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    Studies on the bionomics and molecular insecticide resistance of anophelines in Gorgora, north-west Ethiopia
    (University of Khartoum, 2008-11) Balkew, Meshesha ; M Elhassen, Ibrahim ; E.Ibrahim, Muntaser
    Dembia district (wereda) in Gondar, north Ethiopia is one of the malarious areas in the country. An entomological study was carried out in one of the villages, Gorgora, during the malaria transmission seasons in 2006 and 2007 with the main objectives of documenting the species compositions of prevailing anophelines, study the feeding and resting habits, identify blood meal sources, detect Plasmodium infection, determine the susceptibility/resistance status of the main malaria vectors and detect the mechanisms of resistance. Anophelines were captured using Center for Disease Control (CDC) light trapnets, aspirators, space sprays and pit shelters. Morphological features were used to discriminate anophelines into species, and the polymerase chain reaction (PCR) was employed to screen members of the Anopheles gambiae complex. Sprozoite and blood meal ELISA analysis were carried out to detect infections and identify blood meal sources. Insecticide susceptibility status of An. arabiensis and An. pharoensis to DDT, permethrin, deltamethrin and malathion was evaluated using standard bioassay WHO test kits on adults reared from larvae and pupae. For comparative reasons, An. arabiensis from Sodere in central and Ghibe River Valley in south west Ethiopia were additionally included. Colonies were established from surviving individuals for assessing the occurrence of true as well as cross resistance. Genetic and metabolic resistance mechanisms on An. arabiensis were investigated by conducting PCR and micro plate biochemical assays, respectively. Overall, 5,664 in 2006 and 3,106 in 2007 adult female comprising 9 Anopheles species were captured predominantly from CDC light trap-nets. The dominant species was An. pharoensis Theobald comprising 70% of collections in 2006 and 54% in 2007 followed by An. arabiensis Patton accounting to 20% and 18% in the two consecutive years. The other species made up the remaining 10% and 28% of collections in those years. The density of An. arabiensis was highest from July to September and reduced in October and November, in contrast, An. pharoensis peaked from September to November. III CDC trap-nets collected all gonotrophic stages of An. arabiensis and An. pharoensis. The geometric mean of the blood unfed stages were considered relevant to determine the human biting rate (HBR) which corresponds to mosquitoes/trap/night, and hence, the HBR of An. arabiensis during the five months (July-November) in 2006 ranged from the lowest 0.4 to the highest 0.7 mosquitoes/man/night. The HBR of An. pharoensis for the same period was between 0.3 and 1.4 mosquitoes/man/night. There was no detectable difference in biting rates in 2007. From the CDC trap-net collections, blood meals of 255 An. arabiensis and 206 An. pharoensis were analyzed, out of which 64.7% and 36.9% were found to contain human blood, respectively. The proportion of cattle fed mosquitoes in that order was 27.5% and 44.7%. The rest of the blood meals contained both mixed human and cattle or were non-reactive. Only 4 An. arabiensis were captured from human habitations with aspirator in 2006 and 33 in 2007 using space sprays. Similarly, An. pharoensis was caught in small numbers. Both species were collected from animal shelters and the density of An. arabiesnsis varied from 0 to 7.5 mosquitoes/shelter/day while that of An. pharoensis was 0 to 14.8 mosquitoes/shelter/day at different months. However, the latter species density reached to 234 mosquitoes/shelter/day from animal shelters in Toka Agroindustrial Enterprise. It is, however, difficult to surmise animal shelters in the enterprise serve as resting sites since fresh fed mosquitoes were collected on window screens in the act of egression. From animal shelter collections, 59 An. arabiensis and 86 An. pharoensis blood meals were analyzed. The respective human blood index of the two species was 0.17 (n=1) and 0.58. (n=5). The proportion fed on cattle was 76.3% (n=45) and 50% (n=46), respectively while the rest of the blood meals were unidentified either human or bovine. More than 1000 An. arabiensis and 800 An. pharoensis were examined for parasite infection against circumsporozoite proteins of Plasmodium falciparum and P. vivax 210 and 247 polymorphs.but none was found with parasite infections. Insecticide bioassay test results of both mortality and knockdown time revealed very high DDT resistant An. arabiensis from Ghibe (96.2%) followed by Gorgora (50.5%). DDT IV failed to produce 100% knock-down in