Influenza and respiratory syncytial viruses: Efficacy of different diagnostic assays.

OBJECTIVE: To determine the efficacy of cell culture, immunoflourescence Assay (IFA) and real time polymerase chain reaction (rRT-PCR) in relation to diagnosis of influenza and Respiratory Syncytial Virus (RSV). METHODS: Total 2781 specimens of throat swabs and nasopharyngeal aspirates were obtained from patients suspected of respiratory viruses' infections from January 2009 to December 2011 at Universiti Kebangsaan Malaysia Medical Centre(UKMMC). The specimens were processed by cell culture and immunoflurescence assay (IFA) and (rRT-PCR). RESULTS: Thirty three (1.19%) specimens were positive for influenza virus A and 42 (1.51%) were positive for RSV by cell culture and IFA. On the other hand, rRT-PCR was able to identify 189 of 505 (37.43%) specimens in which 65 were influenza A virus and 124 were RSV. Sensitivity of rRT-PCR was 100% for both influenza A virus and RSV and specificity was 88% and 77% for influenza A virus and RSV, respectively. CONCLUSION: rRT-PCR diagnosed respiratory viruses in shorter time with a high level of sensitivity in comparison to conventional assays - cell culture and IFA. These advantages help in managing patients by saving cost and hospitalization stay.

Helicobacter pylori: molecular detection of vacA gene and vacuolating activity in human gastric adenocarcinoma cells.

BACKGROUND AND AIMS: Helicobacter pylori strains secrete a vacuolating cytotoxin (VacA), plays an important role for the development of peptic ulcer disease and gastro-duodenal diseases. vacA gene is responsible to regulate the activity of the vacuolating cytotoxin. The objective of this study was molecular detection of vacA gene and observes the vacuolating activity on human gastric adenocarcinoma (AGS) cells. MATERIALS AND METHODS: H. pylori vacA gene was determined by polymerase chain reaction. Vacuolation activity of VacA toxin in broth culture filtrates was assessed in AGS cells and quantified by neutral uptake assay. Different concentration dosages of VacA and incubation time were used in the measurement of the vacuolating activity on AGS cells. RESULTS: The results showed that VacA toxin could stimulate vacuolating activity on AGS cells with minimum concentration 1.0 µg/ml from both of s1m1 and s1m2 alleles (vacA gene). The toxin produced optimal reaction at 5.0 µg/ml with significant differences observed between the alleles. The results also showed that both alleles commenced the vacuolating activity at the minimum of 3 hr incubation time, and the activity showed in time-dependent manner. CONCLUSION: Optimal concentration of VacA toxin (s1m1 allele) causes more interaction with AGS cell producing more vacuolating activities. Time-dependent vaculation of both alleles might allow H. pylori for persistent infection without rapid destruction of gastric cells might promote gastro-duodenal diseases. The study provided us better understanding of the pathogenesis of the diseases associated with H. pylori infection which is an emerging problem in developing countries.

Using pulsed-field gel electrophoresis in the molecular investigation of an outbreak of Serratia marcescens infection in an intensive care unit.

INTRODUCTION: Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in immunocompromised patients with severe underlying disease. An outbreak due to S. marcescens infection was detected from 13 to 22 February 2001 at the intensive care unit (ICU) of our institution. We used pulsed-field gel electrophoresis (PFGE) typing to analyse the outbreak strains involved. METHODS: A total of 25 isolates were included in this study: 12 isolates from infected patients, nine isolates from insulin solution, one isolate from sedative solution (midazolam and morphine infusion) and one isolate from frusemide solution. Two isolates from other wards which were epidemiologically-unrelated were also included. RESULTS: The S. marcescens from patients, insulin solution and sedative solution showed an identical PFGE fingerprint pattern. The isolate from the frusemide solution had a closely-related PFGE pattern to the outbreak strain with one band difference. Attempts were made in the present study to identify the environmental reservoir of S. marcescens during the outbreak. We found that the insulin and sedative solutions used by the patients were contaminated with S. marcescens which was proven to be the source of the outbreak. CONCLUSION: Using PFGE, we showed that the outbreak in the ICU of our hospital was due to the clonal spread of a single strain of S. marcescens.

