Bioinformatic analysis of KIT juxtamembrane domain mutations in Syrian GIST patients: jigsaw puzzle completed.

BACKGROUND: The huge number of detected somatic KIT mutations highlights the necessity of in silico analyses that are almost absent in the relevant medical literature. The aim of this study is to report the mutation spectrum analysis of exon 11 encoding the juxtamembrane (JM) domain of the KIT gene in a group of Syrian GIST patients. METHODS: Forty-eight formalin-fixed paraffin-embedded GIST tissue samples, collected between 2006 and 2016, were retrieved from the pathological archives and analyzed for KIT exon 11 mutations by DNA sequencing. Structural/functional impact of detected variants was predicted using several bioinformatic tools. RESULTS: Twenty-one different variants have been detected in intron 10, exon 11, and intron 11 of the KIT gene, eight of which were novel changes. Mutations in exon 11 of the KIT gene were detected in 28 of 48 (58.3%) GIST patients and predicted to be pathogenic and cancer promoting. Specifically, age above 60 was very significantly associated with the negative selection of deletion mutations (p = .007), a phenomenon that points to deletion severity. CONCLUSIONS: Six bioinformatic tools have proved efficient in predicting the impact of detected KIT variations in view of published structural, experimental, and clinical findings.

EBV Plasma Epstein-Barr Virus (EBV) DNA as a Biomarker for Diagnosis of EBV-positive Hodgkin Lymphoma in Syria.

INTRODUCTION: Epstein Barr Virus - positive Hodgkin lymphoma is defined by the presence of Epstein-Barr virus (EBV) in tumor cells. EBV plays an important role in the development and prognosis of Hodgkin's lymphoma. The standard way to detect EBV in Hodgkin lymphoma is immunohistochemistry stains for latent membrane protein-1 (LMP1) in tumor cells. The present study aimed to evaluate plasma Epstein-Barr virus (EBV) DNA as a noninvasive biomarker for diagnosis of EBV-positive Hodgkin lymphoma. METHODOLOGY: The study included 60 newly diagnosed patients with Hodgkin lymphoma, ranging in age from 4 to 60 years, and 55 sex and age-matched controls. (60) Formalin-fixed paraffin embedded blocks of Hodgkin lymphoma tissue samples were used to investigate the EBV by in immunohistochemistry stains for (LMP1) in tumor cells. Plasma EBV DNA was quantified by real-time quantitative polymerase chain reaction (PCR) for all Hodgkin lymphoma patients prior to therapy and for control. RESULTS: The results showed that (25/60, 41.7%) of Hodgkin lymphoma were positive for histological LMP1, whereas plasma EBV DNA was detectable (range from 1.1×103 to 1.5×104 copies/mL, median: 1.1×104 copies/mL) in all EBV-positive Hodgkin lymphoma samples (25/25). EBV DNA was undetectable in all cases of EBV-negative Hodgkin lymphoma (35/35) and all healthy control (55/55). It is worth mentioning that our results demonstrated that the EBV DNA load was high in the EBV associated Hodgkin lymphoma patients suffering poor prognostic state. CONCLUSIONS: Plasma EBV-DNA can be used as a noninvasive biomarker for diagnosis of EBV- positive Hodgkin lymphoma.

Association Between STAT4 rs7574865 Polymorphism and Rheumatoid Arthritis: Debate Unresolved.

BACKGROUND: STAT4 rs7574865 polymorphism has been evidently associated with susceptibility to Rheumatoid Arthritis (RA) in European and Eastern Asian populations, whereas studies in other countries reported otherwise. OBJECTIVE: We investigated the distribution of STAT4 rs7574865 polymorphism in a group of Syrian RA patients. METHODS: Eighty-one RA patients and forty healthy controls were enrolled and STAT4 rs7574865 was genotyped by direct sequencing. RA patients were stratified according to Anti-Citrullinated Protein Antibodies (ACPA) status for analysis. RESULTS: Minor T allele frequencies were 30.4%, 16.7%, and 23.8% in ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls, respectively. No significant differences in STAT4 rs7574865 allele/genotype frequencies were found between ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls (P>0.05). CONCLUSION: STAT4 rs7574865 TT genotype showed a potential impact on ACPA positivity in Syrian RA patients. However, STAT4 rs7574865 effect on RA onset and severity is minor compared to other genetic factors such as HLA-DRB1 shared epitope alleles.

Polymorphism of IFN-γ (+874 T/A) in Syrian patients with chronic hepatitis B.

