Amr Mohamed

Phenotypic and Molecular Typing of Tuberculous and Nontuberculous Mycobacterium Species from Slaughtered Pigs in Egypt

A total of 745 slaughtered pigs were examined during routine meat inspection for suspected tuberculous lesions. Specimens from suspected lesions were collected for conventional mycobacteriologic examinations. Suspected mycobacterial colonies were subjected to molecular typing based on the Mycobacterium species-specific intergenic spacer (IGS) target. The study resulted in detection of suspected lesions in 110 (14.8%) carcasses, from which only 67 specimens produced suspected mycobacterial colonies. Conventional examination was only able to identify 56 isolates as Mycobacterium species, which was confirmed by polymerase chain reaction amplification of the IGS target. Interestingly, out of these, 18 and 12 isolates were Mycobacterium tuberculosis and Mycobacterium bovis, respectively. Sequence analysis of IGS resolved the identities of 10 of the 11 conventionally unidentified isolates as being 4 different nontuberculous Mycobacterium species. The last isolate was proposed as a non-Mycobacterium species and was confirmed by its identification as Rhodococcus equi based on the 16S ribosomal DNA sequence analysis. The study described the isolation of Mycobacterium tuberculosis from pigs and revealed high burden of infection with both tuberculous and nontuberculous mycobacterial species among pigs in Egypt. In addition, the study showed the usefulness of IGS sequence analysis as a reliable molecular tool that would be useful for further epidemiologic and public health studies.

Thymoquinone subdues tumor growth and potentiates the chemopreventive effect of 5-fluorouracil on the early stages of colorectal carcinogenesis in rats

Colorectal cancer (CRC) is one of the most prevalent cancers and has a high mortality rate. Insensitivity and the limited therapeutic efficacy of its standard chemotherapeutic drug, 5-fluorouracil (5-FU), represents an important challenge in CRC treatment. The robust antitumor properties of thymoquinone (TQ), the main bioactive constituent of Nigella sativa, have recently been demonstrated on different cancers. We investigated whether TQ could potentiate the chemopreventive effect of 5-FU to eradicate the early stages of CRC and elucidated its underlying mechanisms. An intermediate-term (15 weeks) model of colorectal tumorigenesis was induced in male Wistar rats by azoxymethane (AOM), and the animals were randomly and equally divided into five groups: control, AOM, AOM/5-FU, AOM/TQ, and AOM/5-FU/TQ. TQ (35 mg/kg/d; 3 d/wk) was given during the seventh and 15th weeks post-AOM injection, while 5-FU was given during the ninth and tenth weeks (12 mg/kg/d for 4 days; then 6 mg/kg every other day for another four doses). At week 15, the resected colons were subjected to macroscopic, histopathological, molecular, and immunohistochemical examinations. Interestingly, 5-FU/TQ combination therapy resulted in a more significant reduction on AOM-induced colorectal tumors and large aberrant crypts foci than treatment with the individual drugs. Mechanistically, 5-FU and TQ remarkably cooperated to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and TBRAS and upregulate the expression of anti-tumorigenesis DKK-1, CDNK-1A, TGF-β1, TGF-βRII, Smad4, and GPx. Overall, our findings present the first report describing the in vivo enhancement effect of combined TQ and 5-FU against early stages of CRC; however, further studies are required to determine the value of this combination therapy in an advanced long-term model of CRC and also to realize its clinical potential.

Evaluation of indirect TaSP enzyme-linked immunosorbent assay for diagnosis of tropical theileriosis in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in Egypt.

The aim of the present study was to evaluate the validity of Theileria annulata surface protein (TaSP)-ELISA, in comparison with traditional microscopic test, for the diagnosis of T. annulata infection among Egyptian baladi cattle (Bos taurus) and water buffaloes (Bubalus bubalis). Molecular confirmation of infection using T. annulata merozoite surface (Tams-1) target amplification by PCR was used as a gold standard. A total of 76 clinically suspected animals including 64 baladi cattle and 12 water buffaloes were investigated in the current study by the three methods. Based on the PCR-confirmed results, the evaluation study revealed higher sensitivity of TaSP-ELISA (72.9% and 75%) as compared to microscopic examination (58.3% and 50%) among cattle and buffaloes, respectively. On the other hand, the specificity of TaSP-ELISA in diagnosis of T. annulata infection was higher (87.5%) in baladi cattle as compared to water buffaloes (37.5%). In conclusion, TaSP-ELISA was shown to be suitable for the diagnosis of T. annulata infection in cattle under field conditions.

Crypto-Giardia antigen rapid test versus conventional modified Ziehl-Neelsen acid fast staining method for diagnosis of cryptosporidiosis.

