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- Volume 3, Issue 1, 2005
Molecular Diagnosis and Vaccines - Volume 3, Issue 1, 2005
Volume 3, Issue 1, 2005
TTV infection among hemodialysis patients in Egypt: relation to schistosomiasis and Hepatitis C virus co-infectionsSource: Molecular Diagnosis and Vaccines 3, pp 1 –9 (2005)More Less
Patients on haemodialysis (HD) are a high-risk group for blood-borne infections. TTV is a new parenterally transmitted DNA virus and little is known about its pathogenic role and its endemic situation. It was our intent to determine the prevalence of TT virus in a population of Egyptian patients on HD, to evaluate its possible route (s) of transmission and its clinical impact especially relation with HCV infection and schistosomiasis as two endemic health problems of a great social and economic impact in Egypt. Seventytwo patients on maintenance HD and 24 healthy blood donors (control group) were tested for: TTV-DNA by heminested PCR using primers NG059, NG061 and NG063 from the ORF1 region. HBsAg, and anti-hepatitis C by ELISA-2. HCV-RNAby nested PCR with primers directed to the highly conserved 5' non-coding region. TTV-DNA was detected in 51.4% (37/72) of patients and in 25% (6/24) of the blood donors (p= 0.03). Eightyone percent (30/37) of TTV-DNA positive patients reported history of blood transfusion compared to 42.9% (10/35) of TTV-DNA negative patients (p= 0.001). Also, patients with TTV-viremia had significantly lower hemohglubin level than patients without TTV-viremia (p= 0.001). No significant difference was found between the two groups as regards age, gender, liver function tests, HCV infection, history of schistosomiasis, or duration of dialysis. TTV was present alone in 25%, of the patients, as a co-infection with HCV in 26.4%, while HCV infection was present alone in 31.9%. Mean ALT level in patients with HCV/TTV co-infection was significantly higher than that in patients with TTV alone (41 32 vs. 23 18, p= 0.04) but not in patients with HCV alone Mean hemoglobin level in patients with HCV/TTV co-infection was significantly lower than that in patients with HCV alone (8.8 vs. 10.1 gm/dl, p= 0.001) but not in patients with TTV alone. No significant differences were detected between patients with mixed TTV/HCV and either single TTV, or single HCV as regards age, history of schistosomiasis, blood transfusion, duration of HCV or duration of dialysis. In conclusions: TTV infection is common among Egyptian patients on HD; its high prevalence is significantly related to blood transfusion suggesting that it is very likely the route of TTV transmission in such patients; TTV infection in Egyptian HD patients is unrelated to either HCV infection of schistosomiasis; TTV alone, does not induce liver function abnormalities. Finally, the real clinical impact and epidemiological relevance are still unclear and in need for further studies.
Association of human T-cell lymphotropic virus type 1 infection with T-cell leukemia and lymphoma in adult Egyptian patientsSource: Molecular Diagnosis and Vaccines 3, pp 11 –22 (2005)More Less
Human T-cell Lymphotropic virus type 1 (HTLV-1) is causally associated with adult T-cell leukemia/lymphoma (ATLL), an aggressive T-cell malignancy with a poor prognosis. The aim of this study is to study the association of HTLV-1 infection with Egyptian cases of T cell leukemia/ lymphoma. The study was conducted on ninety subjects, involved two groups; group I included sixty adult Egyptian patients with T cell leukemia or lymphoma who attended Hematology & Oncology Units at Ain-Shams University, El Maady Military Hospital, and National Institute for Cancers. Group II were thirty apparently healthy individuals as a control group. Enzyme linked immunosorbent assay (ELISA) was done for detection of circulating antibodies against HTLV-1 in