AIM: This study aimed to investigate the association of IFN- γ +874 (T/A) polymorphism with susceptibility to chronic HBV infection in the Syrian population. BACKGROUND: Accumulating evidence indicate that the inadequate immune responses are responsible for HBV persistency. Therefore, polymorphisms in genes encoding the cytokines, which are responsible for regulation of the immune response, can affect the course and outcome of the infection. The IFN-γ +874 T/A polymorphism affects the expression of IFN-γ, which has been shown to be crucial to HBV clearance. METHODS: In this case-control study, 140 samples were collected (70 healthy individuals, 70 chronic HBV patients), and genomic DNA was isolated. Sequencing and ARMS-PCR were performed to genotype the IFN-γ +874 T/A polymorphism. RESULTS: Results of this study showed an association between IFN- γ +874 T/A polymorphism and the susceptibility to chronic HBV infection (P < 0.05). In addition, results showed that the AA genotype increased the risk of chronicity (OR = 3.05, 95% CI = 1.35 - 6.89), whereas the AT and TT genotypes reduced the risk of chronicity (OR = 0.33, 95% CI = 0.150 - 0.753). CONCLUSION: Results of this study conclude that the IFN- γ +874 T/A polymorphism may be associated with the chronic HBV infection, according to the genetic model AA vs. AT&TT.

Seropositivity of Hepatitis B and C among Syrian Multi-transfused Patients with Hemoglobinopathy.

BACKGROUND AND OBJECTIVES: Blood transfusion is a lifesaving therapy for patients with hemoglobinopathies. However, the need of frequent transfusion carries the risk of transmitting hepatitis B and C infections which are intermediately prevalent in Syria. Despite screening blood donations with sensitive methods, the risk of transmission is still present when infectious blood is donated within the window period. This study aimed to investigate the incidence of HBV and HCV seropositivity, and its association with multiple transfusions among Syrian hemoglobinopathies patients. MATERIALS AND METHODS: HBsAg, anti-HBc, anti-HBs and anti-HCV were tested for 159 Syrian multi-transfused patients by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Thirty-nine of 159 (24.5%) multi-transfused patients were HBsAg/anti-HBc or anti-HCV positive, 26 (16%) of which never visited the dentist, and they either tested postsurgically negative for HBsAg and anti-HCV or never underwent a surgical procedure. On the contrary of anti-HCV seropositivity, HBsAg/anti-HBc seropositivity was significantly associated with the number of blood transfusions, number of blood units and age (P < 0.001). CONCLUSION: About one-sixth of our patients most likely acquired HBV/HCV infection via blood transfusion. Administering HBV vaccine, ensuring the immune status, and monitoring hepatitis markers might considerably minimize the incidence of viral hepatitis among multi-transfused patients.

Frequency of BCR-ABL Transcript Types in Syrian CML Patients.

Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P = 0.047). Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

Screening of GJB6 gene large deletions among Syrians with congenital hearing impairment.

BACKGROUND: Research into the genetics of congenital hearing impairment in the Syrian population, where cases are noticeably encountered, is still in its infancy. AIMS: Our goal was to estimate the frequencies of the del(GJB6-D13S1830) and del(GJB6-D13S1854) mutations in a group of Syrians with autosomal recessive nonsyndromic hearing loss (ARNSHL). METHODS: Forty-one unrelated Syrian probands, already screened for exon 2, GJB2 gene mutations, were reanalyzed for del(GJB6-D13S1830) and del(GJB6-D13S1854) mutations by polymerase chain reaction. RESULTS: The del(GJB6-D13S1830) mutation was only found in homozygosity in 1 of 41 probands (2.43%), while the del(GJB6-D13S1854) mutation was not detected in any of the enrolled patients. Coexistence of GJB2 and GJB6 mutations was not encountered in any case. CONCLUSIONS: Our study reports the first case in Syria with the del(GJB6-D13S1830) mutation. This mutation might be considered in the diagnosis and genetic counseling of inherited hearing impairment in the Syrian population.

Prevalence of "anti-HBc alone" among Syrian blood donors.

