Multiplex Polymerase Chain Reaction Technique Research Articles

Application of reverse transcriptase nested, hemi-nested And multiplex polymerase chain reaction techniques For the detection of pv 11, pv 3462 and cvs Strains of rabies virus

Application of reverse transcriptase nested, hemi-nested And multiplex polymerase chain reaction techniques For the detection of pv 11, pv 3462 and cvs Strains of rabies virus

Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.

Aim:This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques.Materials and Methods:Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B.Results:A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54%) were characterized at the same reaction into PM Type A, five isolates (23%) were Type B and the rest five isolates (23%) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B.Conclusion:PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

Occurrence of methicillin-resistant Staphylococcus aureus in raw shellfish at retail markets in Malaysia and antibacterial efficacies of black seed (Nigella sativa) oil against MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is a remarkable pathogen which causes nosocomial diseases worldwide. In this study, raw green mussel and cockle samples in Malaysia were collected and investigated for the occurrence of MRSA. A total of 80 samples were analyzed using phenotypic and molecular approaches. A assembling of the most probable number (MPN) with multiplex polymerase chain reaction (mPCR) technique was used to identify MRSA in the samples. The occurrences of S. aureus and MRSA in shellfish samples were 15% and 8.75%, respectively, with the microbial load from <3 to 12.2 MPN/g. MRSA isolates were tested for their sensitivity to 17 generally used antibiotic agents by using disc diffusion assay. All the isolates were resistant to amikacin and penicillin. However, sensitivity was recorded for cefoxitin, ceftaroline, chloramphenicol, ciprofloxacin, clarithromycin levofloxacin, linezolid, ofloxacin, quinupristin/dalfopristin, tetracycline and trimethoprim-sulfamethoxazole. Moreover, the antimicrobial activities against MRSA were evaluated using minimum inhibited concentration (MIC) and minimum bactericidal concentration (MBC). The findings revealed that the black seed oil acted as a potent inhibitor of MRSA. The lowest value of MIC and MBC of black seed oil against MRSA was 32.8 and 42.2 mg/mL, respectively. The detection of MRSA in this study deserves a public attention because shellfish are commonly eaten raw or partially cooked in Malaysia.

FcrR3A-158 Polymorphism and Stromal Tumor-Infiltrating Lymphocytes and Survival among Patients with Metastatic HER2-Positive Breast Cancer Receiving Trastuzumab-Based Treatment.

PurposeThe prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer has markedly improved since the introduction of trastuzumab. We aimed to evaluate the association between stromal tumor-infiltrating lymphocyte (sTIL) or FcrR polymorphisms and survival among patients with metastatic HER2-positive breast cancer who were treated with trastuzumab.MethodsA total of 56 women with recurrent or metastatic HER2-positive breast cancer who received the trastuzumab-taxane combination as first-line treatment were included in this retrospective analysis. The single-step multiplex allele-specific real-time polymerase chain reaction technique was employed for FcrR3A genotyping. sTILs were identified via immunohistochemical analysis of surgical (n=34, 60.7%) or biopsy specimens of metastatic lesions (n=22, 39.3%).ResultsWe classified patients based on the sTIL level (≤10% [n=44] or >10% [n=12]); high sTIL counts were more commonly observed in patients with hormone receptor-negative tumors than in those with hormone receptor-positive tumors (34.8% vs. 12.1%, p=0.02). There was a significant association between high sTIL levels and longer progression-free survival in comparison to low sTIL levels (median, 28.4 months vs. 16.8 months; p=0.03). With regard to the FcrR3A-158 genotype, patients were classified into the Phenylalanine/Phenylalanine group (23 patients, 41.1%), Phenylalanine/Valine group (23 patients, 41,1%), or Valine/Valine group (10 patients, 17.9%); these classifications were not associated with clinical outcomes.ConclusionHigh sTIL expression may be associated with better efficacy of trastuzumab-containing therapy in patients with metastatic HER2-positive breast cancer. However, this finding warrants further evaluation in the larger population.

Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are food-borne pathogens that cause serious gastrointestinal illness and frequent food safety accidents. This study aimed to develop a practical multiplex polymerase chain reaction (mPCR) technique for the simultaneous detection of these food-borne pathogens in culture broth and artificial food matrix. Pathogen-specific DNA sequences in the rfbE, nuc, and invA genes were used as targets to design primers for the identification of E. coli O157:H7, S. aureus, and Salmonella, respectively. As expected, the method produced species-specific bands of amplified products without any contaminating non-specific bands. The highest species specificity was established with primer concentrations of 0.1, 0.2, and 0.4μM for E. coli O157:H7, S. aureus, and Salmonella, correspondingly. The detection sensitivity of this assay was 103 CFU/mL in culture broth, and the limit of detection was consistent with singleplex PCR in the food sample. The mPCR assay proposed here is an easy and convenient detection method, which will be valuable for microbial epidemiology and food safety investigations.

Association between polymorphism of GSTP1, GSTT1, GSTM1 and CYP2E1 genes and susceptibility to benzene-induced hematotoxicity

Occupational exposure to benzene has been associated with leukemia, anemia, leukopenia, and thrombocytopenia. Genetic susceptibility to benzene toxicity in humans may be related to variations in benzene metabolizing genes. The main objective of this study was to ascertain whether polymorphism of GSTP1, GSTM1, GSTT1 and CYP2E1 genes might influence susceptibility to the adverse effects of benzene among employees of a petrochemical plant. In this cross-sectional study, 124 employees of a petrochemical plant who had been occupationally exposed to benzene and had one or more abnormal hematological parameter (cases) and 184 subjects with a similar exposure scenario, free from any abnormal hematological parameters (referent) were studied. Atmospheric concentrations of benzene were measured and GSTM1 and GSTT1 genotypes were evaluated using the multiplex polymerase chain reaction (PCR) technique. Additionally, GSTP1 and CYP2E1 genotypes were determined by PCR- restriction fragment length polymorphism (PCR–RFLP). The frequency of null GSTT1 genotype in cases was significantly higher than that of referent group (32.3 vs. 18.5%, OR 2.1, 95% CI 1.23–3.56, p = 0.004). The mean value of platelets in subjects with null GSTT1 genotype was significantly lower than that of individuals with positive GSTT1 genotype (p = 0.015). Conversely, the mean value of leukocytes was significantly higher in subjects with null GSTM1 genotype as compared to those with positive GSTM1 genotype (p = 0.026). Logistic regression analysis showed that, subjects with null GSTT1 genotype had a significantly higher risk for hematological disorders, as compared to those with positive GSTT1 genotype (OR 2.1, 95% CI 1.23–3.56). Moreover, subjects with both null GSTT1 and GSTM1 genotypes had a significantly higher risk for hematological disorders as compared to subjects with positive GSTT1 and GSTM1 genotypes (OR 2.35, 95% CI 1.14–4.8). The results of this study showed that, individuals carrying null GSTT1 or both null STT1 and GSTM1 genotypes had a higher risk and were more susceptible to benzene-induced hematological disorders.

Association of genetic variants of hemostatic genes with myocardial infarction in Egyptian patients

Hemostatic genes polymorphisms are well known to be associated with venous thrombosis, but their association with arterial thrombosis especially myocardial infarction (MI) remains to be clarified. We investigated the role of three hemostatic gene polymorphisms, prothrombin G20210A, factor XIII (FXIII) Val34Leu (G/T), and fibrinogen-β-455G/A and their coexistence in Egyptian patients with MI. The possible correlation of these polymorphisms with plasma fibrinogen level was also evaluated. The study included 120 patients with MI and 60 healthy volunteers. Gene polymorphisms were tested using multiplex polymerase chain reaction and reverse-hybridization technique. Plasma fibrinogen level was determined by ELISA. Our study showed an increased risk of MI with fibrinogen β-455G/A heterozygosity as well as FXIII Val34Leu homo and heterozygosity. In addition, the FXIII T allele (Leu34) and fibrinogen β-455A allele were significantly associated with MI. Conversely, the prevalence of prothrombin mutation did not differ between patients with MI and controls. Combined carriers of FXIII Leu34 and fibrinogen-β455A alleles were at higher risk of MI, whereas combined FXIII Val34Leu and prothrombin 20210A polymorphisms did not show increased risk for MI compared with controls. Plasma fibrinogen levels were significantly higher in patients with MI than controls. In MI patients, plasma fibrinogen levels were significantly higher in those with FXIII GT/TT or fibrinogen β-455 GA, while were significantly lower in those with prothrombin 20210 GA compared with patients with wild type genotypes. In conclusion, our results suggest a possible thrombotic predisposition of FXIII Val34Leu, fibrinogen β-455G/A polymorphisms and their coexistence for MI. These polymorphisms may add complexity to disease pathology by increasing plasma fibrinogen level. Extended studies are needed to confirm our results; nevertheless, these data may be implicated in genetic counseling and screening of high-risk individuals.

