Detection of Human Torovirus Like Particles and Adenovirus Type F in Children Attending to Babylon Maternity and Children Hospital

Toroviruses are enteric viruses belonging to the Nidovirales order that infect different animal species and human . Torovirus-like particales (TVLPs) that are immunologically related to BRV have been reported as etiological agents of gastroenteritis in humans. The lack of “in vitro” culture systems for toroviruses, except  for the prototype Berne virus or BEV, isolated originally from an infected horse, has hampered their study and the development of diagnostic assays. This  study  describes a real time RT-PCR method to detect  human  torovirus- like particles  (TVLPs) RNA in clinical  stool  samples using primers corresponding to the gene coding for  the nucleocapsid protein which are conserved in all (TVLPs) strains known to date. During this study, the CT value  measured  during real-time PCR analysis was used as an indication of the viral load found in the stool  sample . The assay was evaluated with 72 stool samples from children attending the Babylon maternity and children hospital. Fifty tow out of 72 (72.2%) children were shedding virus at  the time of sample collection, indicating a high incidence of TVLPs  infection  in Babylon Province. This  is the first study  attempted  for   estimating  the presence of TVLPs  in Iraq. The real time RT-PCR assay described in this study  provides a rapid, highly sensitive, specific and reliable detection and quantization  method enabling future TVLPs  epidemiological studies. In addition to that  the study included the development of real-time PCR assays for the detection of group F Adenovirus in 250 stool samples of pediatric subjects  exhibiting symptoms of diarrhea and/ or vomiting  which  were examined. PCR results of 10 positive Adenovirus group F diarrheic stool samples were confirmed by electron microscopy  examination which gave clear positive Adenovirus appearance . Till now there was no successful virus  culture growth for  isolation of diarrhegenic type 40 and 41 grow in routine cell culture . The result of this study by real time reverse transcription  – PCR  assay reflected in 72 .2 % and 58 % torovirus and adenovirus group F respectively. The genotyping results of adenoviruses(genotype 40 and 41)  highlight the significance of rapid molecular methods for the routine screening of stool samples in diagnostic laboratories to provide rapid and efficient methods . Keywords: Human Torovirus, Adenovirus, RT-PCR, Electron Microscopy

Rapid Identification of Human Adenoviruses and Cytokine Estimation among Patients with Epidemic Keratoconjunctivitis in Babylon Governorate, Iraq

One hundred seventy five conjunctival swabs were collected from patients with keratoconjunctivitis. All swabs were cultured on Blood agar and MacConkey agar , 82 positive bacterial culture isolates were excluded and 93 negative bacterial culture isolates were taken as a study samples. All 93 swabs were subjected to rapid immunochromatography test (IC) test. The results of rapid identification revealed that 79 specimens (84.9%) were found positive for human adenoviruses , whereas 14 specimens (15.1%) were found negative for human adenoviruses. In addition , the results indicated that the positive rate of the IC test for specimens obtained within 1–5 days after of the onset of illness 59 (74.7%) were significantly higher than that for specimens obtained within 6–10 days after of the onset of illness 20 (25.3%) at P-value < 0.05.Tear samples were also collected from both (30) patients and (25) controls to estimate the tear levels of some immune parameters Interleukine-6  (IL-6), Interlukine-8 (IL-8), and tumor necrosis factor alpha (TNF-α) by ELISA (Enzyme linked Immunosorbent Assay) method .The cytokine profile showed that there is a significant increase (P<0.05) in the mean tear concentration of each (IL-6, IL-8 and TNF-α) between EKC patients and control groups , and the result showed that IL-8 was the predominant cytokine released and followed by IL-6 and TNF-α . Keywords: Adenovirus, Keratoconjunctivitis, Immunochromatography test, Cytokin

Detection of Human Cytomegalovirus (HCMV) among Infertile Male in AL-Najaf governorate.

