kroniese inflammatoriese siekte van die sentrale
senuweestelsel. Oor die algemeen word aanvaar dat VS geassosieerd is met
outoimmuniteit en dat infektiewe agente en oorerflike faktore ’n rol speel. Die hoër
herhalingsrisiko van VS in families (4-5%) in vergelyking met die voorkoms in die
algemene populasie (0.1%) dui op 'n genetiese basis. Alhoewel volledige analise van
die mensgenoom om gene onderliggend aan VS te identifiseer aangedui het dat
verskeie gene waarskynlik bydra tot vatbaarheid vir die siekte, is die aard van die gene
wat betrokke is grootliks onbekend.
In hierdie studie is kandidaatgene betrokke by ystermetabolisme en immunulogie vir die
eerste keer geanaliseer binne die konteks van beide outoimmuun en infektiewe siekte
vatbaarheid, ten einde die rol van genetiese en virale faktore in die patogenese van VS
te ondersoek.
Die Z-DNS herhalingsvolgorde polimorfisme in die promotor area van die SLC11A1
geen was betekenisvol geassosieerd met VS (P<0.01) in die geneties homogene
Afrikaner populasie van Suid-Afrika. ’n Soortgelyke assosiasie kon egter nie aangetoon
word in die Duitse en Franse populasies deur gebruik te maak van onderskeidelik ‘n
gevalle-kontrole studie en transmissie-koppelings-disekwilibrium benadering nie.
Betekenisvolle verskille in die genotipe verspreiding is egter tussen Duitse VS pasiente
met ‘n sekonder- en primer progressiewe verloop van die siekte (P<0.05), en tussen die
Duitse pasiente met terugvallende en primere progressiewe VS aangetoon (P<0.05).
Hierdie bevinding verskaf verdere bewyse dat die SLC11A1 geen geassosieerd is met
VS, heel waarskynlik weens die rol van die geen in yster-homeostase.
Ten einde die invloed van virusse in die etiologie van VS te ondersoek is serum en
witbloedselle van VS pasiente, naby-verwante familielede en nie-verwante kontroles
getoets vir die teenwoordigheid van die VS-geassosieerde retrovirus (MSRV) en twee
herpesvirus (HHV-6 en EBV) geenvolgordes. Die pol geen uitdrukking van MSRV was teenwoordig in die serum RNA van 69% van die Suid-Afrikaanse VS pasiente en in 70%
van hul ongeaffekteerde naby-verwante familielede, terwyl dit afwesig was in 39 nieverwante
kontrole individue (P<0.001). Dit dui daarop dat virusse waarskynlik die risiko
vir VS meer in die populasie verhoog as in families. HHV-6 was ook teenwoordig teen ‘n
beduidende laer frekwensie (P<0.04) in nie-verwante kontroles (5%) in vergeleke met
VS pasiente.
‘n Puntmutasie (77C-G) in die geen wat kodeer vir die proteien tirosien fosfatase
reseptor tipe C (PTPRC), wat belangrik is vir aktivering van T- en B-helperselle, is
vroeer gevind om geassosieerd te wees met VS in die Duitse populasie. Analise van die
Afrikaner en Duitse populasies in ons studie het egter geen bewyse gelewer dat die
PTPRC geen ‘n rol speel in VS nie. Dit egter is moontlik dat hierdie mutasie bydra tot die
uitdrukking van VS, aangesien die mees geaffekteerde VS pasient in een van die Suid-
Afrikaanse families met twee geaffekteerde susters positief getoets het vir die mutasie.
Die mutasie mag dus van belang wees in die prognose van VS.
Die multidissiplinere studie-benadering en stapsgewyse insameling van wetenskaplike
inligting het gelei tot ’n nuwe perspektief ten opsigte van die siekteproses onderliggend
aan VS.
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Disease dynamics in patients with drug-resistant tuberculosis residing in a high incidence communityVan Rie, Annelies 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Drug-resistant tuberculosis poses a threat to global tuberculosis control by the
WHO DOTS strategy. Studies in the United States and Europe have shown (i) that
drug-resistant tuberculosis is present in every country; (ii) that, by contrast to previous
dogma, drug-resistant bacilli are virulent and can be transmitted, especially in
institutional settings and to immunocompromised patients; and (iii) that the majority
of cases arise by acquisition of drug resistance due to errors in the management of TB
cases. (iv) Furthermore, it has been shown that the extremely high case fatality rates
of the 1980s and early 1990s can be reduced by individualized, but costly treatment.