Virulence and extended-spectrum β-lactamase encoding genes in Escherichia coli recovered from chicken meat intended for hospitalized human consumption.

Aim:This study describes the prevalence of Escherichia coli in frozen chicken meat intended for human consumption with emphasis on their virulence determinants through detection of the virulence genes and recognition of the extended-spectrum β-lactamase (ESBL) encoding genes (blaOXA and blaTEM genes).Materials and Methods:A total of 120 frozen chicken meat samples were investigated for isolation of E. coli. All isolates were subjected to biochemical and serological tests. Eight serotypes isolated from samples were analyzed for the presence of various virulence genes (stx1, stx2, and eae A genes) using multiplex polymerase chain reaction (PCR) technique. Moreover, the strains were evaluated for the ESBL encoding genes (blaTEM and blaOXA).Results:Overall, 11.66% (14/120) chicken meat samples carried E. coli according to cultural and biochemical properties. The most predominant serotypes were O78 and O128: H2 (21.5%, each), followed by O121: H7 and O44: H18. Molecular method detected that 2 strains (25%) harbored stx1, 3 strains (37.5%) stx2, and 3 strains (37.5%) both stx1 and stx2, while 1 (12.5%) strain carried eae A gene. Particularly, only O26 serotype had all tested virulence genes (stx1, stx2, and eae A). The results revealed that all examined 8 serotypes were Shiga toxin-producing E. coli (STEC). The ESBL encoding genes (blaTEM and blaOXA) of STEC were detected in 4 (50%) isolates by multiplex PCR. The overall incidence of blaTEM and blaOXA genes was 3 (37.5%) and 2 (25%) isolates.Conclusion:The present study indicates the prevalence of virulent and ESBL-producing E. coli in frozen chicken meat intended for hospitalized human consumption due to poor hygienic measures and irregular use of antibiotics. Therefore, the basic instructions regarding good hygienic measures should be adapted to limit public health hazard.

Foodborne disease outbreaks studied by molecular techniques

To apply a multiplex real-time polymerase chain reaction (PCR) technique to detect Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica as a diagnostic support tool for the surveillance of foodborne disease outbreaks. Molecular methodology was applied on clinical samples taken from individuals who were associated with foodborne disease outbreaks in two departments of Colombia. The results were compared with the data obtained by conventional culture methodology. In addition, the clonal relation of the isolations was evaluated using the Pulsed Field Gel Electrophoresis (PFGE) technique. 123 cases of foodborne disease were determined, of which 45 biological samples were confirmed by laboratory and 88 by epidemiological link. The molecular methodology detected 35/45 positive samples versus 17/45 positive samples detected by conventional methodology. PFGE demonstrated a clonal relation during each outbreak. The results of the study demonstrate the applicability of the molecular technique as a useful diagnostic support tool to characterize foodborne disease outbreaks, allowing a timely and reliable response.

Glutathione S-Transferase Gene Polymorphisms and the Development of New-Onset Diabetes After Liver Transplant.

The association between the glutathione S-transferase polymorphisms and the development of new-onset diabetes mellitus after liver transplant was studied. Peripheral blood samples were collected from 106 liver transplant patients divided into 2 groups: 52 with new-onset diabetes mellitus and 54 without new-onset diabetes mellitus; 169 healthy individuals with no clinical evidence of diabetes mellitus were selected as a control group. The multiplex polymerase chain reaction technique was used for genotyping GSTM1 and GSTT1 genes, using the cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) gene as an internal control. The genotype of GSTP1 was determined using the restriction fragment length polymorphism-polymerase chain reaction technique. The frequency of both GSTM1 null and GSTT1 null genotypes was not significantly different in liver transplant patients with new-onset diabetes mellitus compared with the control group (P = .11 for GSTM1; P = .71 for GSTT1). Also, there was no statistically significant association between the frequency of the GSTP1 genotypes in the liver transplant patients with new-onset diabetes mellitus compared with controls. Neither GSTM1 nor GSTT1 null genotypes were associated with the risk of developing new-onset diabetes mellitus (P = .22 for GSTM1; P = .56 for GSTT1). However, the frequency of the heterozygous mutation (AG) in the A313G GSTP1 polymorphism in patients with new-onset diabetes mellitus was significantly higher than in patients without new-onset diabetes mellitus (55.8% vs 7.4%; P = .00). Thus, the risk of developing new-onset diabetes mellitus was significantly higher in patients presenting with heterozygous GSTP1 genotypes (odds ratio = 15.76; 95% confidence interval = 4.53-60.28; P = .00). The GSTP1 AG genotype was associated with an increased susceptibility to the development of new-onset diabetes mellitus after liver transplant.