INTRODUCTION: We aimed to evaluate the prevalence of "anti-HBc alone" among Syrian blood donors, highlighting the possibility of representing occult HBV infection. METHODOLOGY: Sera of 3,896 healthy blood donors were tested for both HBsAg and anti-HBc. HBsAg-negative, anti-HBc-positive samples were further tested for the antibodies to HBsAg (anti-HBs), and "anti-HBc alone" sera were tested for HBV DNA. RESULTS: Of 3,830 HBsAg-negative donors, 63 were "anti-HBc alone" donors, five of whom were HBV DNA positive. CONCLUSIONS: Greater consideration should be given to the "anti-HBc alone" serological profile in blood screening, premarital testing, organ transplantation tests, and other HBV transmission-related procedures in Syria.

Hepatitis B splice-generated protein antibodies in Syrian chronic hepatitis B patients: incidence and significance.

BACKGROUND: Previous studies have suggested hepatitis B splice-generated protein (HBSP), when expressed, is involved in the pathogenesis of HBV infection. OBJECTIVES: We aimed to evaluate anti-HBSP incidence and association with several HBV infection parameters in a group of Syrian chronic hepatitis B patients. PATIENTS AND METHODS: Eighty treatment-naïve HBsAg-positive adult chronic hepatitis B patients' sera were included in our prospective targeted study. Liver function, virological and histological tests results were obtained from patients' medical files. Three variants of a 20-mer HBSP-derived peptide were designed based on HBV genome sequences obtained from Syrian patients' sera (GenBank Accession No. JN257148-JN257217). Microtiter plate wells were coated with the synthetic peptides and used to detect anti-HBSP antibodies by an optimized indirect enzyme-linked immunosorbent assay (ELISA). Samples were considered positive when showed optical density (OD) values higher than the cut-off value for at least one peptide variant. RESULTS: Seven out of eighty (9%) CHB patients were positive for anti-HBSP antibodies. Mean OD values were not significantly different between HBeAg-positive and -negative patients (P > 0.05). OD values showed weak positive correlation with ALT and AST values (P < 0.05), and weak to moderate positive correlation with liver biopsy staging ranks (P < 0.05). No significant correlation was revealed with viral load values or liver biopsy grading ranks (P > 0.05). CONCLUSIONS: We introduced an anti-HBSP antibodies ELISA, designed for locally circulating HBV strains. Correlation observed of Anti-HBSP with liver fibrosis staging regardless of viral replication and liver inflammation suggests anti-HBSP antibodies as possible indicator for HBV-associated liver fibrosis.

HLA-DRB1 allele association with rheumatoid arthritis susceptibility and severity in Syria.

INTRODUCTION: Rheumatoid arthritis (RA) is a complex multifactorial chronic disease. The importance of human leukocyte antigen as a major genetic risk factor for RA was studied worldwide. Although it is widely distributed in different Syrian areas, studies of human leukocyte antigen (HLA) alleles' role are absent. OBJECTIVE: The aim of our study was to determine the association of HLA-DRB1 alleles with the susceptibility and severity of RA in Syria. PATIENTS AND METHODS: Eighty-six RA patients and 200 healthy controls from Syria were genotyped using polymerase chain reaction with sequence-specific primer (PCR-SSP). Anti-CCP antibodies were measured by ELISA. Rheumatoid factor (RF), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and disease activity score 28 (DAS-28) values were obtained from patients' medical records. DAS-28 was used to assess the clinical severity of the patients. RESULTS: The HLA-DRB1*01, *04, and *10 frequencies showed a strong association with the disease susceptibility (OR = 2.29, 95% CI = 1.11-4.75, P = 0.022; OR = 3.16, 95% CI = 2.0 -4.8, P < 0.0001; OR = 2.43, 95% CI = 1.07-5.51, P = 0.029 respectively), while the frequencies of HLA-DRB1*11, and *13 were significantly lower in RA patients than in controls (OR = 0.49, 95% CI = 0.3-0.8, P = 0.004; OR = 0.32, 95% CI = 0.15-0.69, P = 0.002, respectively). The other HLA-DRB1 alleles showed no significant difference. The frequency of anti-CCP antibodies was higher in shared epitope (SE) positive patients compared with SE-negative patients (OR = 5.5, 95% CI = 2-15.1, P = 0.00054). DAS-28 of RA patients didn't show significant difference between the SE negative and the SE positive groups. CONCLUSION: Our results indicate that HLA-DRB1*01, *04, and *10 alleles are related with RA, while HLA-DRB1*11 and *13 protect against RA in the Syrian population.

High prevalence of genetic abnormalities in Middle Eastern patients with idiopathic non-obstructive azoospermia.

