Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR

Objectives: Emergence of methicillin resistant Staphylococcus aureus [MRSA] is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information

Methods: We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors

Results: We found 38.33% isolates as methicillin resistant, a-toxin, the major cytotoxic factor, was detected in 40% whereas 6-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible

Conclusions: These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures

Relationship of oxidative stress with elevated level of DNA damage and homocysteine in cardiovascular disease patients

Amounts of DNA damage and homocysteine [Hcy] in heart patients blood may have strong function in the causation of cardiovascular disease [CVD]. The main objective of this work was to know experimentally the role of total oxidants [produced by Reactive Oxygen species [ROS], clinical biochemical indices, their oxidized products and total antioxidant status [TAS] among such patients to find the association of homocysteine, total oxidation status [TOS] and oxidative DNA damage with other clinical parameters in sixty positive CVD patients compared with those of 60 normal subjects. As compared to healthy individuals, CVD patients had significantly higher concentrations of homocysteine [p<0.0001], total oxidants stress [TOS] [p<0.0001], serum total lipids [p<0.04], malondialdehyde [MDA] [p<0.001], high density lipoprotein-cholesterol [HDL-C] [p<0.0001], and low density lipoprotein cholesterol [LDL-C] [p<0.01], than those of healthy individuals. Plasma Hcy content, TOS and amount of DNA were positively and significantly associated with cholesterol, triglycerides, systolic blood pressure, urea, and albumin [p values<0.01]. TOS, Hcy and oxidative DNA damage were negatively correlated with HDL-c, TAS and proteins. It is suggested that these parameters have pivotal role in diagnostic process of determining severity in CAD patients. Oxidized products of macromolecules in blood of CVD patients impart major functions in causing CVD disease

Nested PCR based diagnosis of salmonella enterica serovar paratyphi a directly from blood samples

Development of a rapid, reliable PCR - based method for molecular identification of Salmonella enterica serovar Paratyphi A directly from blood samples. S. Paratyphi A isolates were used for regular PCR targeting specific region of fliC-a gene. New primers were designed and conditions were optimized for a nested PCR that could be directly applicable on blood samples. The procedure was tested on 70 blood samples from suspected cases of typhoidal infection and comparison made with blood culture. Blood culture was able to diagnose only four patients as infected with S. Paratyphi A. Regular PCR was unable to detect S. Paratyphi A directly from blood where as nested PCR detected S. Paratyphi A in blood of thirteen patients. S. Paratyphi A, which is emerging as a major pathogen can be detected with better sensitivity by nested PCR as compared with blood culture

Differentiation of common gram negative pathogens by PCR- ribotyping

Gram negative bacteria especially members of family Enterobacteriaceae are among the most frequently isolated organisms from the clinical specimens. Rapid diagnosis of the pathogen in a clinical sample is always very important. Conventional methods are time-consuming. Among molecular techniques, PCR is very useful but unless very specific primers are used, non-specific amplifications are a problem. PCR-ribotying is a technique that gives very specific multiple bands by use of a single primer set. This study was designed to establish patterns for five common pathogens of Enterobacteriaceae, namely Escherichia coli, Salmonella enterica serovar Typhi [Salmonella Typhi], Proteus vulgaris, Klebsiella aerogenes, and Cirtobacter freundii along with another very common and problematic gram negative pathogen Pseudomonas aeruginosa. Each species gave a specific ribotyping pattern. Escherichia coli gave four amplification products of 1200, 850, 800, and 700 bps. Four amplification products of different sizes were also observed in Citrobacter freundii [3000, 850, 700, and 580 bps], Proteus vulgaris [900, 800, 750 and 700 bps], and Klebisella aerogenes [3000, 870, 700 and 520 bps]. More discrimination with five amplification products was seen in Salmonella Typhi [3000, 1200, 900, 850, and 700 bps]. On the other side of spectrum was Pseudomonas aeruginosa only a single amplification product of 750 bps was observed. PCR-ribotyping can very efficiently and specifically differentiate between opportunistic gram negative human pathogens

Significant incidence of typhoid in hepatitis C patients

Only proven way of transmission of Hepatitis C is through blood. The origin is unknown in nearly half the cases. Pollution is suspected as a cause but it is impossible to prove this relationship directly. We thought that typhoid being a proven pollution related disease, determination of its confection in Hepatitis C patients representing same Socio-economic group would be of interest. A typhoid in Hepatitis C patients can easily be overlooked because symptoms like fever and abdominal discomfort are present in both diseases. Blood samples were collected from three groups of study as mentioned in materials and methods. These samples were processed for 4[th] generation HCV ELISA. PCR for HCV, PCR for typhoid. Blood culture for typhoid and widal test as required [details are given in methodology]. Finally the data thus obtained was analysed and conclusions were drawn. Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering [NIBGE] and Millat Laboratory Faisalabad. April 2004 to Oct 2004. The parameters included were PCR, blood culture and widal test. There were three groups of study, PCR and ELISA positive patients of Hepatitis C [105] - further subdivided into two groups, with history of exposure to known causes of spread of HCV in last one year [65] and those without such history [40]; clinically diagnosed cases of typhoid [30]; and healthy controls [50]. In the three groups, PCR was positive in 9.5[7.7 and 12.5], 63.3, and 2.0% cases respectively. Figures for blood culture were 4.7[3.1 and 7.5], 33.3, and 0% in the same order, and the respective figures for widal test were 34.2[33.8 and 35.0], 56.6, and 24.0%. The increase in PCR and blood culture positivity in Hepatitis C cases as compared with normal subjects is statistically significant [P< 0.05]. These results clearly suggest that the source of infection for the two diseases is same in many cases, and therefore, provides a strong indication of a relationship between pollution and Hepatitis C