POLYMORPHISM OF CYTOCHROME P450, SUPERFAMILY19, POLYPEPTIDE 1 GENE AND RELATED TO AROMATASE ENZYME ACTIVITY IN ACROMEGALY IRAQI PATIENTS

Objective: The objective of the present work was to investigate the association between aromatase enzyme activity and polymorphisms, rs2236722, in exon 2 of cytochrome P450 (CYP), superfamily19, polypeptide 1 (CYP19A1gene) in patients with active acromegaly.Methods: A total of 120 males and females (age 20-60 years) were enrolled in this study, 60 patients with active acromegaly who have attended the National Diabetes Centre and and Specialist Center for endocrinology and diabetes, Baghdad, from December 2015 to June 2016 and 60 healthy individuals with matches as a control group. From the whole blood, genomic DNA was extracted to perform genotyping analysis and single-nucleotide polymorphism, rs2236722, in the CYP19A1 gene using multiplex polymerase chain reaction technique, and serum aromatase levels were determined using a solid-phase enzyme-linked immunosorbent assay based on sandwich method.Results: The results show that the aromatase activity levels of patients were a highly significant decrease when contrasted with healthy control groups in both sexes (p≤0.01), and there were summarized positive results of an allele in single-nucleotide polymorphism, rs2236722, were highly significant in TT allele when contrasted with healthy control groups. While revealed that there was non-significant difference in TC allele when contrasted with healthy control groups (p&gt;0.01).Conclusion: we conclude that there was associated between CYP19A1 gene polymorphism (rs2236722) with aromatase activity and related to acromegaly patients.

GENETIC POLYMORPHISMS OF GLUTATHIONE STRANSFERASE (M1 AND T1) AND PARAOXONASE 1 (PON1) AND SUSCEPTIBILITY TO CHRONIC KIDNEY DISEASE OF UNKNOWN ETIOLOGY IN PESTICIDE EXPOSED PATIENTS AT ZAGAZIG UNIVERSITY HOSPITALS

Background: Pesticide compounds are commonly used worldwide and in Egypt. Chronic low level exposure of humans to these compounds has resulted in many health adverse effects. There are many reports relating chronic kidney disease of unknown etiology (CKDu) epidemic to pesticide exposure including organophosphates (OP). Glutathione-S-transferase mu (GSTM1) and glutathione-S-transferase theta (GSTT1) are phase II xenobiotic metabolizing enzymes. Paraxonase 1 (PON1) is an enzyme linked to high-density lipoprotein. It can prevent oxidation of low-density lipoproteins and participates in organophosphate detoxification. Objective: To investigate the association of genetic polymorphism of GST (M1, T1) and PON1 Q192R with CKDu in pesticide exposed patients presented to Nephrology Unit, Internal Medicine Department, Zagazig University Hospitals, Sharkia Governorate. Methods: Gas chromatography- mass spectrometry (GC-MS) was used for detection of dialkyl phosphate metabolites in urine samples of subjects included in the study then genomic DNA isolated from the blood samples of 48 CKDu patient chronically exposed to pesticides giving positive result for dialkyl phosphate metabolites and matched control group was used to detect the presence or absence of the GSTM1 and GSTT1 genes (gene deletion) by multiplex polymerase chain reaction technique (multiplex PCR) and PON1 polymorphisms using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Conclusion: This study suggested that there is a correlation between chronic exposure to organophosphates and the risk of CKDu due to GSTM1 and GSTT1 genes deletions and PON1 gene polymorphisms.

Study of thrombophilic gene polymorphisms in Egyptian patients with deep venous thrombosis using in situ hybridization technique.