human sera in patients and control groups. It was found that seven (11.7 %) out of 60 patients and one control person (3.3%) were serologically positive by ELISA technique. All serologically positive samples (n=8) and randomly selected negative samples from both patients (n=33) and control groups (n=9) were confirmed by polymerase chain reaction (PCR) for the detection of HTLV-1 proviral DNA in the peripheral blood mononuclear cells (PBMNCs). Three serum samples (3/40; 7.5%) from patients with T cell leukemia/ lymphoma were determined to be positive for HTLV-1 proviral DNA by PCR, while none of the control group samples showed reactivity to PCR. The specificity of ELISA was 90.2% and sensitivity was 100%, while specificity and sensitivity of PCR was 100%. The three patients positive for HTLV-1 proviral DNA were males, their ages ranged from 46-60 (mean 48), two of them (66.7%) were diagnosed as T-cell lymphoma of ATLL-type and one (33.3%) was acute Leukemia of ATLL-type. Two of them (2/3; 66.7%) had a history of blood transfusion. We conclude that Human T -cell Lymphotropic virus type 1 (HTLV-1) is probably associated and may be causally related to some cases of adult Egyptian patients afflicted with T cell leukemia /lymphoma and testing for HTLV-1 may be of help in establishing such a relation, in assessing the prognosis and risk stratification for optimum management. Although ELISA is a sensitive, reliable, and cost-effective technique for diagnosis of HTLV-I infection, positive ELISA results should be confirmed by PCR. Further epidemiological and interval studies to nationally assess HTLV-1 infection should be initiated and preventive measures to decrease the spread and transmission of HTLV-1 are warranted in Egypt.
Genotypes and serological markers of Hepatitis B virus among patients with chronic Hepatitis B virus infection in Taif, Saudi ArabiaSource: Molecular Diagnosis and Vaccines 3, pp 23 –35 (2005)More Less
Hepatitis B virus (HBV) infection is endemic in the Kingdom of Saudi Arabia (KSA). This study highlights the distribution of HBV serological markers and its genotypes in patients with chronic hepatitis B infection Serum samples from 232 chronically HBV infected patients were collected from Taif, KSA during the period from 2001 to 2004. Hepatitis B surface antigen (HBsAg), antibodies to hepatitis B core antigen (anti-HBc), hepatitis B e antigen (HBeAg), and antibodies to hepatitis B e antigen (anti-HBe) were assessed by ELISA. Existence of HBV-DNA was determined by polymerase chain reaction (PCR). HBV genotypes were determined using the INNO-LiPA assay kit. Positive HBsAg was highly prevalent (71.6%) followed by HBeAg (18.1%). The prevalence among Saudi patients was 27.6% for HBsAg, 10.3% for HBeAg, 1.3% for anti-HBc, and 0.9% for anti-HBe, as while for non-Saudi patients it was 44.0, 7.8, 1.7, and 6.5% respectively. Prevalence of HBsAg among non-Saudi nationalities showed a high rate among Filipino (16/16; 100%), followed by Bangladeshi (19/21; 90.5%), Pakistani (21/28; 75%), Egyptians (11/18; 61.1%) then Indians (21/35; 60%). In all groups the prevalence of HBsAg was higher in males than females. HBV-DNA was detected in only 31.5% Saudi and 46.6% non-Saudi patients with chronic hepatitis B infection. However, HBV genotype distribution in the 181 viremic patients was as follows; in Saudi population B, 63 (34.8%); C, 8 (4.4%) and A, 2 (1.1%), while that in non-Saudi patients was B, 74 (41.4%); C, 22 (12.2%); A, 5 (2.8%), and E, 4 (2.2%). Type B was mainly expressed in patients with positive HBsAg and HBeAg marker (77.2%, and 72.7% respectively), followed by patients with positivity for anti-HBc and anti-HBe (66.7, and 62.5% respectively). In conclusion, HBsAg and genotype B are the predominant marker and genotype respectively in patients with chronic HBV infection in Taif, Saudi Arabia.