However, the majority of drug-resistant TB cases reside in the developing world.
Data on disease epidemics in less developed parts of the world are scarce. The aim of
this thesis was to study the disease dynamics of drug-resistant TB in a developing
country where TB is endemic.
All cases of drug-resistant TB during a 5-year period in two communities with
poor socioeconomic living conditions were included for this observational study.
Three different methods were used: restriction fragment length polymorphism
(RFLP), mutation detection analysis by dot-blot hybridisation technique and a
Geographic Information System. Results of RFLP analysis and mutation detection
analysis showed that community outbreaks of drug-resistant Mycobacterium
tuberculosis strains occur, even without the involvement of immunocomprimised
patients. Infection with a drug-resistant strain occurred in new patients (primary drug
resistance) as well as in patients treated before (exogenous reinfection). Exogenous
reinfection was also shown to be an important mechanism of recurrence after previous
cure for drug-sensitive TB. Transmission of drug-resistant strains occurred more
frequent in areas with lower socioeconomic living conditions. The relative
contribution of transmission differed substantially between the group of multi drugresistant
(two thirds of cases) and single-drug-resistant (no cases) cases, which
probably reflects the prolonged infectiousness of multi drug-resistant cases. To stop
the growing epidemic of multi drug-resistant TB, prevention of acquisition as well as
transmission of drug-resistant tuberculosis will be required. This will only be possible
in areas where a DOTS strategy is well functioning and with a modification of central
elements of the standard DOTS mechanism: a "DOTS-plus" strategy. Early and accurate diagnosis of drug resistance is essential for effective management. Diagnosis
based on two direct smear tests might have to be replaced by routine drugsusceptibility
tests at diagnosis. Because the routine performance of phenotypic drugsusceptibility
tests was inferior to the performance of genotypic tests, the
development of an affordable commercial kit testing a limited number of mutations
conferring resistance could be of great value in the global fight against multidrugresistant
TB. Because of the importance of early diagnosis, selective active contact
tracing for multidrug-resistant cases, additional to the routine passive contact tracing,
could prove to be cost-effective. Individualized treatment regimens are effective in
reducing the failure rate, mortality and probably transmission of multidrug-resistant
TB.
Multidrug-resistant tuberculosis is a problem confronting the efforts for global
tuberculosis control. Efficient strategies to turn the tide exist, but international
political commitment and financial support will be essential. / AFRIKAANSE OPSOMMING: Middel weerstandige tuberkulose hou 'n bedreiging in vir globale tuberkulose
kontrole deur die WGO DOTS strategie. Studies in die Verenigde State en Europa het
getoon (i) dat middel weerstandige tuberkulose in alle lande voorkom; (ii) dat, in
teenstelling met vorige dogma, middel weerstandige bakterieë virulent is en oorgedra
kan word, veral in inrigtings en aan immuun-onderdrukte pasiënte; en (iii) dat die
meeste gevalle ontstaan deur die verwerwing van middel weerstandigheid a.g.v. die
foutiewe hantering van tuberkulose gevalle. (iv) Bykomend is getoon dat die
ontsettende hoë mortaliteit syfers van die 1980s verlaag kan word deur geindividualiseerde,
maar duur behandeling.
Die meeste middel weerstandige tuberkulose gevalle woon egter in die
ontwikkelende wêreld. Data oor siekte epidemies in minder ontwikkelde dele van die
wêreld is skaars. Die doel van hierdie tesis was om die siekte dinamiek van middel
weerstandige tuberkulose te bestudeer in 'n ontwikkelende land waar tuberkulose
endemies is.
Alle gevalle van middel weerstandige tuberkulose gedurende 'n 5-jaar periode in
twee lae sosio-ekonomiese gemeenskappe, is in hierdie studie ingesluit. Drie
verskillende metodes is gebruik: restriksie fragment lengte polimorfisme (RFLP),
mutasie analise deur dot-blot hibridisasie en 'n Geografiese Inligting Stelsel.