Abstract:     The current study aimed to detect human cytomegalovirus among infertile individual. Blood and seminal fluid collected from 300 infertile male randomly , ELISA technique was used to know the activity of humoral immunity among them through measuring anti-HCMV IgM and IgG antibodies , the result revealed that 31 (11%) were positive for  IgM result and 234 (78%) were positive for IgG. The PCR technique showed that out of 300 samples only 22 (7.4%) gave positive results,    In case of control group, results were negative for anti- HCMV IgM antibodies in ELISA test and PCR technique. While IgG gave a weak positive results.Aim of the study:The present study was to study the relationship between the virus and infertility through detection of human cytomegalovirus (HCMV) infection in infertile male in AL-Najaf governorate by screening of anti-human cytomegalovirus IgM and IgG in the serum and detection of human cytomegalovirus DNA in  seminal fluid, using PCR technique.Keywords:  HCMV, Infertile males, ELISA, PCR.

Molecular and Immunological Study for Detection of BK Human Polyoma Virus in Patient with Hemorrhagic Cystitis in some Iraq People

يعتبر ألتهاب المثانة الدموي من المضاعفات المهمة لأشكال التهابات الجهاز البولي السفلي والتي تظهر بشكل عسر بول او بول دموي من جراء نزف من المثانة. ان  ألتهاب المثانة الدموي يمكن ان يشاهد بول مصحوبا بالدم لفتره كنتيجة للإصابة الفيروسية . التهاب المثانة الفيروسي يمثل شكل اخر من التهابات الجهاز البولي غير البكتيرية والتي تصيب البالغين و الأطفال على حد سواء.بالإضافة للدراسة الحالية   BKتوجد فايروسات أخرى مثل الفايروس HAdVS CMV,  هدف الدراسة إيجاد علاقة الأنماط المصلية للفايروس BK  بالمتلازمة الحادة للبول الدموي و دراسة هذه الأنماط المصلية من الناحية الجزئية و تتابع السلسلة الجزيئي لمثل هذا الفيروس والتأكيد على العوامل المناعية المرتبطة به لدى الأشخاص المصابين. أجريت الدراسة الحالية على مئتين وثلاثون نموذج بول حاوي على دم ومئتين وثلاثون دم جمعت من ٢٣٠ مريض تم اختيارهم للتخلص من الأسباب الأخرى لوجود الدم للادرار، مجموعة المرضى شملت ١٧٠ذكر و ٦٠ انثى تراوحت أعمارهم بين ٦الى ٦٥ سنوات هؤلاء المرضى يراجعون بعض من المستشفيات      . عينات المرضى (الادرار و الدم) و التي جمعت للتحري السريع والمتقدم عن فايروس BK بأستخدام الفحص السايتولوجي السريع للأدرار وتفاعل أنزيم البلمرة . جميع نماذج الادرار المصحوبة بالدم تمت زراعتها على المزارع الاعتيادية لغرض التفريق بين الإصابة البكتيرية و فايروس BK . الدراسة الحالية وجدت ١٥٤ عينة (٦٧%) موجبة للزرع البكتيري و قد تم استبعادها و ٧٦ عينة (٣٣%) كانت سالبة للزرع البكتيري والتي من المتوقع ان تكون كمسبب فايروسي للادرار الدموي .كل الحالات السالبة للزرع البكتيري  صنفت اعتمادا على راي أخصائي الجراحة البولية كالآتي (%٥٫٢٦)٤مرضى التهاب الكبيبات الكلوي و (١٩٫٧٦%)١٥فشل كلوي مزمن و (١٨٫٤٢%)١٤التهاب الجهاز البولي ، (٣٥٫٥٢%)٢٧زرع الكليه، (٢١٫٥%)١٦التهاب المثانه. الفئه العمرية أكثر من خمسين سنه ظهرت كأعلى نسبه مئوية  (%٢٧٫٦) ٢١بالمقارنة بالمجاميع العمرية الأخرى بالإضافة إلى النسبة المئوية للذكور اعلى من الاناث. كل بول المرضى ٧٦ عينة مع ٢٥ عينة بول مجموعة سيطرة تم تحليلها بواسطة تقنية تفاعل انزيم البلمرة بالإضافة للتقنيات الأخرى المستخدمة لتشخيص الفايروسات في عينات أدرار الدراسة الحالية تناولت بعض العوامل المناعية لتقييم الجهاز المناعي مثل CD4,IFN-α,IFN-γ,TNF-α  CD8, لتقييم الحالة المناعية. نتيجة CD4,CD8,IFN-α,IFN-γ أظهرت مستويات معنوية  عالية بالمقارنة مع عينات السيطرة في قيمة  (اقل من ٠.