INTRODUCTION: Our objective is to detect the frequency and types of major genetic abnormalities of idiopathic non-obstructive azoospermia (NOA) to give appropriate genetic counseling before assisted reproductive techniques (ART) in Middle East and to compare the frequencies with other regions of the world. MATERIAL AND METHODS: A total of 880 Middle Eastern patients with NOA were recruited in this multicenter study for genetic evaluation prior to use of ART. Karyotyping was performed on peripheral blood lymphocytes according to standard G-banding methods, polymerase chain reaction (PCR) was performed to screen the microdeletions in the AZF region of the Y chromosome. RESULTS: The present study shows that the total prevalence of genetic abnormalities is 28.41 %, including 184 patients (20.91 %) with chromosome disorder and 66 patients (7.5 %) with Y chromosome microdeletions. The most prevalent chromosome abnormality is Klinefelter's syndrome, which includes 161 patients (18.3 %), 7 patients had XX reversal male sex (0.8 %), 2 patients had 47XYY (0.23 %) and 2 patients had 45XO/46XY (0.23 %). Structural abnormalities occurred in 12 patients (1.36 %). CONCLUSIONS: The high prevalence of genetic abnormalities (28.41 %) in our study strongly suggests the need for routine genetic testing and counseling prior to assisted reproduction in such population with idiopathic infertility, as a result may help determine the prognosis, as well as the choice of ART. Moreover it allows specific pre-implantation genetic testing to minimize the risk of transmitting genetic defects to offspring.

Associação do alelo HLA-DRB1 com suscetibilidade a artrite reumatoide e gravidade da doença na Síria / HLA-DRB1 allele association with rheumatoid arthritis susceptibility and severity in Syria

INTRODUÇÃO: A artrite reumatoide (AR) é uma doença crônica multifatorial complexa. A importância do sistema de antígenos leucocitários humanos (HLA) como fator significativo de risco genético para AR foi estudada no mundo. Embora amplamente distribuídos em diferentes áreas na Síria, faltam estudos sobre o papel dos HLA. OBJETIVO: O objetivo de nosso estudo foi determinar a associação dos alelos HLA-DRB1 com a suscetibilidade a AR e sua gravidade na Síria. PACIENTES E MÉTODOS: Foram genotipados 86 pacientes com AR e 200 controles normais, usando-se reação em cadeia da polimerase com sequência de primer específico (PCR-SSP). Anticorpos anti-CCP foram determinados por ELISA. Fator reumatoide (FR), proteína C-reativa (PCR), velocidade de hemossedimentação (VHS) e o índice de atividade da doença (DAS-28) foram obtidos nos registros médicos e utilizados para avaliar a gravidade clínica dos pacientes. RESULTADOS: Os alelos HLA-DRB1 *01, *04 e *10 mostraram forte associação com suscetibilidade à doença (OR = 2,29, IC 95% = 1,11-4,75, P = 0,022; OR = 3,16, IC 95% = 2,08-4,8, P < 0,0001; e OR = 2,43, IC 95% = 1,07-5,51, P = 0,029, respectivamente), enquanto a frequência dos alelos HLA-DRB1 *11 e *13 foi significativamente mais baixa nos pacientes com AR do que nos controles (OR = 0,49, IC 95% = 0,3-0,8, P = 0,004; OR = 0,32, IC 95% = 0,15-0,69, P = 0,002, respectivamente). Os outros alelos HLA-DRB1 mostraram diferença significativa. A frequência dos anticorpos anti-CCP foi maior em pacientes epítopo compartilhado (EC) positivos do que em pacientes EC-negativos (OR = 5,5, IC 95% = 2-15,1, P = 0,00054). O índice DAS-28 de pacientes com AR não mostrou diferença significativa entre os grupos EC-negativo e EC-positivo. CONCLUSÃO: Nossos resultados indicam que os alelos HLA-DRB1 *01, *04 e *10 estão relacionados com AR, enquanto os alelos HLA-DRB1 *11 e *13 protegem a população síria contra a AR.