OBJECTIVE To evaluate the validity of Crypto-Giardia antigen rapid test (CA-RT) in comparison with the conventional modified Ziehl-Neelsen acid fast (MZN-AF) staining method for the diagnosis of cryptosporidiosis. METHODS Fifteen preserved stool samples from previously confirmed infections were used as positive controls and 40 stool samples from healthy people were used as negative control. A total of 85 stool samples were collected from suspected patients with cryptosporidiosis over 6 months during the period from January till June, 2011. The study was conducted in the department of parasitology, central laboratory, Alnoor Specialist Hospital, Makkah, Saudi Arabia. All samples were subjected to CA-RT and conventional MZN-AF staining method. Validation parameters including sensitivity (SN), specificity (SP), accuracy index (AI), positive predictive value (PPV), and negative predictive value (NPV) were evaluated for both tests. RESULTS Out of 15 positive controls, CA-RT detected 13 (86.7%) while MZN-AF detected 11(73.3%) positive cases. However, CA-RT detected no positive case in 40 normal controls but MZN-AF detected 2(5%) as positive cases. Based on the results, the SN, SP, AI, PPV and NPV were high in CA-RT than MZN-AF staining method, ie., 86.7%vs. 73.3%, 100%vs. 95%, 96.4%vs. 89.1%, 100%vs. 84.6% and 95.2%vs. 90.5%, respectively. Out of a total of 85 suspected specimens, CA-RT detected 7(8.2%) but MZN-AF detected 6(7.1%) cases as positive. CONCLUSIONS CA-RT immunoassay is more valid and reliable than MZN-AF staining method.

Paricalcitol Enhances the Chemopreventive Efficacy of 5-Fluorouracil on an Intermediate-Term Model of Azoxymethane-Induced Colorectal Tumors in Rats

Colorectal cancer is a common cancer with high mortality rate. Despite being the standard anti–colorectal cancer drug, 5-fluorouracil (5-FU) exhibits only limited therapeutic benefits. Herein, we investigated whether paricalcitol, a synthetic vitamin D analogue with potential antitumor properties, would enhance the chemopreventive efficacy of 5-FU on an intermediate-term (15 weeks) model of colorectal tumors induced by azoxymethane (AOM) in rats. After AOM injection, 5-FU was administered during the 9th and 10th weeks (12 mg/kg/day for 4 days, then 6 mg/kg every other day for another 4 doses), whereas paricalcitol (2.5 μg/kg/day; 3 days/week) was given from the 7th to the 15th week. At week 15, the animals were euthanized and their resected colons were examined macroscopically and microscopically. Quantitative RT-PCR was used to measure the transcription activities of Wnt, β-catenin, DKK-1, CDNK-1A, NF-κB, and COX-2 genes, and ELISA was used to quantify the protein levels of β-catenin, COX-2, HSP90, and VEGF. IHC was additionally used to measure β-catenin, HSP90, and inducible nitric oxide synthase (iNOS). Compared with their individual therapy, combination of 5-FU and paricalcitol showed more significant reducing effect on numbers of grown tumors and large aberrant crypts foci. Mechanistically, paricalcitol and 5-FU had cooperated together to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and HSP-90 more, and to upregulate the expression of antitumorigenesis DKK-1 and CDNK-1A, compared with their monotherapies. Our findings suggest that combined use of paricalcitol with 5-FU exhibits an augmenting chemopreventive effect against colorectal tumors, and might potentially be useful for chemoprevention in colorectal cancer patients. Cancer Prev Res; 9(6); 491–501. ©2016 AACR.

Predominance and association risk of Blastocystis hominis subtype I in colorectal cancer: a case control study

BackgroundBlastocystis, a genetically diverse intestinal parasite with controversial pathogenic potential, has increasingly been incriminated for diarrheal illness in immunocompromised individuals including colorectal cancer (CRC) patients. The aim of the current study was to assess the possible association between Blastocystis infection and CRC condition in Makkah, Saudi Arabia (KSA).MethodsStool samples were collected from 80 non-cancer (NC) and 138 cancer subjects including 74 CRC patients and 64 patients with other cancers outside gastrointestinal tract (COGT). Molecularly confirmed Blastocystis isolates were genetically grouped and subtyped using multiplex polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and sequence-tagged site primers-based PCR (PCR-STS), respectively.ResultsBlastocystis hominis were confirmed in 29.7, 25 and 15% among CRC, COGT and NC patients, respectively. Obtained Blastocystis isolates were initially categorized into 2 groups (A and C), which were subsequently subtyped into 3 different subtypes; subtype-I (38%), subtype-II (44%) and subtype-V (22%). Interestingly, subtype-I was the most predominantly detected subtype (54.5%) among CRC patients with a significant association risk (COR 7.548; 95% CI: 1.629–34.987; P = 0.004).ConclusionTo the best of our knowledge, the current study is the first to provide genetic insights on the prevalence of Blastocystis hominis among CRC patients in Makkah, KSA. Moreover, the study suggests for a possible association between subtype-I of Blastocystis hominis and CRC, which could indicate a potential influence of Blastocystis on CRC condition. Further studies are required to confirm this association risk and to investigate the possible underlying mechanism of postulated carcinogenic influence of Blastocystis hominis subtype-I.