٥)P بينما نتيجة TNF-α لم تظهر اختلاف معنوي بالمقارنة مع مجموعة السيطرة مع ملاحظة  CD4و CD8وعلاقتها بالفئات العمرية وجدت بأن المرضى في العمر اكبر من 50سنة ايضا تناولت الدراسة جانب من استخدام برامج البايوانفورماتك  لتصميم بعض البرايمرات للجينات كما اثبتت النتائج بوجود سلالات جديدة من خلال تقنيات sequence DNA technology  وتم تسجيل بعض الجينات لفاريوس BK اعتمادا على جينات ال VP1,Agno وسجلت  العزلات  الجديدة  في Nucleotide/Blast  في  بنك الجين العالمي الامريكي باعتبارها عزلات جديدة.Hemorrhagic cystitis (HC) is considering one of the important complications in lower urinary tract hematuria, hemorrhage and dysuria are most common symptom .In hemorrhagic cystitis, short-term hematuria can also be seen in bladder infections as a result of viral infection. Viral cystitis represents another form of non-bacterial urinary tract infection affected adult and children. Human polyomavirus BK type I and V, Adenovirus types 21 and 11 and CMV viruses also can be cause of hemorrhagic cystitis. The aim of the present study work was to find out the relation of polyoma virus BKVS with acute syndrome consists of hematuria and studying the sequence and molecular genotyping of such virus with certain immunological factors among infected patients. This study was performed on two hundred and thirty bloody urine and blood specimens were collected from patients that  were selected to eliminate other causes of hematuria, the patients group comprised 170 male and 60 female age group 6 -65 years.  Patient samples (Urine and serum) were collected for detection of Human polyoma virus (BKVS) by using conventional PCR technique and immunological parameters. All bloody urine cultured on ordinary media to differentiate between viral and bacterial infection. The result of  current  study was found that 154 samples (67%) have positive bacterial culture which excluded and the other 76 samples(33%) give negative bacterial culture were included as suspected  a viral cause of hematuria. All negative culture cases was classified  as  4(5.26%) patients with glomerulonephritis ,15 (19.73%) chronic renal failure,14(18.42%)urinary tract infection ,27(35.5%) kidney transplantation ,16(21.5%) cystitis. The age range distribution mostly in age group more than 50 years at 21 (27.6 %) as higher percent in comparison with other age groups as well as the male percentage higher than female. All urine patients 76 with 25 urines samples of control were analyzed by Convention PCR for detection BVS. It was found only 9(11.8%) as a positive result. There were different immunological parameters used to evaluate function immune system such as IFN-α, IFN-ɤ, TNF-α, CD4 and CD8, to maintain the disease status. The result of IFN-α IFN-ɤ, CD4, CD8 showed a higher significant level in comparison with control samples at P value (< 0.05), while the ‘result of TNF-α showed no significant difference in comparison with control. It is  noted that the result of CD4 and CD8 in relation to age group showed that the age > 50 years have higher level of infection  than other. The percentage of BKVS is more dominant in adult age in both sexes, mostly at age more than 50 years. Bioinformatics was used for specific primers designed from the data base sequence information, for capsid protein genes (vp1, and agno) genes in (NCBI) and Sequencing of DNA And Sequencing analysis were used to diagnosis genes of BKVS . Conclusion: This study showed that the detection of Human polyoma virus in bloody urine could be applicable in hospitals laboratories by rapid decony cell.  It  reported the application of qPCR assay for rapid specific and highly sensitive for confirmative detection of BKVS as causative agents in hemorrhagic cystitis. The elevation level of immunological  factors(CD4,CD8,TNF-α,IFN-α and IFN-ɤ) act as monitor the  immune response  to effectiveness of BK virus on Hemorrhagic cystitis  patients  by ELISA assay. Sequencing and phylogenetic analysis reported that  Baghdad and Al-Najaf population are infected with hemorrhagic cystitis induced by BKVS is more predominantly with newly genotype