Comparative evaluation of mecA gene detection with phenotypic conventional testing for methicillin resistant Staphylococcus aureusAuthor Nagwa M. ShawkySource: Molecular Diagnosis and Vaccines 3, pp 37 –45 (2005)More Less
Early detection of methicillin resistant Staph. aureus (MRSA) is critical for both the management of infected patients and the timely institation of appropriate infection control measures. To compare conventional phenotypic methods for detection of MRSA in routine laboratory practice with the wellestablished molecular method, mecA gene detection, this study was conducted on 50 patients with pyogenic infections. Samples were cultured on selective media and isolated Staph. aureus was identified using standard methods. MRSA isolates were identified using disc diffusion, salt agar screen test and BBL crystal MRSA ID system. Polymerase Chain Reaction (PCR) with primers specific for mecA gene was done for all isolates. MRSA was identified in 33.3% of the isolates by mecA-PCR as compared to 25%, 27.8% and 27.8 by the disc diffusion and agar screen and BBL crystal methods respectively. When mecA was used as gold standard the sensitivity was 75%, 83.3% and 83.3% for the disc diffusion, salt agar screen and BBL crystal method respectively and the specificity was 100% for all. It is concluded that although mecA-PCR is a reliable method for diagnosis of methicillin resistance Staph. aureus, the rapid and cheap conventional tests offer an acceptable alternative for smaller, non reference laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation.
Source: Molecular Diagnosis and Vaccines 3, pp 47 –55 (2005)More Less
In most human cells , the average length of telomere repeats at the ends of chromosomes provides indirect information about their mitotic history, and in haematopoietic stem cells it may serve as an indicator of hematopoietic ageing. Telomere length (TL) is maintained by a balance between replication rate and telomerase activity. The aim of this study is to explore telomere length and telomerase activity and their association with the clinical severity in patients with acquired aplastic anemia (AA) and to correlate them with the clinical factors of the disease. Thirty one cases with acquired AA were studied. They included eight cases with severe AA (sAA) (5 males and 3 females , their mean ages 7.1 0.8 years), 13 cases with moderate AA (mAA) (8 males and 5 females, their mean ages 7 0.7 years ) and 10 cases in complete remission (CR) (6 males and 4 females, their mean ages 7.2 0.6 years), were studied. Ten age and sex matched healthy children were taken as control group. .All patients were subjected to through history taking , clinical examination,complete blood picture, bone marrow aspiration and/or biopsy, measurement of Telomere length by flowcytometry using fluorescent in situ hybridization and fluorescin conjugated PNA probe and measurement of Telomerase activity (TA) by PCR-ELISA technique. The results of this study revealed significant decrease in telomere length in cases of AA than that of controls (p<0.001). The TL in sAA and mAA was significantly decreased compared with that of normal controls. Moreover, TL in patients with sAA was significantly shorter than patient with mAA(p<0.02). The TL in CR also tended to be shorter than that of normal controls, although the difference was not statistically significant. Telomerase activity was significantly high in patients with aplastic anemia (p<0.001). A positive correlation was found between absolute neutrophil count (ANC), hemoglobin (Hb), platelet count and TL(p<0.001), while negative correlation between these parameters and TA was found. Inverse correlation was observed between TL and TA (P<0.001). Our findings suggest that haematopoietic stem cells in patients with AA rapidly lose telomere length and show elevated telomerase activity. The telomere shortening further accelerated, while telomerase activity further reduced depending on the severity of stem cell pool contraction. Defective telomerase suggests that treatments directed at correction of telomerase activity might benefit patients who do not respond to conventional therapy. Further studies of telomere length and TA are needed to address the relationship with late clonal disorders and for the development of new therapeutic protocol for the disease.