INTRODUCTION: Rheumatoid arthritis (RA) is a complex multifactorial chronic disease. The importance of human leukocyte antigen as a major genetic risk factor for RA was studied worldwide. Although it is widely distributed in different Syrian areas, studies of human leukocyte antigen (HLA) alleles' role are absent. OBJECTIVE: The aim of our study was to determine the association of HLA-DRB1 alleles with the susceptibility and severity of RA in Syria. PATIENTS AND METHODS: Eightysix RA patients and 200 healthy controls from Syria were genotyped using polymerase chain reaction with sequencespecific primer (PCR-SSP). Anti-CCP antibodies were measured by ELISA. Rheumatoid factor (RF), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and disease activity score 28 (DAS-28) values were obtained from patients' medical records. DAS-28 was used to assess the clinical severity of the patients. RESULTS: The HLA-DRB1*01, *04, and *10 frequencies showed a strong association with the disease susceptibility (OR = 2.29, 95% CI = 1.11-4.75, P = 0.022; OR = 3.16, 95% CI = 2.0 -4.8, P < 0.0001; OR = 2.43, 95% CI = 1.07-5.51, P = 0.029 respectively), while the frequencies of HLA-DRB1*11, and *13 were signifi cantly lower in RA patients than in controls (OR = 0.49, 95% CI = 0.3-0.8, P = 0.004; OR = 0.32, 95% CI = 0.15-0.69, P = 0.002, respectively). The other HLA-DRB1 alleles showed no signifi cant difference. The frequency of anti-CCP antibodies was higher in shared epitope (SE) positive patients compared with SE-negative patients (OR = 5.5, 95% CI = 2-15.1, P = 0.00054). DAS-28 of RA patients didn't show signifi cant difference between the SE negative and the SE positive groups. CONCLUSION: Our results indicate that HLA-DRB1*01, *04, and *10 alleles are related with RA, while HLA-DRB1*11 and *13 protect against RA in the Syrian population.

Rethinking therapeutic decisions for hepatitis B infection in Syria: insights into molecular monitoring.

INTRODUCTION: Hepatitis B virus patients are usually treated in Syria with alpha interferon and nucleos(t)ide analogues. Genotypic viral factors causing inadequate response or relapse following initial response are not routinely investigated. This study aimed to explore and discuss local therapeutic decisions from a molecular perspective. METHODOLOGY: Fifty patients with hepatitis B from Syria were tested for HBV genotyping and drug-resistance mutations by DNA sequencing. RESULTS: All patients had genotype D, which is characterized by relatively low response to interferon-based therapy. Drug-resistant viral mutant variants were detected in one fifth of the enrolled patients, and distributed similarly in both nucleos(t)ide analogues-naïve and -treated patients. However, nucleos(t)ide analogues-based therapy was associated with the existence of more mutations and hence increased resistance. CONCLUSIONS: Investigating HBV genotypes and drug-resistance mutations to support treatment decisions is critically needed for efficient therapy and patients' survival.

Brucellosis laboratory tests in Syria: what are their diagnostic efficacies in different clinical manifestations?

INTRODUCTION: Diagnosis of brucellosis in Syria is based on the presence of compatible symptoms in addition to positive agglutination results. This study investigated the potential of culture, ELISA and real-time PCR to support the diagnosis in different clinical manifestations of brucellosis. METHODOLOGY: Peripheral blood samples from 34 suspected brucellosis patients and 42 probable chronic or relapsed brucellosis patients were tested by agglutination tests, culture, ELISA and real-time PCR. RESULTS: Among 34 samples collected from suspected cases, 18/34 (53%) were agglutination tests positive, 12/34 (35%) were culture positive, 12/34 (35%) were Brucella IgG positive, and 10/34 (29%) were real-time PCR positive. Three out of 34 patients were positive by real-time PCR but not by agglutination tests or culture. Among 42 samples obtained from probable chronic or relapsed patients, 27/42 (64%) were agglutination tests positive, 26/42 (62%) were Brucella IgG positive, 4/42 (10%) were culture positive, and 1/42 (2%) was real-time PCR positive. CONCLUSION: To rule in or rule out the diagnosis of brucellosis, a combination of several tests should be applied. Agglutination tests should be performed first considering their high sensitivity. If the agglutination test is negative, real-time PCR, and/or ELISA, and/or culture should be performed. When relapse or chronic brucellosis are suspected, agglutination tests and/or ELISA are recommended.

Utility of an interferon-gamma release assay as a potential diagnostic aid for active pulmonary tuberculosis.

