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Babiker, Amira Mustafa Ahmed (2014) Genetic Diversity of Sudanese Leishmania Parasites and Possible Correlation to Clinical Signs. [Tesi di dottorato]

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Abstract (inglese)

Introduction: Leishmaniasis forced itself upon medical attention as an increasingly significant problem over the last decade hence it is associated with poverty in developing countries and considered as imported or re-emerging diseases in Europe. Leishmaniasis which is a globally widespread group of diseases is caused by obligatory, Intracellular pathogen of the immune system targeting macrophages and dendritic cells. In humans the infection is transmitted through the bites of female sand flies. The symptoms ranging from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes to fatal visceral disease affecting the haemopoetic organs. It is endemic in the tropical and subtropical regions of 88 countries, sixteen are developed countries, 72 are developing countries, 13 of them are among the least developed. Sudan is one of the greatest foci worldwide where Leishmaniasis represents a serious health problem causing high number of fatalities. Leishmania donovani complex and L.major are known to be the etiologic agents, however, the Leishmania donovani complex parasites that causes different clinical forms, i.e. visceral (VL), cutaneous (CL), muco-cutaneous (ML) and post kala-azar dermal Leishmaniasis (PKDL) are genetically considered as a homogenous group.
Objectives: To understand the correlation between the genetic diversity and the clinical manifestations.
Specific Objectives 1) To genetically characterize isolates from Sudanese patients diagnosed with different types of leishmaniasis to understand the correlation between different clinical manifestations and the genetic profile of Leishmania.2) Investigate presence or absence of mixed infections and/ or hybrids. 3) To build up a reliable picture of the various phylogenetic subdivisions with relation to virulence, using the Internal Trascribed Spacer (ITS) gene, the Lyshmanolysin (GP63) and the cytochrome Oxidase sub- unit II gene.
Materials and Methods: Comparative genome based approach was used to analyse the three genes, Cytochrome Oxidase II (COII) gene, the Internal Transcribed Spacer (ITS) and the GP63 (both gp63 3/4 and MSPC). 111 samples from 72 Sudanese patients suspected of different Leishmaniasis were used. Firstly a Real Time PCR was developed to pick up positive samples. Secondly, to detect the interspecific polymorphism at species level, Cytochrome Oxidase II (COII) gene was amplified for all Real Time PCR positive samples. The methodology encompasses the use of direct sequencing and cloning techniques. Thirdly, to detect the intraspecific polymorphism at strain level and the under types of some positive isolates, (ITS) and the GP63 (both gp63 3/4 and MSPC) genes were amplified , The methodology encompasses the use of cloning techniques.
Results and Discussion: 40 patients out of 72 (55.6%) and 58 samples out of 111 (52.3%) were confirmed by the Real Time PCR as Leishmania positive. Five CL samples from five patients have been examined, three of them have been concomitantly infected with both L.donovani and L.major as confirmed by the cloned COII and ITS genes. The ITS sequence data analysis of L. major related colonies in the three CL samples revealed the presence of more than one strain /hybrid of L. major as different polymorphic sites were detected within and between patients. The other two CL samples were found to be caused merely by L.donovani, but different sequence patterns have been detected by the ITS gene.
One patient possessed L. infantum similar COII sequence, the GP63 sequence data analysis of the same patient confirmed some colonies as L. infantum, while two out of the six identified polymorphic sites in the ITS gene were similar to those characteristic for L.infantum different zymodemes.
The number of PKDL and ML samples was not sufficient to draw a conclusion., However, COII and ITS data pointed L.donovani as the causative agent. ITS data showed considerable different sequence patterns between colonies from the two samples. The same results were found regarding other samples attributed to VL.
Gp63 sequence data analysis of some colonies attributed to VL samples (namely 10 HBM, 3HBM and 11HBM) has identified two genetic groups, both within the L. donovani complex but different from L. infantum and L. donovani .
Conclusions: The study concludes that 1) affirmed the presence of mixed infection with L. donovani and L. major from a single cutaneous aspirate in CL patients. To the best of our knowledge this is the first report of L. donovani and L. major mixed-infection in Sudan. Additionally no other cases of such mixed-infection from the same cutaneous sample were reported elsewhere. 2) the study assessed the circulation of more than one strain/hybrid of L. major and L. donovani between Sudanese patients. 3) the presence of L. infantum as an etiological agent of VL in Sudan has been affirmed by CO II and ITS genes. 4) Leishmania isolates with mixed species or strains might be subjected to selective pressure upon parasite culture, so direct extraction from biological materials is important for characterization.
These findings have important implications regarding the diagnosis, the choice of the most appropriate therapy and the possibility of developing drug resistance and at the same time set the stage for future studies as the prevalence of different species and vectors and the competence of Phlebotomus spp. in transmitting different Leishmania.

Abstract (italiano)