all test mosquitoes. An. arabiensis from Ghibe was also greatly resistant to permethrin (69.3%) and deltamethrin (69.7%), while the strain from Gorgora was less resistant (15-19%). Permethrin was less effective to cause complete knock-down in the vector from Ghibe while deltamethrin took an elevated KT50 of 37.6 minutes. The respective KT 50 values of the two pyrethroids on An. arabiensis from Gorgra were 19.6 and 25.3 minutes. The strain from Sodere can be considered moderately resistant (20%) to DDT and highly susceptible to the pyrethroids if mortality data are only taken into consideration, however, failure of DDT to knockdown all test mosquitoes and higher KT50 of 17.8 minutes for permethrin and 21.9 minutes for deltamethrin indicate presence of considerable resistance which needs regular follow up. The existence of cross resistance between DDT and permethrin was confirmed on DDT selected An. arabiensis from Gorgora, where as, it is absent between DDT and deltamethrin. The West African type kdr mutation was found in the vector population from Gorgora and Ghibe, and this molecular mechanism probably confers resistance to DDT and the pyrethroids in addition to metabolic enzymes. Biochemical assays on DDT selected An. arabiensis from Sodere revealed more glutathione-S-transferase and general estrases activities than the Debrezeit susceptible strain. DDT resistance in An. pharoensis from Gorgora was very high reaching up to 93.8%. The knock-down time for the two pyrethroids is longer than reference susceptible strains. Both species are susceptible to malathion
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    Molecular Characteristics and Epidemiology of Plasmodium falciparum Susceptibility to Sulfadoxine-Pyrimethamine and Chloroquine in Haj Yousif and Gedarif Town, Sudan
    (University of Khartoum, 2008-01) Tagelsir, Nawal ; Suliman, Nageeb ; Ibrahim, Muntaser E.
    Malaria is the world's most important tropical parasitic disease. The problem of malaria was aggravated due to the emergence of resistance to antimalarials and insecticides in the absence of a suitable malaria vaccine. The present study was carried out in two areas during two years (1998 to 2001), with the objectives of assessment of resistance to chloroquine and sulphadoxine-pyrimethamine as first-line antimalarials. Another purpose was the validation of molecular markers related to chloroquine and sulphadoxinepyrimethamine resistance for prediction of treatment failure to these antimalarials; and evaluation of the contribution of host and parasite factors in the development of resistance to antimalarial drugs. The study clearly demonstrated that chloroquine was no longer effective in the treatment of falciparum malaria. High levels of chloroquine resistance were detected in both areas; it was 77.1% and 56.3% in Gedarif and Haj Yousif respectively. High levels of sulphadoxine-pyrimethamine susceptibility were observed in Haj Yousif (95.6% and 98% in the first and second malaria seasons respectively) compared to 76.7% in Gedarif. Estimation of genuine levels of resistance to both antimalarials in Haj Yousif was carried out by genotyping of polymorphic genes msp1, msp2 and glurp. The mutant allele of pfcrt K76T was predominant in both study areas. In Haj Yousif, all pre-treatment specimens were found to be carriers of the mutant allele 76T regardless of their response to chloroquine. Also, in Gedarif all pre-treatment specimens were found to be carriers of the mutant allele 76T either as a pure mutant allele (96.2%) or a mixed allele (3.8%) regardless of their response to chloroquine. Lack of a significant association between mutant allele of Pfmdr1 N86Y and chloroquine treatment failure in both areas was detected. In Haj Yousif the frequency of mutant allele 86Y, wild allele N86 and mixed allele was 82.9%, 10% and 7.1% respectively. All post-treatment specimens were carrying mutant allele 86Yexcept one which had a wild one. In Gedarif the frequencies of mutant allele 86Y, wild allele N86 and mixed allele were 68.4%, 7.6% and 24.1% respectively. Similarly the mutations in dhfr and dhps genes had no significant role on sulphadoxine-pyrimethamine resistance in both areas. The association was observed in dhps K540E in Haj Yousif first cross-sectional study. The mutant allele dhfr S108N III and dhfr N51I were predominant in both areas. Less mutation was observed in dhps gene. The pattern of dhfr and dhps mutations in Haj Yousif second cross-sectional study was similar to that of the first study except in dhfr C59R and dhps K540E where the mutant allele has completely disappeared in the second study. Single, double and quadruple mutations in both genes were detected in the first and the second study while the triple mutation