Source: Molecular Diagnosis and Vaccines 3, pp 57 –61 (2005)More Less
The involvement of immune mechanisms in the etiology of pre-eclampsia has been suggested in a number of publications. Basically, a state of tolerance to the half-foreign antigens of the fetus during normal pregnancy is widely accepted to explain the success of pregnancy and non-rejection. In pre-eclampsia, this immunological tolerance might not occur. Free HLA molecules or the so called soluble HLA (sHLA) molecules are known to have an immunomodulatory function. As sHLA antigens are present in the seminal plasma, they might cause tolerance in the mother to paternal antigens. In order to test this hypothesis, we used western blotting technique to qualitatively investigate whether sHLA class I and/or sHLA-II antigens are present in seminal plasma(sp) of husbands of pre-eclamptic women and compared the results with those from a matching group of husbands of wives with normal pregnancy. We also measured the levels of sHLA class I and sHLA class II molecules in sp from husbands of pre-eclamptic and normal pregnant controls by ELISA. sHLA class I and sHLA class II were detected in all pre-eclamptic and control groups. However, the levels of sHLA class I and sHLA class II were significantly lower in seminal plasma of husbands of preeclamptic women as compared to those of normal pregnancy control group.
Source: Molecular Diagnosis and Vaccines 3, pp 63 –68 (2005)More Less
Chlamydia pneumoniae (C. pneumoniae) has recently been implicated in the development of atherosclerosis. The aim of work was to investigate the role of C. pneumoniae in the pathogenesis of atherosclerotic plague. Endarterectomy and vascular wall specimens from seventy-five patients (mean age, 59 10; 55 male, 20 female) were studied. They included 50 specimens from patients with ischemic vascular disease (20 patients with coronary artery bypass grafting, 18 patients with carotid endarterectomy, and 12 patients with surgery of the abdominal aorta for atherosclerotic obstructive lesions), and 25 control specimens from healthy regions of the ascending aorta from non ischemic patients. The presence of C. pneumoniae DNA in endarterectomy specimens was assessed by nested polymerase chain reaction (PCR). Positive PCR was found in 14 (28%) of the 50 atherosclerotic plaque specimens but in none of the healthy vascular-wall specimens (P < 0.001). These findings support the idea that C. pneumoniae might has a role in the development of atherosclerosis in Egypt, where infection is prevalent and where conventional risk factors fail to explain the high prevalence of atherosclerotic vascular disease.
Source: Molecular Diagnosis and Vaccines 3, pp 69 –76 (2005)More Less
The TT virus (TTV) is a DNA virus which was first identified in patients with non-A to -G hepatitis following blood transfusion. Sera from 100 attendees of hemodialysis (HD) unit of Al-Hussain University Hospital were tested for the presence of anti-HCV, HCV-RNA using nested polymerase chain reaction ""PCR"" with primers directed to the highly conserved 5' non-coding region of the HCV genome and TTV genome by nested-PCR using the primers of the open reading frame 1 (ORF). The overall prevalence of anti-HCV was 38 % from which only 22% were positive for HCV-RNA. TTV viremia was found in 26%, the prevalence of TTV infection was higher in patients with previous blood transfusion (18/26) and in those with diabetes mellitus (23/26). No significant relationship was found between TTV viremia and hepatitis C virus, age, sex, and duration of HD treatment. These findings support that TTV viremia is widespread among hemodialysis patients and can be detected in 26 % since no viremic patient had clinical or biochemical signs of liver disease.
Characterization of Mycobacterium tuberculosis complex isolates from sputum of patients in Cairo, EgyptSource: Molecular Diagnosis and Vaccines 3, pp 77 –84 (2005)More Less
We characterized 45 (11 multi-drug resistant MDR and 34 drug susceptible) Mycobacterium tuberculosis complex (MTC) isolates from sputum samples of Egyptian patients with pulmonary tuberculosis. One M. bovis and 44 M. tuberculosis (MTB) isolates were identified by allele-specific PCR analysis of the oxyR gene. Among the 44 M. tuberculosis isolates, 36 unique strain types (from 5-16 copies of IS6110) and 4 clusters (with 2 isolates each) were found by IS6110 restriction fragment length polymorphism (RFLP) analysis. None of the 11 MDR isolates were clustered. 22 (50 %) were in spoligotype clusters compared to the 8 out of 44 isolates (18.18%) in RFLP clusters. The spoligotypes included at least 5 that have been previously reported from other countries and from other studies in Egypt. We demonstrated evidence of diversity particularly -resistant strains consistent with low-incidence countries.