Up to 70% of thrombotic patients with no identifiable risk factors were termed idiopathic. Now, molecular diagnostics combined with existing laboratory techniques allow accurate classification of at least half of patients with inherited thrombotic disorders. However, the previously studied genetic variants explain only a fraction of all thromboembolic events, so the aim of the present study was to expand the genetic prevalence determination to include also more extensive thrombophilic gene polymorphisms in patients with DVT compared to healthy subjects in Egyptian population. This study was conducted on 75 patients with DVT and 45 age and sex matched healthy subjects as control group. The diagnosis of DVT was based on patient’s history, clinical findings, D-dimer test, and confirmed by Doppler ultrasonography. Both groups were assessed for thrombophilic gene polymorphisms using multiplex polymerase chain reaction and reverse-hybridization technique through CVD strip assay. It was found that FV Leiden G1691A (P=0.001), Factor V H1299R (P=0.02), MTHFR A1298C (P=0.02), β-fibrinogen−455 G/A (P=0.01), PAI-1 4G/5G (P=0.03) and ACE (P=0.03) polymorphisms were all significantly associated with an increased risk of DVT. Our data are of extreme importance in clinical practice for introducing the extended CVD panel into routine molecular diagnostic tests to allow management of thrombotic patients and screening, thromboprophylaxis and genetic counselling for high-risk individuals.

Molecular Characterization and Distribution of Class 1 Integron-Bearing Methicillin Resistant Staphylococcus aureus Strains in Burn Patients, Tehran, Iran

Background: Methicillin resistant Staphylococcus aureus (MRSA) is an increasingly common hospital pathogen in burn patients, which is known to cause over 50% of burn related deaths. One of the serious threats associated with clinical isolates of MRSA is multi-drug resistance, which is associated with integrons. Objectives: The aim of this study was to determine the distribution and molecular types of MRSA in burn patients and their carriage of integrons. Methods: During a 7-month period, 106 MRSA isolates were collected from burn wounds of patients admitted to a referral burn hospital in Tehran. Antimicrobial susceptibility testing (AST) was performed for 12 antimicrobial agents. Polymerase chain reaction (PCR) was used to detect nucA, mecA, pvl and tsst-1 genes, and class 1 and 2 integrons. Multiplex PCR technique was used to determine the Staphylococcal cassette chromosome mec (SCCmec) types of MRSA strains. All isolates were genotyped by staphylococcal protein A (spa) typing. Results: AST showed the lowest rate of resistance to quinupristin-dalfopristin (19.8%), mupirocin (31.3%), and rifampicin (37.7%). All isolates were susceptible to vancomycin, teicoplanin, and linezolid. Multi-drug resistance was observed in 97% of isolates. The most SCCmec type was SCCmec type III (98.1%) while only 2 (1.9%) MRSA isolates harbored SCCmec type IV. SCCmec types I, II, and V were not detected. The study revealed the presence of class 1 integron in 58 (54.7%) isolates and class 2 integron in 3.8% of isolates. Six different spa types of t030 (66%), t037 (14.2%), t065 (9.4%), t1358 (4.7%), t937 (3.8%), and t084 (1.9%) were identified amongst the isolates. Conclusions: The study revealed a high prevalence of multi-drug resistance (MDR), class 1 integron, SCCmec type III, and spa type t030 amongst MRSA associated with burn wounds in an Iranian hospital. The existence of SCCmec type III in burn patients emphasizes the nosocomial origin of these strains.

Geographic distribution of canine heartworm (Dirofilaria immitis) infection in stray dogs of eastern Romania

A survey was conducted in the eastern part of Romania to assess the prevalence and geographical distribution of Dirofilaria immitis in dogs. Plasma samples were collected from 458 stray dogs hosted in shelters in 8 counties and tested serologically for the presence of heartworm. In addition, 45 blood samples from dogs of a shelter in Galati city were examined by the modified Knott and multiplex polymerase chain reaction (PCR) techniques. The immmunological assay showed a heartworm infection prevalence of 8.9% in the dogs. Optical density results for positive samples ranged between 0.217 and 2.683. Geographical information systems (GIS) were used to produce overlays of distribution maps of D. immitis prevalence and predictive maps based on temperature suitability. High prevalence of D. immitis was found in the central East up to the northern border of the country, i.e. Galati county (60%), followed by the counties of Vaslui (12.0%) and Iasi (7.7%). Out of 45 samples examined using the Knott test, 23 were positive for circulating microfilariae (51.1%), while 19 dogs were positive for D. immitis and 4 for both D. immitis and D. repens with the multiplex PCR test. The high prevalence for D. immitis shown in dogs in the Southeast (Galati, 42.2%) also by multiplex PCR gave strong support to the results achieved by the serological tests. The present study confirms the ability of GIS to predict the distribution and epidemiology of dirofilariosis in different geographical territories as has been already demonstrated by the empirical epidemiological data obtained at the continental, national and intraregional levels.

