Department of Parasitology and Medical Entomology

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    Molecular Detection of Cytotoxin Associated Gene A and E of Helicobacter pylori Isolated from Patients Undergoing Upper GI Endoscopy in Khartoum State
    (University of Khartoum, 2020) Esraa Hassan Osman Almobark
    Abstract Background: Helicobacter pylori cytotoxin associated gene pathogenicity island (cag-PAI) is one of the strain-specific genes (they do not exist in all strains). It is a deoxyribonucleic acid (DNA) fragment contains 31 genes, including; cagA, cagE, virB11 genes, in addition to other genes that code for functional components of a type IV secretion system. These genes are involved in inducing inflammation, ulceration, and carcinogenesis. This study aimed to detect and characterize cagA and cagE virulence genes of H. pylori strains from Sudanese patients with gastric discomfort in Khartoum State. Methods: This was a descriptive cross-sectional study conducted during the period from 2018 to 2020. A total of 288 gastric biopsies were collected endoscopically by the gastroenterologist in different Khartoum hospitals, using 1.0 ml brain heart infusion broth with 15% glycerol as transport media. Gastric biopsies were homogenized and plated on H. pylori selective media and incubated in standard microbiological conditions. DNA was extracted from the biopsies using the guanidine chloride extraction method and stored at -70˚C until use. The polymerase chain reaction was used for the detection of H. Pylori (using specific primers targeting 16S rRNA and glmM genes) and two virulence factor encoding genes (cagA and cagE genes). Five cagA and 21 cagE amplified gene products were sequenced and the sequences were analyzed and compared with reference sequences (JF798705.1& MK074991.1 for cagA, AB191082.1, and AY153111.1 for cagE). Data were analyzed using the Statistical Package for Social Science (SPSS version 11.5) computer program. Frequencies were calculated. The Chi-square test was used. Results: Out of 288 specimens, 98 (34%) were positive for H. pylori (glmM and/or 16S rRNA positive). Fifty-one percent of the H. pylori-positive patients were females and 49% were males. The majority of the H. pylori-positive patients were adults 97% and 3% were adolescents. There was no statistically significant association between the presence of H. pylori, epidemiological findings (geographical distribution of patients (P-value= 0.257), gender (P-value= 0.076) and age groups (P-value= 0.784)) and endoscopic findings (P-value= 0.440). The isolation of H. pylori by culture was failed and no bacterial growth in all 288 specimens. The cagA gene was present in 40/98 (41%) specimens, their clinical diagnosis as followed: the majority of them 29 (72.5%) from patients with gastritis, 6 (15%) duodenal ulcer, 2 (5%) esophagitis, and 3 (7.5%) were diagnosed as normal by endoscopy. The cagE gene was present in 38/98 (39%) specimens, their clinical diagnosis as followed: 28 (73.7%) from patients with gastritis, 1 (2.6%) gastric ulcer, 4 (10.5%) duodenal ulcer, 2 (5.3%) esophagitis and 3 (7.9%) were diagnosed as normal. Thirteen specimens (13%) were positive for cagA only, 11/98 (1%) were positive for cagE only, 27/98 (28%) were positive for both genes, 51/98 (52%) were positive for one gene at least, 47/98 (48%) were negative for both genes. There was no statistically significant association between the presence of these virulence genes and the clinical diagnosis (P-value= 0.305). The alignment of 5 cagA and 21 cagE protein sequences with JF798705.1 and AY153111.1 reference sequence respectively revealed a substitution of one amino acid in two cagA protein sequences, these two sequences have proline (P) instead of alanine (A), with 100% identity to MK074991.1 reference sequence. In addition to that one cagE protein sequence revealed a substitution of one amino