Resultate van die RFLP analise het getoon dat uitbrake van middel weerstandige
Mycobacterium tuberculosis stamme in die gemeenskap voorkom, selfs sonder die
aantasting van immuun-onderdrukte pasiënte. Infeksie met middel weerstandige
stamme het voorgekom in nuwe pasiënte (primêre middel weerstandigheid) en ook in
pasiënte wat reeds voorheen behandel is (eksogene herinfeksie ). Daar is ook gevind
dat eksogene herinfeksie 'n belangrike meganisme was van herhaalde tuberkulose na
vorige genesing van middel sensitiewe tuberkulose. Die oordrag van middel
weerstandige stamme het meer dikwels voorgekom in areas met laer sosioekonomiese
omstandighede. Die relatiewe bydrae van oordrag het merkwaardig
verskil tussen multi-middel weerstandigheid (twee derdes van gevalle) en enkelmiddel
weerstandigheid (geen gevalle). Dit weerspieël waarskynlik die verlengde
periode van infektiwiteit van die multi-middel weerstandige gevalle. Die bekamping
van die groeiende epidemie van multi-middel weerstandige tuberkulose, vereis die
voorkoming van verworwe sowel as oorgedraagde middel weerstandige tuberkulose. Dit sal slegs moontlik wees in areas waar 'n DOTS strategie reeds goed funksioneer
en met 'n aanpassing van die sentrale elemente van die roetine DOTS meganisme: 'n
"DOTS-plus" strategie. Vroeë en akkurate diagnose van middel weerstandigheid is
essensieël vir effektiewe hantering. Diagnose gebaseer op twee direkte sputum smeer
toetse mag moontlik vervang moet word deur roetine middel sensitiwiteit bepalings
by diagnose. Die roetine fenotipiese middel sensitiwiteit bepaling is gevind om
minderwaardig te wees in vergelyking met die genotipiese toetse. Die ontwikkeling
van 'n bekostigbare toetsstelsel wat die mees algemene mutasies vir middel
weerstandigheid sal opspoor, kan van groot waarde wees in die stryd teen mutimiddel
weerstandige tuberkulose. Aangesien vroeë diagnose so belangrik is, kan
aktiewe kontak opsporing koste-effektief wees. Ge-individualiseerde
behandelingskedules is effektief om die sukses van behandeling en oorlewing te
verbeter, en moontlik ook om die oordrag van multi-middel weerstandige tuberkulose
te verminder.
Multi-middel weerstandige tuberkulose is 'n probleem vir die globale kontrole van
tuberkulose. Effektiewe strategieë om die vloed te stuit, bestaan, maar politieke
verbintenis en geldelike ondersteuning sal essensieël wees.
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The epidemiology and management of drug-resistant tuberculosis in childhoodSchaaf, Hendrik Simon 12 1900 (has links)
Thesis (MD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Resistance to antituberculosis agents became evident soon after antituberculosis
treatment was introduced for the first time. Combined drug therapy seemed to resolve
this problem. Animal experimental studies, which showed that isoniazid (INH)-resistant
strains of Mycobacterium tuberculosis were less infectious and pathogenic than drugsusceptible
strains, gave further reassurance that drug resistance was not a major issue.
Transmission of INH- and multiple-drug-resistant strains did, however, occur.
Studies in children, who develop mainly primary drug resistant tuberculosis (TB),
showed that drug resistance in adults was followed by a similar rise in drug-resistant
(TB) in children, and that tuberculous infection rates in childhood contacts of INHresistant
and drug-susceptible adult TB cases were the same.
It was however, only after the significant rise in the incidence of TB and large
outbreaks of multidrug-resistant (MDR) TB cases in developed countries (mainly
because of the human immunodeficiency virus epidemic) in the early nineties that
sufficient attention was again focussed on the problem of drug-resistant TB. Drugresistant
tuberculosis, and more in particular MDR TB, posed a serious threat to global
TB control programmes.
Despite this renewed interest, childhood drug-resistant TB remained neglected. The
incidence of drug-resistant TB among children, which could give a good indication of
currently circulating strains in a community, is hardly known. The management of
childhood contacts of adults with infectious MDR TB or children with MDR TB has also
not been studied prospectively. All confirmed childhood TB cases from a specific geographic drainage area over a
3.5-year period were prospectively included in a drug resistance surveillance study. The
incidence of drug resistance in children was comparable to the incidence of initial
(primary plus undisclosed previous treatment) drug resistance documented in adults in
the same area. The findings show that the incidence of drug-resistant TB in children in
the Western Cape province is low, and probably reflects the level of primary drug
resistance amongst organisms currently circulating in this community.