Multi-drug resistant mycobacterium tuberculosis from Abbasia Chest Hospital, Cairo, Egypt: molecular identification by DNA based methods of mutations associated with antituberculosis drug resistanceSource: Molecular Diagnosis and Vaccines 3, pp 85 –95 (2005)More Less
From a collection of 155 Mycobacterium tuberculosis isolates from patients in Abbasia Chest hospital, Cairo, Egypt in 1999, 25 (16%) had multidrug resistance (MDR), to at least rifampin (RIF) and isoniazid (INH), with additional resistance to streptomycin (STR), ethambutol (EMB) and pyrazinamide (PZA) in 18, 16, and 2 of them respectively. Thirteen (10.74%) MDR isolates were from newly diagnosed untreated cases and 12 (35.29 %) were re-treated cases (p<0.05). Restriction fragment length polymorphism (RFLP) analysis revealed that the MDR isolates were unique types (strains). We further evaluated the MDR strains for mutations associated with resistance in rpoB, katG, rpsL, and embB by single strand conformational polymorphism (SSCP) and DNA sequence analyses. Mutations in studied genes did not account for all RIFr, INHr, STRr, and EMBr strains, since 24%, 53%, 44.5%, and 63.7% of the strains respectively were wild-type respectively. All INHr strains were resistant to between 0.2 and 1 ?g/ml INH, none were resistant to 5 ?g/ml and 47.1% of them showed katG315 amino acid substitutions. All STRr strains were resistant to between 2 and10 ?g/ml, and 22.2 % and 33.3 % of them showed rpsL43 43 and rspL88 amino acid substitutions respectively. No major differences were found in the frequency of mutation or type of amino acid substitution between newly diagnosed untreated cases and re-treated cases. The sensitivity of SSCP was 100% when compared to DNA sequence analysis. A silent mutation was found in one RIFr strain. Our data underscore the need for continuous drug resistance monitoring due to recent increases in MDR-TB in Egypt. Early detection of resistance and characterization of resistance mechanisms in M. tuberculosis may facilitate the control of tuberculosis in Egypt.
Direct differentiation between Mycobacterium bovis and Mycobacterium tuberculosis in sputum specimens from smear positive pulmonary tuberculosis casesSource: Molecular Diagnosis and Vaccines 3, pp 97 –103 (2005)More Less
An oxyR allele-specific polymerase chain reaction (oxyR-PCR) was adopted for direct differentiation between Mycobacterium tuberculosis and Mycobacterium bovis in sputum from smear +ve pulmonary tuberculosis patients. The assay is based on one genetic polymorphism at the position 285 in the oxyR gene of both organisms. A ttal of 126 smear positive pulmonary tuberculosis-under treatment patients were studied. All of them were subjected to molecular diagnosis of tuberculosis (M-TB complex) using the IS6110-PCR. An internal control DNA fragment was sensitized and utilized in each reaction. Of the 116 culture positive cases, 111 (95.68%) were IS6110 +ve, 3 (2.58%) were IS6110 ve and 2 (1.72%) showed PCR inhibition, as indicated by the absence of amplification of the internal control DNA fragment. Of the 10 culture negative cases, 6 (60%) were IS6110-PCR positive, and 4 (40%) were negative. The specimens which showed no PCR inhibition in the IS6100-PCR were further subjected to oxyR PCR. All culture +ve/IS6110 +ve cases (n=111) were oxyR positive, and of them only one was identified as M. bovis (1/124=0.8%), and the remaining 110 were M. tuberculosis. On the other hand, 6 and 8 out of 10 culture negative cases were IS6110 and oxyR positive for M. tuberculosis respectively, denotying that oxyR PCR is more sensitive than the IS6110-PCR. In conclusion, oxyR PCR has the capacity to directly differentiate between M. bovis and M. tuberculosis in the sputum specimen. Pulmonary tuberculosis caused by M. bovis still occurs in Egypt and can easily be diagnosed by allele specific oxyR-PCR.