acid; this sequence has valine (V) instead of isoleucine (I), with 100% identity to AB191082.1 reference sequence. Conclusion: H. pylori low prevalence in this study (34%) indicates that Sudan is in the low-risk region for infection. There is high dissemination of cagA and cagE genes in H. pylori from Sudanese patients. Still, they have reduced virulence. The cagA and cagE protein sequences have synonymous amino acid variations. المستخلص الخلفية: جينات جرثومة المعدة المصاحبة للجينات المسببة للأمراض الجينية (cag-PAI) هي واحدة من الجينات الخاصة بالسلالة (لا توجد في جميع السلالات). إنها جزء من حمض النووي الريبوزي منقوص الأكسجين (DNA) تحتوي على 31 جينًا، بما في ذلك؛ جينات cagA و cagE و virB11 بالإضافة إلى الجينات الأخرى التي ترمز للمكونات الوظيفية لنظام الإفراز من النوع الرابع. تشارك هذه الجينات في إحداث الالتهاب والتقرح والسرطان. الهدف من هذه الدراسة هو الكشف عن جينات الضراوة cagA و cagE لسلالات جرثومة المعدة من المرضى السودانيين الذين يعانون من اضطرابات في المعدة في ولاية الخرطوم. الطرائق: كانت هذه دراسة وصفية مقطعية أجريت خلال الفترة من 2018 إلى 2020.تم جمع 288 خزعة معدة بمنظار المعدة من قبل أخصائي أمراض الجهاز الهضمي في مستشفيات مختلفة في الخرطوم، باستخدام 1.0 مل brain heart infusion broth مع 15٪ جلسرين كوسيط نقل. تم تجانس خزعات المعدة. وتزريعها على وسائط انتقائية لجرثومة المعدة و حضنت في ظروف مايكروبيولوجية قياسية .تم استخراج الحمض النووي من الخزعات باستخدام طريقة استخراج كلوريد الغوانيدين و تخزينها في -70 درجة مئوية حتى الاستخدام. تم استخدام تفاعل البلمرة المتسلسل للكشف عن جرثومة المعدة (باستخدام بادئات محددة تستهدف جينات 16S rRNA و glmM) واثنين من جينات ترميز عامل الضراوة (جينات cagA و cagE). تم تسلسل 5 منتجات جينية مضخمة cagA و 21 cagEو تم تحليل التسلسلات و مقارنتها مع التسلسلات المرجعية (JF798705.1 و MK074991.1 لـ cagA و AB191082.1 و AY153111.1 لـ cagE). .تم تحليل البيانات باستخدام البرنامج الحاسوبي للحزمة الإحصائية للمعلومات الاجتماعية (SPSS الإصدار 11.5). تم حساب الترددات. تم استخدام اختبار .Chi-Square النتائج: من أصل 288 عينة، كانت 98 (34٪) موجبة لجرثومة المعدة (glmM و/ أو 16S rRNA). كان واحد وخمسون بالمائة من المرضى المصابين بجرثومة المعدة من الإناث و 49٪ من الذكور. غالبية المرضى المصابين بجرثومة المعدة كانوا بالغين 97٪ ، و 3٪ كانوا مراهقين. لم تكن هناك علاقة ذات دلالة إحصائية بين وجود جرثومة المعدة و النتائج الوبائية (التوزيع الجغرافي للمرضي (قيمة = P 0.257)، الجنس ( قيمة= P 0.076) والفئات العمرية (قيمة = P 0.784)) و النتائج بالمنظار (قيمة = P 0.440) .فشل عزل جرثومة المعدة عن طريق التزريع ولم يكن هناك نمو للبكتريا في جميع العينات البالغ عددها 288. كان الجين cagA موجودًا في 40/98 (41٪) عينة، تشخيصهم السريري على النحو التالي: غالبيتهم 29 (72.5٪) من مرضى التهاب المعدة، 6 (15٪) قرحة الاثني عشر، 2 (5٪) التهاب المريء، و 3 ( 7.5٪) كانت طبيعية وقد تم التشخيص عن طريق منظار المعدة. كان جين cagE موجودًا في 38/98 (39٪) عينة، تشخيصهم السريري على النحو التالي:: 28 (73.7٪) من مرضى التهاب معدي، 1 (2.6٪) قرحة معدية ، 4 (10.5٪) قرحة الاثني عشر، 2 ( 5.3٪) التهاب المريء و 3 (7.9٪) كانت طبيعية. كانت ثلاث عشرة عينة (13٪) موجبة لـ cagA فقط ، 11/98 (1٪) كانت موجبة لـ cagE فقط ، 27/98 (28٪) كانت إيجابية لكلا الجينين ، 51/98 (52٪) كانت موجبة لجين واحد على الأقل،47/98 (48٪) كانت سلبية لكلا الجينين. لم تكن هناك علاقة ذات دلالة إحصائية بين وجود جينات الضراوة و التشخيص السريري (قيمة = P 0.305). كشفت محاذاة 5 تسلسلات cagA و 21 cagE مع التسلسلات المرجعيةJF798705.1 و AY153111.1 على التوالي عن استبدال حمض اميني واحد في تسلسل بروتينين cagA ، يحتوي هذان التسلسلان على البرولين (P) بدلاًمن الألانين (A) ، مع تطابق 100٪ لـلتسلسل المرجعي MK074991.1 ، بالاضافة الى ان تسلسل بروتين cagE كشف عن استبدال حمض اميني واحد ، هذا التسلسل يحتوي على فالين (V) بدلاً من (isoleucine (I ، مع تطابق 100 ٪ للتسلسل AB191082.1. الخلاصة: إنخفاض انتشار جرثومة المعدة في هذه الدراسة (34٪) يشير إلى أن السودان في منطقة منخفضة الخطورة للعدوى. هناك انتشار كبير لجينات cagA و cagE في جرثومة المعدة من المرضى السودانيين، ومع ذالك لديها ضراوة منخفضة. تسلسلات بروتين cagA و cagE لهما اختلافات مترادفة في الأحماض الأمينية.