The short- and long-term outcome of children <5 years of age in contact with
infectious adult MDR TB cases was determined by prospective follow-up for 30 months.
The initial evaluation showed an infection rate significantly higher in MDR TB contacts
compared with contacts of drug susceptible cases, but the disease rate was lower. On
follow-up, many more children became infected or developed disease. The finding that
90% of those who developed disease did so within the first 12 months, indicates that
follow-up beyond 12 months is probably not cost-effective in resource poor countries.
The results demonstrate that MDR TB is not less infectious than drug susceptible TB.
Despite the fact that some children received chemoprophylaxis, 24% of the children
eventually developed disease. This is not different from the expected prevalence of
disease in childhood contacts <5 years of age of infectious drug-susceptible adult
pulmonary TB cases.
Restriction fragment length polymorphism analysis confirmed transmission from an
adult source case to a child contact in 5 of 6 adult-child pairs in whom both isolates were
available. If therefore an isolate of M tuberculosis for susceptibility testing cannot be
obtained from a child in close contact with an infectious MDR TB case, the child should therefore be treated according to the drug susceptibility pattern of the source case's
strain.
Treatment of children with confirmed and probable MDR TB included 2 or 3 drugs
to which the adult source case's isolate was susceptible in addition to pyrazinamide and
high-dose INH. Duration of treatment ranged from 6 to 12 months depending on the
severity of the disease. INH was included in the treatment regimen because low-level
resistance to INH was present in about half the cases of primary INH resistance. The
pharmacokinetics of INH in children confirmed that an adequate concentration and
exposure time could be achieved for this purpose. Ethionamide often caused
gastrointestinal adverse events, but these could be overcome in most cases by temporary
dose adjustments. The fluoroquinolones, which are not generally recommended for use in
children, possibly caused arthralgia in 1 of the17 children treated for ~6 months. This is
in accordance with previous reports of the safety of these drugs in children for short- and
medium-term treatment.
TB disease occurred significantly less often in children who received appropriate
chemoprophylaxis (according to the drug susceptibility pattern of the adult source case's
isolate). Although this was not a randomised controlled trial, the group that received
chemoprophylaxis was at higher risk for developing disease. This implies that prevention
of TB in MDR contacts is possible. A prospective, randomised controlled study is
necessary to evaluate the best drug combinations and the optimal duration of such
chemoprophylactic regimens. / AFRIKAANSE OPSOMMING: Middelweerstandigheid het na vore gekom kort nadat antituberkulose behandeling
vir die eerste keer in gebruik geneem is. Die gekombineerde gebruik van middels het
klaarblyklik die probleem oorkom. Diere eksperimente wat getoon het dat isoniasied
(INH)-weerstandige stamme van Mycobacterium tuberculosis minder infektief en
patogenies IS as vatbare stamme, het verdere gerustelling gegee dat
middelweerstandigheid nie 'n groot probleem is nie.
Die oordrag van INH- en multi-middelweerstandige stamme het egter wel
plaasgevind. Studies in kinders, wat hoofsaaklik primêre middelweerstandige tuberkulose
(TB) ontwikkel, het getoon dat middelweerstandigheid in volwassenes gevolg is deur 'n
soortgelyke toename in middelweerstandige TB in kinders en dat die voorkoms van
tuberkuleuse infeksie in kinderkontakte van INH-weerstandige en middelvatbare
volwasse TB gevalle dieselfde is.
Dis egter eers toe daar 'n beduidende toename in die insidensie van TB en groot
uitbrake van multimiddelweerstandige (MDR) TB gevalle in die ontwikkelde lande
(hoofsaaklik as gevolg van die menslike immuungebrek virus epidemie) in die vroeë
negentigerjare was dat daar opnuut aandag aan die probleem van weerstandige TB
geskenk is. Middelweerstandige TB, en in besonder MDR TB, hou 'n ernstige bedreiging
vir globale TB beheerprogramme in.
Tenspyte van die nuwe belangstelling in middelweerstandige TB is die probleem in
kinders steeds afgeskeep. Die insidensie van weerstandige TB in kinders is onbekend
alhoewel dit 'n goeie weergawe van die huidig sirkuIerende stamme in 'n gemeenskap sou gee. Die hantering van kinderkontakte van volwassenes met infektiewe MDR TB of
kinders met MDR TB is ook nog nie prospektiefbestudeer nie.