Introduzione: Negli ultimi anni Leishmaniosi si imposta all'attenzione medica come un problema sempre più rilevante. Le malattie sono associate con la povertà nei paesi in via di sviluppo e considerate importati o riemergenti in Europa. Leishmaniosi è un gruppo di malattie globalmente diffusa causata da un patogeno intracellulare del sistema immunitario targeting i macrofagi e le cellule dendritiche. Nell'uomo l'infezione si trasmette attraverso le punture dei flebotomi. I sintomi variano dalle lesioni cutanea e muco-cutanea che possono causare vasta distruzione delle membrane mucose alle malattie mortale viscerale che colpisce gli organi haemopoetici. leishmaniosi è endemica nelle regioni tropicali e subtropicali di 88 paesi, sedici sono paesi sviluppati, 72 sono in via di sviluppo, 13 di loro sono tra i meno sviluppati.
Il Sudan è conosciuto fra i più grandi focolai a livello mondiale dove Leishmaniosi rappresenta un grave problema di salute causando elevato numero di vittime. Leishmania donovani complex e Leishmania major sono noti per essere gli agenti eziologici, tuttavia, i parassiti della Leishmania donovani comlex che causano diverse forme cliniche, i.e viscerale (VL), cutanee (CL), muco-cutanea (ML) e post kala-azar Leishmaniosi (PKDL) sono considerati geneticamente un gruppo omogeneo.
Obiettivi: Per comprendere la correlazione tra la diversità genetica e le manifestazioni cliniche.
Obiettivi specifici: 1) geneticamente caratterizzare isolati da pazienti Sudanesi diagnosati di diversi tipi di leishmaniosi per capire la correlazione tra diverse manifestazioni cliniche e il profilo genetico di Leishmania.2) Indagare sulla presenza o assenza di infezioni miste e / o ibridi. 3) Per costruire un quadro attendibile delle varie suddivisioni filogenetici in relazione alla virulenza, utilizzando i gene Internal Transcribed Spacer (ITS) Lyshmanolysin (GP63) e il citocromo ossidasi II
Materiali e metodi: è stato utilizzato un’approccio basato sul genoma comparativo per analizzare i tre geni, citocromo ossidasi II (COII), Internal Transcribed Spacer (ITS) e il GP63 (sia gp63 3/4 che il MSPC). Sono stati utilizzati 111 campioni provenienti da 72 pazienti sudanese sospettati di diversi Leishmaniosi.In primo luogo, un real time PCR è stato sviluppato per raccogliere campioni positivi. In secondo luogo, per rilevare il polimorfismo interspecifico al livello di specie, citocromo ossidasi II (COII) gene è stato amplficato per tutti i campioni positivi nel real time PCR. Il metodo comprende l'uso di sequenziamento diretto e tecniche di clonazione. In terzo ed ultimo luogo, per rilevare il polimorfismo intraspecifico al livello del ceppo e dei sotto tipi di alcuni isolati positivi, (ITS) e il GP63 (entrambi gp63 3/4 e MSPC) geni sono stati amplificati. Il metodo comprende l'uso di tecniche di clonazione
Risultati e Discussione: 40 pazienti su 72 (55,6%) e 58 campioni su 111 (52,3%) sono state confermate dal Real Time PCR come Leishmania positivi. Cinque campioni cutanei provenienti da cinque pazienti sono stati esaminati, tre di loro sono stati infettati in concomitanza sia con L.donovani che con L.major come è confermato dal clonaggio del COII e ITS geni.
L’ analisi dei dati ITS delle colonie correlate a L. major nel tre campioni cutanee ha rivelato la presenza di più di un ceppo / ibrido di L.major come è stato dimostrato dai siti polimorfici rilevati all'interno dei pazienti e tra di loro. Gli altri due campioni cutanei sono stati causati soltanto da L.donovani, ma differenti tipi di sequenza sono stati rilevati dal ITS gene.
Un paziente possedeva sequenza simile di L. infantum COII, il GP63 analisi di sequenza dallo stesso paziente ha confermato alcune colonie come L. infantum, mentre, due dei sei siti polimorfici individuati nel ITS gene erano simili a quelli caratteristici per L.infantum diversi zymodemi.
Il numero di campioni PKDL e ML non era sufficiente per trarre una conclusione. Tuttavia, COII e ITS dati hanno indicato L.donovani come l’agente causale, ITS dati ha mostrato notevoli tipi di diversi sequenze tra le colonie dei due campioni. Gli stessi risultati sono stati trovati per quanto riguarda altri campioni attribuiti al VL.
Gp63 analisi dei dati di sequenza di alcune colonie attribuite ai campioni VL (ossia 10 HBM, 3HBM e 11HBM) ha individuato due gruppi genetici all'interno della L. donovani complex, ma diversi da L. infantum e L. donovani.
Conclusioni: Lo studio conclude che 1) ha affermato la presenza di infezione mista con L. donovani e L. major da un unico aspirato cutaneo. a nostra conoscenza questo è il primo rapporto di L. donovani e L. maggiore mista-infezione in Sudan. In aggiunta altri casi di tale infezione mista dallo stesso campione cutanea non sono stati riportati altrove. 2) lo studio ha valutato la circolazione di più di un ceppo / ibrido di L. major e L. donovani tra pazienti sudanesi. 3) la presenza di L. infantum come agente eziologico di VL in Sudan è stato affermato da CO II e ITS geni. 4) Leishmania isolati con le specie o ceppi misti potrebbero essere sottoposti a pressione selettiva durante la cultura, l’estrazione diretta da materiali biologici è importante per la caratterizzazione.
Questi risultati hanno implicazioni importanti per quanto riguarda la diagnosi, la scelta della terapia più appropriata e la possibilità di sviluppare resistenza ai farmaci e allo stesso tempo, porre le basi per futuri studi come la prevalenza delle diverse specie e vettori e la competenza del Phlebotomus spp. nel trasmettere diversi Leishmania.

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Tipo di EPrint:Tesi di dottorato
Relatore:CAPELLI, GOIA
Correlatore:HASSAN, MOAWIA MUKHTAR
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > SCIENZE VETERINARIE > SANITA' PUBBLICA E PATOLOGIA COMPARATA
Data di deposito della tesi:30 Gennaio 2014
Anno di Pubblicazione:30 Gennaio 2014
Parole chiave (italiano / inglese):L.donovani, Mixed infection, Sudan
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Biomedicina Comparata ed Alimentazione
Codice ID:6719
Depositato il:04 Nov 2014 15:15
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