Molecular Evaluation of Conventional Microscopic Method Versus Fecal Antigen Capture Enzyme-Linked Immunosorbent Assay and Rapid Immunochromatographic Assay for Diagnosis of Cryptosporidium Infection

BackgroundCryptosporidium has emerged as an important cause of diarrheal illness worldwide, especially among young children and patients with immune deficiencies. AimThe present study aimed to investigate the frequency of Cryptosporidium infection among suspected versus nonsuspected patients and to evaluate the performances of conventional microscopic assay, Cryptosporidium antigen detection enzyme-linked immunosorbent assay (CAD-ELISA), Crypto-Antigen Rapid Test (CA-RT), for diagnosis of Cryptosporidium infection using Cryptosporidium-specific polymerase chain reaction (CS-PCR) as criterion standard test. MethodsA total of 181 diarrheic stool samples including 105 from suspected patients and 76 from nonsuspected ones were collected. ResultsThe results revealed higher rate of infection among suspected patients (25% among diarrheic children, 24.2% among elderly/immune suppressed patients) as compared with nonsuspected patients (6.6% among immune-competent apparently healthy adults). Furthermore, conventional microscopic assay exhibited higher sensitivity (83.3%) as compared with CAD-ELISA (75%) and CA-RT (45.8%). On the other hand, higher specificity was shown for CA-RT (99.4%) and CAD-ELISA (98.7%) as compared with conventional assay (92.9%). Meanwhile, CA-RT and CAD-ELISA showed higher positive predictive values (91.7%, 90%) and lower negative predictive values (92.3%, 96.9%) than conventional assay (64.5%, 97.3%). ConclusionsNo single test is perfect for diagnosis of cryptosporidiosis, and it is worth mentioning that a significant percentage of Cryptosporidium infections would have been missed if any of these assays had been the sole method of diagnosis. It is suggested to include an antigen detection immunoassay, preferably ELISA, in addition to microscopy for better diagnosis of Cryptosporidium infection.

Evaluation of Fibronectin-Binding Protein Ag85-B as Target for Serodiagnosis of Swine Mycobacteriosis in Living Animals

The aim of the current study was to evaluate the fibronectin-binding protein Ag85-B as a potential antigen for ELISA-based serodiagnosis of swine mycobacteriosis in living pigs. The validity of Ag85-B, in comparison with tuberculin purified protein derivatives (PPD), for the serodiagnosis of swine mycobacteriosis was investigated in the current study. A total of 89 serum samples from previously confirmed Mycobacterium and non-Mycobacteriuminfected slaughtered pigs were used for the evaluation study. These included 66 serum samples from confirmed cases with swine mycobacteriosis and 23 samples from non-Mycobacterium infected cases. Obtained ELISA results revealed an overall accuracy index, sensitivity, specificity, positive predictive value, and negative predictive value of 87.6%, 87.9%, 86.9%, 95.1%, and 71.4%, respectively for Ag85-B as compared to 80.9%, 86.4%, 65.2%, 87.7%, and 62.5%, respectively for tuberculin PPD. In conclusion, the study revealed the reliability of Ag85-B as a potential candidate for ELISA-based serological assay that would provide for early and rapid diagnosis of swine mycobacterial infections in living animals.

Seroprevalence of Asymptomatic Dengue Virus Infection and Its Antibodies Among Healthy/Eligible Saudi Blood Donors: Findings From Holy Makkah City:

Background: Threat to blood transfusion–transmitted dengue virus (DENV) and its antibodies has recently emerged worldwide. Dengue fever is an endemic disease in Saudi Arabia, particularly in its Western region. The aim of this study was to estimate the seroprevalence of asymptomatic DENV infection and its antibodies among eligible Saudi blood donors. Methods: Serum samples from 910 healthy/eligible adult male Saudi blood donors, who reside in Holy Makkah City of Saudi Arabia, were collected between March 2015 and August 2016 and screened for the detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM and IgG antibodies using commercial enzyme-linked immunosorbent assay kits (Panbio, Brisbane, QLD, Australia). Results: Among the tested donors, 48 (5.3%) were seropositive for DENV-NS1 antigen, whereas 50 (5.5%) and 354 (38.9%) were seropositive for anti-DENV IgM and IgG antibodies, respectively. Seropositivity for DENV-NS1 antigen and/or anti-DENV IgM antibody among the tested donors reflects their ongoing asymptomatic viremic infectious stage with DENV during their donation time, whereas high prevalence of anti-DENV IgG seropositivity reflects the high endemicity of dengue disease in this region of Saudi Arabia. Conclusions: These results show high prevalence of asymptomatic DENV infection and its antibodies among Saudi blood donors, raising the importance of establishing blood screening for dengue disease at different blood donation services and units in Saudi Arabia to improve the guarantee of blood transfusions and to control DENV dissemination.