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    Accuracy of Rapid Diagnostic Tests Compared with Microscopy, Polymerase Chains Reaction and Placental Histology in Diagnosing Pregnancy Associated Malaria in Gadarif State ,Sudan,Microscopic,Chains Reaction,Gadarif State ,Sudan
    (University of Khartoum, 2015-09-15) Kashif, Awad Alla Hamza Osman ; Ishag Adam Ahmed ; Medical Parasitology and Entomology
    Background: Diagnosing malaria during pregnancy and placental malaria are a great challenge for clinicians because of the absent or low density of parasites in the peripheral blood due to sequestration of parasites in placenta. Microscopy which considered as the gold standard method for malaria diagnosis fails to detected malaria parasites in these cases. Nevertheless, few data on the use of malaria rapid diagnostic tests (RDTs) during pregnancy have been published. Therefore the main objective of this study was to evaluate the accuracy and reliability of rapid diagnostic tests (RDTs) in diagnosing malaria during pregnancy and placental malaria in an area of unstable malaria transmission in Gadarif State eastern Sudan compared with microscopy, polymerase chains reaction (PCR) and placental histology. Methods: This was a comparative (analytical) cross- sectional study conducted at the Gadarif Teaching Hospital, Eastern Sudan during the period from March 2012 to December 2013. Malaria infections were assessed in 156 febrile pregnant women by rapid diagnostic test RDT (based on detection of histidine rich protein-2 and parasite lactate dehydrogenase enzyme), microscopic examination of their Giemsa stained blood smears and Nested – PCR. In addition, at the same time other study group consist of 150 women were assessed at the time of delivery by investigations; RDT, microscopy and Nested – PCR added to placental histology (sections were stained with haematoxylin and eosin). The SD Bioline for detection falciparum and vivax species (Pf, Pv), was RDT kit evaluated in this study manufactured by (Bio Standard Diagnostics, Gurgaon, Korea). Results: Among the 156 febrile pregnant women 17 (11.0%), 26 (16.7%) and 18 (11.5%) positive cases of P. falciparum were detected by microscopy, RDT and PCR respectively. The sensitivity, specificity, positive predictive value and negative predictive value for the RDT when compared with PCR as gold standard were 83.3%, 92.0%, 57.7% and 97.7% respectively. The corresponding values for RDT where microscopy considered as gold standard were 88.2%, 92.1%, 57.6% and 98.4%. Moreover, the sensitivity, specificity, positive predictive value and negative predictive value for the microscopic analyses were 94.4%, 100%, 100% and 99.3% respectively where PCR was used as gold standard. Whereas, in other study group there were no cases of malaria detected by microscopic examination of blood smears, 27 (18.0%), 21(14.0%) and 46 (30.7%) out of the 150 parturient women investigated had P. falciparum as judged by RDT, PCR, and histology, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for the RDT kit were 17.4%, 81.7%, 29.6% and 69.6%, respectively where histology was used as gold standard. The equivalent values when the RDT compared with PCR as gold standard were 14.3%, 81.4%, 11.1% and 85.4%. Likewise, the sensitivity, specificity, positive predictive value and negative predictive value for PCR were 6.5%, 82.7%, 14.3% and 66.7% where histology was used as gold standard. Data were analysed using SPSS (Statistical Package for the Social Sciences) soft ware version 19.0. The percentages of sensitivity, specificity positive predictive value (PPV) and negative predictive value (NPV) were calculated by using 2 X 2 tables. Conclusions: The RDT kit used in this study has poor performance for detection peripheral and placental P. falciparum malaria in this setting. Placental histology has obvious superiority over microscopy, RDT and PCR in diagnosing placental malaria. The findings of current study goes with findings of previous studies in different African countries (Uganda, Ghana, Tanzania, Mali, Malawi, Cameroon and Burkina Faso) conducted at the time of birth or/and during pregnancy throughout a few past years. Furthermore the findings of this study were in disagreement with others studies. This disappointing somewhat difficult to explain but it may be due to differences in; endemicity of malaria in areas, type of RDT kits used or PCR techniques applied.
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    Assessment Of Different Stains And Staining Procedures For Microscopic Detection Of Malaria Parasites
    (University of Khartoum, 2015-03-30) Hamza, Awad Alla ; Eldirdieri Salim Ahmed ; Medical Parasitology)
    This study was carried out during the period from July 2002 to September 2003 in order to assess the use of different stains and staining procedures in microscopic diagnosis of malaria. A total of 203 individuals were included in the present study, Field-stained thick blood smear, Giemsa-stained thick and thin blood smears and Saponin lysed Giemsastained smears were prepared from each individual. Three different technicians examined stained blood smears independently. The result showed superiority of Giemsa-stained thick blood smears in detecting malaria parasites over others stained smears. Using Giemsa-stained thick blood smears malaria parasites were detected in 31, 32.5, 33 % whereas it was detected in 28.6, 29.5,30%, by Saponin lyzed venous blood smears and only in 26.1, 27.1%, 27.1% of Field stained smears as examined by three technicians. Further, three stocks solutions of Giemsa stain prepared by different methods were evaluated in staining blood smears, the finding showed a comparable result between the three Giemsa stain stock solutions. Unsatisfactory results were obtained when higher concentrations of Giemsa working solutions (15% & 20%) were used for shorter staining period versus convential concentration (10%). Adequate results were obtained by working solutions of Giemsa stain prepared using distilled water instead of phosphate buffered solutions, whereas poor results were obtained when a tap water was used. The study findings showed that it is possible to obtain Giemsa working solution with satisfactory staining properties, which did not decrease during the first 3 hours of preparation.