INTRODUCTION: Sensitivity, specificity, early confirmation and obtaining an optimal specimen are challenging problems in active tuberculosis (TB) diagnosis. Interferon-gamma release assay (IGRA) is a good indicator for latent TB but can it be useful as a diagnostic tool for active TB? This study was designed to address these challenges and assess the potential of IGRA as a diagnostic indicator of active pulmonary TB by comparing it with other MT diagnostic conventional methods and molecular methods. METHODOLOGY: The study was conducted on 91 patients with suspicion of pulmonary active TB. QuantiFERON-TB-Gold In-Tube, a commercial IFN-gamma assay, was compared with Ziehl Neelsen (ZN) smear, Lowenstein Jensen's (LJ) egg-based culture, and real-time polymerase chain reaction. The final clinical diagnosis was the standard comparator of the study. RESULTS: Active pulmonary TB was confirmed in 48/91 (52.7%) patients. Sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were 72.9%, 100%, 100%, 76.78% for ZN smear, 77.1%, 97.67%, 97.36%, 79.24% for LJ culture, 89.9%, 67.4%, 75.4%, 85.3% for IGRA, and 66.6%, 95.3%, 94.1%, 71.9% for real-time PCR, respectively. CONCLUSION: Albeit confounding in the case of latent TB infected patients presenting with non-TB pulmonary disease, IGRA was more sensitive than the other conventional and molecular methods, so it may improve diagnostic accuracy when used in combination with other standard methods. High NPV of IGRA for the diagnosis of active TB proposed an additional role of this test to exclude the infection with active TB.

The subgingival microbiota of Papillon-Lefèvre syndrome.

BACKGROUND: There is little information about the microbiologic profiles of periodontal lesions in Papillon-Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA-based Human Oral Microbe Identification Microarray (HOMIM). METHODS: Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. RESULTS: The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high-frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. CONCLUSIONS: The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.

Are human herpes viruses associated with autoimmune thyroid disease?

INTRODUCTION: Autoimmune diseases are complex diseases with genetic, endogenous and environmental etiologies. Viral infections have been postulated as one of the factors that may be the trigger of autoimmune diseases. METHODOLOGY: Thyroid peroxidase (TPO) and thyroglobulin (TG) antibodies were measured before thyroidectomy in 100 subjects by chemiluminescence method, 50 of whom were autoimmune thyroid diseases (AITD) patients and 50 of whom were multinodular goiter (MNG) patients used as a control group. Fresh thyroid samples were collected from all 100 subjects after thyroidectomy to investigate the DNAs of herpes simplex viruses types 1 and 2 (HSV-1, HSV-2), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV) and human herpes virus type 6 (HHV-6) by PCR. RESULTS: The DNA of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 were detected in neither the patient group nor in the control group. The mean values of anti-TPO and anti-TG antibodies ranged within 9.5-2000 units/ml (527.8 ± 617.4) and 108-5000 units/ml (1458.2 ± 1774.1) in the AITD patients group, respectively. A statistically significant difference of the mean level of anti-TPO and anti-TG antibodies among the two groups was found (p value < 0.05). CONCLUSIONS: The possible role of human herpes viruses in the pathogenesis of AITD is not supported by our study; hence our raised question stays open for more investigation on more patients and in different parts of the country using different sizes and sites of biopsies.

Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993-2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

Is low birth weight a risk indicator for congenital cytomegalovirus infection?

BACKGROUND: Congenital cytomegalovirus infection is currently the leading cause of congenital infection in 0.2-2.2% of live births worldwide leading to variable serious sequalae. The aim of the study was to determine if low birth weight is an indicator of CMV congenital infection evidenced by detecting CMV-DNA in umbilical cord blood at the time of delivery. METHODOLOGY: CMV-IgG and IgM antibodies and CMV-DNAemia were assessed in umbilical cord blood of two hundreds newborns, one hundred of whom had birth weight < or = 2700 gram and/or head circumference < or = 32 cm. RESULTS: CMV-IgM was not detected, while CMV-IgG was positive in 80-90% of the two hundreds tested newborns. CMV-DNA was detected in four out of the 200 newborns. One of them was over the adopted weight limit (> 2700 gram). CONCLUSIONS: CMV-IgM and IgG antibodies assessment was not a potential discriminative test to identify congenitally infected newborns. In addition, low birth weight and small head circumference at birth failed to predict congenital CMV infection. CMV-DNA detection in umbilical cord blood at the time of delivery using real-time PCR of all newborns is recommended as decisive, rapid and non-invasive test.

Optimization of human cytomegalovirus LightCycler real-time PCR.

BACKGROUND: Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use. METHODOLOGY: Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl2 and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products. RESULTS: Optimal values for most studied primers were found to be 3 mM for MgCl2 concentration, 0.5 microM and 0.6 microM for primers concentration, and 55 degrees C for annealing temperature. CONCLUSION: Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.