was detected only in the first study. In Gedarif study the frequency of mutations in dhfr gene was similar to the pattern in Haj Yousif study. Also, less mutation was observed in dhps gene compared to dhfr gene. Double, triple, quadruple and quintuple mutations in both genes were detected in the area. Although a similar frequency of dhfr mutations was observed in both study areas, the frequency of dhps mutations was significantly higher in Gedarif compared to Haj Yousif. A high frequency of mixed allele in Pfmdr1, dhfr and dhps was observed in Gedarif area indicating the prevalence of multiplicity of infections in the area which were reported from previous studies. . It was found that the assessment of antimalarial efficacy using 14 days followup protocol underestimated the resistance level and therefore it is recommended to use the 28 days follow-up protocol or longer. The contribution of gender, age and initial parasitaemia to the development of treatment failure was investigated. The significant effect of age as a factor for acquisition of immunity against treatment outcome was confirmed in the chloroquine study in both study areas (P< 0.01). There was a significant reverse relationship between age and parasite density. This validated the value of age as an immunological marker. Initial parasitaemia has a significant effect on development of chloroquine resistance in Haj Yousif. The contribution of gender as a factor in treatment failure was ruled out. There was no significant association between gender and treatment failure in both areas. It was concluded that, further clinical studies were needed to define the usefulness of Pfcrt, Pfmdr-1, dhps and dhfr as molecular markers for chloroquine and sulfadoxinepyrimethamine resistance and identify host factors that may influence the clinical outcome of drug treatment and explain the clearance of parasitaemia of parasites carrying mutant Pfcrt Pfmdr-1, dhps and dhfr allele by some patients. The quality of antimalarial products and patient adherence to dosage regimens are important determinants of drug effectiveness, and should be measured alongside the clinical efficacy.
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    Molecular Typing and antimicrobial sensitivity of Mycobacteria isolated from Yemeni Tuberculous Patients
    (University of Khartoum, 2008-02) Ahmad, Anas ; Mahgoub, Elsheikh ; M. Mukthar, Moawia
    The aim of the present study was to characterize Mycobacteria isolated from Yemeni pulmonary TB patients to the species level and determine antibiotic sensitivity and resistance of isolated strains to routine antituberculosis drugs. One hundred seventy early morning sputum samples were collected from the Yemeni pulmonary TB patients referred to the National Tuberculosis Institute (NTI) in Sana’a – Yemen. Out of the 170 of Pulmonary TB cases 158 (93%) were new cases and 12 (7%) were old cases. Samples were processed by the Ziehel-Nelseen stain then the positive samples were inoculated in the special Ogawa’s TB medium and cultivated at 37◦C for 4-6 weeks. After successful cultivation the isolates were tested for sensitivity against the routine antibiotics treatment in Lowenstain-Jenseen medium. Few colonies (1 – 2 colonies) from positive inoculation L-J culture media were taken from the TB cultures and processed with the Phenol- Chloroform heating method to lyse the cell wall of the Mycobacteria and extract the DNA. All those procedures were done in the research microbiology laboratory in the NTI Sana’a Yemen. The extracted DNA were isolated and preserved in special microcenterfiuge tubes and transported to the Institute of Endemic Diseases (IEND), Khartoum University – Sudan for further molecular analysis because molecular techniques are not available till now in Yemen. 15 In the (INED) firstly small portion of isolated DNA were run in the Gel-Electrophoresis to be insured from the availability of DNA, then DNA samples were processed by PCR machine to detect and amplify the Mycobacteria DNA using specific primers complementary to the rpoB gene on the Mycobacteria DNA . After successful amplification of the target sequence of the DNA the PCR product were digested with specific restriction Enzyme by technique called Restriction Fragment Length Polymorphism (RFLP) then the digested DNA was electrophoresed in 2% agarose gel. DNA from All drug resistance isolates were amplified again separately and sent to the Korean Tuberculosis Institute (KTI) for doing DNA sequencing analysis. Results of our study showed that TB was present in all Yemen area and the highest rates of incidence were rerecorded in Hajah (18%), Alhodidh (14%), and Almaharh (12%) which are characterized by its hot weather and rural crowded provinces. Regarding