A major methicillin-resistant Staphylococcus aureus clone predominates in Malaysian hospitals.

This study was conducted to determine the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysian hospitals. A total of 264 MRSA isolates from eight hospitals were subjected to typing by pulsed-field gel electrophoresis (PFGE) of SmaI restricted DNA. Antibiotic disk susceptibility testing was also carried out to determine their resistance patterns. Thirty-one PFGE pattern types were identified. Three major pattern types A, ZC and K were found with type A the predominant profile in c. 80% of strains and present in all hospitals. Unlike type A, other DNA pattern types were unique to the hospitals in which they were isolated. PFGE type A also consisted of strains that were multiply antibiotic resistant. The presence of a single predominant PFGE type in Malaysian hospitals is an important finding which suggests that inter-hospital spread of MRSA had occurred frequently and regularly.

DNA fingerprinting of methicillin-resistant Staphylococcus aureus (MRSA) by pulsed-field gel electrophoresis (PFGE) in a teaching hospital in Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) has been prevalent in our hospital over the last three years. Differentiation among MRSA strains by DNA typing in addition to antibiotic resistance pattern surveillance is crucial in order to implement infection control measures. The aim of this study was to characterize MRSA isolates from patients admitted to Hospital Universiti Kebangsaan Malaysia (HUKM) by phenotypic (analyses of antibiotic susceptibility pattern) and genotypic (PFGE) techniques to determine the genetic relatedness of the MRSA involved and to identify endemic clonal profiles of MRSA circulating in HUKM. Seventy one MRSA strains collected between January to March 2000 from patients from various wards in HUKM were tested for antimicrobial resistance and typed by pulsed-field gel electrophoresis (PFGE). Four major types of PFGE patterns were identified (A, B, C and D) among MRSA strains. Two predominant PFGE types were recognised, Type A (59.2%) and Type B (33.8%). Most of these strains were isolated from ICU, Surgical wards and Medical wards. MRSA strains with different PFGE patterns appeared to be widespread among wards. Strains with the same antibiotype could be of different PFGE types. Most of isolates were resistant to ciprofloxacin, erythromycin, gentamicin and penicillin. One isolate with a unique PFGE pattern Type D and susceptible to gentamicin was identified as a different clone. Some isolates obtained from the same patient showed different PFGE subtypes suggesting that these patients were infected/colonized with multiple MRSA strains. PFGE analysis suggests that MRSA strains with different PFGE types was propagated within our hospital. The relationship between antibiotic susceptibility and PFGE patterns was independent. The ability of PFGE technique in differentiating our MRSA strains make it a method of choice for investigating the source, transmission and spread of nosocomial MRSA infection, and thus an appropriate control programme can be implemented to prevent the spread of MRSA infection.

Contamination of breast milk obtained by manual expression and breast pumps in mothers of very low birthweight infants.

The objective of this study was to compare the rates of bacterial contamination of expressed breast milk (EBM) obtained by manual expression and breast pumps in mothers of very low birthweight (VLBW) infants (<1501 g). This was a randomized, controlled study carried out on 28 mothers of such babies and 92 specimens of EBM were collected: 41 specimens from 13 mothers assigned to the manual group and 51 specimens from 15 mothers in the breast-pump group. EBM was cultured quantitatively by the Miles and Misra method. Breast milk expressed by breast pumps (86.3% or 44/51 specimens) had a significantly higher rate of bacterial contamination than milk expressed by the manual method (61.0% or 25/41 specimens) (P= 0.005). When breast milk was expressed in the hospital, there was no significant difference in contamination rates between the two methods. When breast milk was expressed at home, the rates of bacterial contamination by staphylococci (P= 0.003) and Gram-negative bacilli (P= 0.002) were significantly higher in the breast-pump group than the manual group. In conclusion, the rate of bacterial contamination of EBM of mothers of VLBW infants was high, especially when EBM was obtained by the breast pump or when expression was carried out at home.