Alle bevestigde kinder-TB gevalle van 'n spesifieke geografiese gebied is oor 'n
3.5 jaar tydperk prospektief in 'n middelweerstandige waarnemingstudie ingesluit. Die
insidensie van middelweerstandigheid in kinders was vergelykbaar met die insidensie
van inisiële (primêre weerstandigheid plus onbekende vonge behandeling)
middelweerstandigheid in volwassenes van dieselfde gebied. Die bevindinge toon dat die
insidensie van middelweerstandige TB in kinders in die Weskaap provinsie laag is. Dit
weerspieël waarskynlik die vlak van primêre middelweerstandigheid in organismes wat
tans in hierdie gemeenskap sirkuleer.
Die kort- en langtermyn uitkoms van kinders <5 jaar oud wat in kontak met
infektiewe volwasse MDR TB gevalle was, is prospektief tydens 'n 30-maande opvolg
bepaal. Die aanvanklike evaluasie het 'n beduidend hoër infeksiekoers in die MDR TB
kontakte in vergelyking met kontakte van middelvatbare gevalle getoon, maar die
siektekoers was laer. Tydens die opvolgperiode het baie meer kinders infeksie of siekte
ontwikkel. Aangesien 90% van dié wat siekte ontwikkel het, dit gekry het binne die
eerste 12 maande, is opvolg ná 12 maande waarskynlik nie koste-effektief in hulpbronbeperkte
lande nie. Die bevindinge toon dat MDR TB nie minder infektief is as
middelvatbare TB nie. Tenspyte daarvan dat sommige kinders chemoprofilakse ontvang
het, het 24% van die kinders uiteindelik siekte ontwikkel. Dit verskil nie van die
verwagte siekte-insidensie van kinderkontakte <5 jaar oud wat in kontak met infektiewe
volwasse middelvatbare pulmonale TB was nie. Restriksie fragment lengte polimorfisme analise het oordrag van volwasse
brongeval na kinderkontak in 5 uit 6 volwasse-kind pare, van wie beide isolate
beskikbaar was, bevestig. Indien daar dus nie 'n isolaat van M. tuberculosis vir
vatbaarheidstoetse van 'n kind met nabye kontak met 'n infektiewe MDR TB geval
beskikbaar is nie, behoort die kind volgens die middelvatbaarheidspatroon van die
brongeval se stam behandel te word.
Behandeling van kinders met bevestigde of waarskynlike MDR TB het 2 tot 3
middels waarvoor die volwasse brongeval se isolaat vatbaar was, ingesluit, tesame met
pirasinamied en hoë-dosis INH. Die duur van behandeling het gewissel van 6 tot 12
maande op grond van die omvang van die siekte. INH is in die behandeling ingesluit
omdat dit getoon is dat ongeveer die helfte van die gevalle met primêre INHweerstandigheid
lae-vlak weerstandigheid het. Die farmakokinetika van INH in kinders
het bevestig dat genoegsame vlakke en blootstellingstyd aan INH vir hierdie doel bereik
kan word. Etionamied het dikwels gastrointestinale newe-effekte veroorsaak, maar dit
kon in die meeste gevalle oorkom word. Die fluorokwinolone, wat nie oor die algemeen
in kinders aanbeveel word nie, het moontlik artralgie veroorsaak in 1 uit 17 kinders wat
vir ~6 maande behandel is, wat vorige verslae oor die veiligheid van hierdie middels in
kort- en medium-termyn behandeling bevestig.
TB-siekte het beduidend minder dikwels voorgekom in kinders wat toepaslike
chemoprofilakse (volgens die middelvatbaarheidspatroon van die volwasse brongeval se
isolaat) ontvang het. Alhoewel dit nie 'n ewekansig gekontroleerde studie was nie, het
die groep wat chemoprofilakse ontvang het die hoogste risiko vir die ontwikkeling van
siekte gehad. Dit dui daarop dat voorkoming van TB in MDR TB kontakte moonlik is. 'n Prospektiewe, ewekansig gekontrolleerde studie is nodig om die beste middel
kombinasies en die optimale duur van so 'n chemoprofilaktiese behandeling te bepaal.
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