gender our findings showed that the incidence of disease was high (69%) among males than females (31%) therefore the difference was significant. Infection was noted in patients at any age group but the disease was high (39%) among patients whose age ranged between 20 – 30 years. The antibiotics sensitivity results showed that; (91.2%) were sensitive to the four antibiotics: Isoniazid, Rifampicin, Streptomycin, and Ethambutol; but 15 (8.8%) were resistant to one or Multi drug resistant (MDR). Out of 15 resistant isolates 5/15 (33.3%) were resistant to only one type 16 of antibiotic "mono-resistant" whereas 10/15 (66.7%) were multi drug resistant MDR-TB. The highest percent of MDR-TB isolates were resistant to Isoniazed 14/15 (93%) followed by Rifampicin 8/15 (53%), Ethambutol 6/15 (40%) and Streptomycin 5/15 33%. According to the history of disease 9/15 (60%) of those MDR-TB were new cases and 6/15 (40%) were old cases. The molecular PCR thermocyclear results showed that the DNA sequence of rpoB gene was amplified successfully using (TB-MF and MR) primers. But the RFLP results showed the PCR-RFLP using (Hind11) restriction enzyme gave very clear, more reliable, and easier in the interpretation of the result than the (Msp1) enzyme. Out of 120 samples 118 (98.3%) were Mycobacterium tuberculosis complex (MTC) and two samples (1.3%) were Mycobacterium other than tuberculosis complex (MOTT). Our findings showed that MOTT strains were not sensitive to all antibiotics. Both MOTT strains isolates were resistant to three types of antibiotics TB treatment; Isoniazid, Rifampicin, and Streptomycin and sensitive only to Ethambutol. In addition the DNA sequencing analysis results showed that the alignment of nucleic acid of DNA in MOTT is different from that of MTC. Also DNA sequencing alignment shows that one isolate of MDR-TB which was collected from a TB patient comes from Somalia and harbored various nucleic acid sequence which suggested that this strain may be M.africanum.
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    Prevalence of Echinococcus granulosus in Southern Libya: (Immuno-epidemiological Study)
    (University of Khartoum, 2006) Abugrara, Abdulkadir
    Human cystic hydatidosis due to infection with the larval stage of Echinococcus granulosus is a serious public health problem in many countries. The disease is highly endemic in Libya, with previous studies that concentrated on the north-east, north-central and north-west. This is cross-sectional and community-based study aimed at determining the prevalence of Echinococcus granulosus in Murzuk province in southern Libya. Following informed consent a total of 1274 individuals from 25 villages in the province were interviewed and screened serologically. ELISA (Gold-standard) and a devised Latex agglutination (devised) techniques were used to detect anti-echinococcal antibodies. Biochemical profiles of the sera were examined using Reflotron Machine (Blood dry chemistry). Demographic and laboratory data were entered, checked and verified using the EpiInfo 2004 software package. Twenty nine individuals (29/1274; 2.3%) were sero-reactive using ELISA techniques. The seroprevalence was significantly different between different areas of the province and ranged from 1.9% to 4.1% (p=0.0006). The Latex agglutination technique was positive in 28/1274 individuals. Females constituted 68.0% of sero-reactive group [Female:Male=20:9] (p=0.008). Sero-reactive individuals has normal urea, creatinine (p>0.05). AST, ALT and alkaline phosphatase were universally increased (p=0.0000). The seroprevalence was commonly seen in the age group 7-50 years (p=0.006). The sensitivity, specificity, positive predictive value and negative predictive value of the Latex were 96.6%, 99.9%, 95.8% and 99.9% respectively. In conclusion: Echinococcus granulosus infection is prevalent in Southern Libya with a prevalence that ranges from 1.9% to 4.1%. The devised Latex agglutination is easy to prepare and cheap test showed high sensitivity, specificity, positive predictive and negative predictive values. The exposure rates was 6.8%.
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    In vivo and in vitro Cellular and Humoral Immune Responses in Sudanese Patients with Type-II Diabetes Mellitus.
    (uofk, 2009-08) Khalid, Mariam ; Awad, Altahir
    Diabetes mellitus is becoming a major health problem in the Sudan with an estimated incidence of (7%). Malaria and tuberculosis are the major health problem in the Sudan.. It is well known that infections in patients with diabetes mellitus are more severe, although there is controversy for increased susceptibility to them. One of the possible causes for this increased prevalence of infections is derangements in immunity.