Association of Hemostatic Gene Polymorphisms With Early-Onset Ischemic Heart Disease in Egyptian Patients.

The association between hereditary thrombophilia and venous thrombosis is well established but controversial data exist with respect to arterial thrombosis. We performed a pilot study on 31 patients with acute myocardial infarction (AMI), 21 patients with unstable angina (UA), and 20 healthy volunteers to investigate the role of various hemostatic gene polymorphisms in young Egyptian patients, who survived their first ischemic heart disease (IHD). Thrombophilic gene polymorphisms were tested using multiplex polymerase chain reaction and reverse-hybridization technique. We showed an increased risk of AMI with factor V (FV) Leiden and prothrombin G20210A heterozygosity. The increased risks of UA was associated with GA and A allele of fibrinogen β-455G→A polymorphism. Conversely, factor XIII (FXIII) Val34Leu GT and T allele were protective in the UA group. Nevertheless, the prevalence of FV H1299R, plasminogen activator inhibitor 1 4G/5G, glycoprotein IIIa C1565T, 5,10-methylenetetrahydrofolate reductase C677T, and A1298C mutations did not differ between patients with IHD and controls. The data have clinical implications regarding screening and thromboprophylaxis in high-risk individuals younger than 40 years.

Identification of Vibrio parahaemolyticus in Seafood by Multiplex PCR

ABSTRACTVibrio parahaemolyticus is a human pathogen frequently found in seafood. Once the seafood is contaminated by V. parahaemolyticus, it can become a vehicle for foodborne illness. The conventional culture methods for detection of V. parahaemolyticus are time-consuming and cannot differentiate pathogenic strains from nonpathogenic ones. In this study, a multiplex polymerase chain reaction (PCR) technique was investigated for detecting tdh, chiA, and toxR of V. parahaemolyticus. The sensitivity of the multiplex PCR was determined by testing 28 strains of V. parahaemolyticus, 15 non-V. parahaemolyticus strains, and fresh seafood spiked with cells of V. parahaemolyticus. All the strains were analyzed for production of thermostable direct hemolysin (TDH) and chitinase. This study showed that both the chiA and toxR are excellent markers for detecting V. parahaemolyticus strains, and a multiplex PCR targeting chiA and tdh genes can be applied to simultaneously detect environmental and pathogenic V. parahaemolyticus.

Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M.tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M.tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

Fecal contamination of drinking water in Kericho District, Western Kenya: role of source and household water handling and hygiene practices.

Inadequate protection of water sources, and poor household hygienic and handling practices have exacerbated fecal water contamination in Kenya. This study evaluated the rate and correlates of thermotolerant coliform (TTC) household water contamination in Kericho District, Western Kenya. Culture and multiplex polymerase chain reaction (PCR) techniques were used to characterize TTCs. The disk diffusion method was used for antibiotic susceptibility profiling of pathogenic Escherichia coli. Out of the 103 households surveyed, 48 (46.6%) had TTC contaminated drinking water (TTC levels of >10 cfu/100 mL). Five of these households were contaminated with pathogenic E. coli, including 40% enteroaggregative E. coli, 40% enterotoxigenic E. coli, and 20% enteropathogenic E. coli. All these pathogenic E. coli strains were multidrug resistant to sulfamethoxazole/trimethoprim, ampicillin, tetracycline and ampicillin/sulbactam. Rural household locality, drinking water hand contact, water storage container cleaning practice, hand washing before water withdrawal, water source total coliforms <10 cfu/100 mL, temperature, and free chlorine levels were associated with TTC contamination of household drinking water. Significant proportions of household drinking water in Kericho District are contaminated with TTCs including with pathogenic multidrug-resistant E. coli. Source and household hygiene and practices contribute significantly to drinking water contamination.

Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position.