Clinical utilization of carbapenems remains under threat with the emergence of acquired carbapenemase-producing bacteria, particularly metallo-β-lactamases (MBL). Rapid detection of MBL-producing Gram-negative bacilli is essential to prevent their widespread dissemination. However, no standardized detection method is available for routine laboratory use. The purpose of the study was to evaluate a chelating-agent based double disk synergic test and disk potentiation test for MBL-producing strain detection and to determine the isolation rate of MBL-producing Pseudomonas aeruginosa and Acinetobacter from clinical samples in our tertiary teaching hospital. A total of 22 and 66 imipenem-resistant P. aeruginosa and Acinetobacter isolates respectively were tested with ceftazidime (CAZ) disk by modified double disk synergic test and disk potentiation test using ethylenediaminetetraacetic acid (EDTA) and 2-mercaptopropionic acid (as chelating agents) to detect MBL production. The tests were compared with EDTA-phenanthroline-imipenem (EPI) microdilution MIC test as gold standard. MBL positive strains were detected in 17 (77.3%) P. aeruginosa and 2 (3.5%) Acinetobacter isolates. The disk potentiation test with 2-mercaptopropionic acid (2-MPA) dilution of 1:12 provided the most acceptable sensitivities and specificities (88.2% sensitivity and 100% specificity in P. aeruginosa; 100% sensitivity and specificity in Acinetobacter) compared to other screening methods used in this study. This study provided useful information on the local prevalence of MBL-producing P. aeruginosa and Acinetobacter in our hospital. Disc potentiation test with CAZ/2-MPA disc appears to be reliable and convenient MBL detection method in the routine clinical laboratory.
Acinetobacter baumannii, genomic species 3 and 13TU are being increasingly reported as the most important Acinetobacter species that cause infections in hospitalized patients. These Acinetobacter species are grouped in the Acinetobacter calcoaceticus- Acinetobacter baumannii (Acb) complex. Differentiation of the species in the Acb-complex is limited by phenotypic methods. Therefore, in this study, amplified ribosomal DNA restriction analysis (ARDRA) was applied to confirm the identity A. baumannii strains as well as to differentiate between the subspecies. One hundred and eighty-five strains from Intensive Care Unit, Universiti Malaya Medical Center (UMMC) were successfully identified as A. baumannii by ARDRA. Acinetobacter genomic species 13TU and 15TU were identified in 3 and 1 strains, respectively. ARDRA provides an accurate, rapid and definitive approach towards the identification of the species level in the genus Acinetobacter. This paper reports the first application ARDRA in genospecies identification of Acinetobacter in Malaysia.
Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months-79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture-the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard-bacterial culture-of any positive test, there was low sensitivity for each test (25-44%) but high specificity (93-98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.
Burkholderia pseudomallei the causative agent of melioidosis, is being increasingly recognized as an important cause of morbidity and mortality in South East Asia. Biofilm formation of B. pseudomallei may be responsible for dormancy, latency and relapse of melioidosis. Based on the colonial morphology of the bacteria on B. pseudomallei selective agar medium, seven distinct morphotypes were identified. This study was conducted to assess the in vitro biofilm produced by B. pseudomallei and to investigate possible correlation between B. pseudomallei morphotypes with biofilm forming abilities of the isolates. Using a standard biofilm crystal violet staining assay, comparison was made between the biofilm forming ability of 76 isolates of B. pseudomallei and Burkholderia thailandensis ATCC 700388. Amongst the blood isolates, 30.2% were considered as high biofilm producers and 27.9% were low producers, 33.3% of the pus isolates were considered as high and 16% low biofilm producers. Most of the isolates were identified as morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. However, we did not find any correlation of B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Additional studies are needed to identify internal and external factors which contribute to the high and low biofilm formation of B. pseudomallei.
Acinetobacter baumannii is an emerging nosocomial pathogen that is resistant to many types of antibiotics, and hence, a fast, sensitive, specific, and economical test for its rapid diagnosis is needed. Development of such a test requires a specific antigen, and outer membrane proteins (OMPs) are the prime candidates. The goal of this study was to find a specific OMP of A. baumannii and demonstrate the presence of specific IgM, IgA, and IgG against the candidate protein in human serum. OMPs of A. baumannii ATCC 19606 and 16 other clinical isolates of A. baumannii were extracted from an overnight culture grown at 37 °C. Protein profiles were obtained using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot analysis was performed to detect the presence of IgM, IgA, and IgG against the OMP in host serum. An antigenic 34.4-kDa OMP was uniquely recognized by IgM, IgA, and IgG from patients with A. baumannii infection, and it did not cross-react with sera from patients with other types of infection. The band was also found in the other 16 A. baumannii isolates. This 34.4-kDa OMP is a prime candidate for development of a diagnostic test for the presence of A. baumannii.
A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.
Toxinotype of Clostridium perfringens (CP) isolates collected from the Bernam River, Selangor River and Tengi Canal between April 2007 and January 2008 were determined by Polymerase Chain Reaction (PCR) using published primers. All the 147 isolates were toxinotype Type A, harbouring the alpha toxin gene. In addition, 5 of the isolates also had the enterotoxin (CPE) gene.
Molecular-based techniques are becoming desirable as tools for identification of infectious diseases. Amongst the Burkholderia spp., there is a need to differentiate Burkholderia pseudomallei from Burkholderia cepacia, as misidentification could lead to false treatment of patients. In this study, conventional PCR assay targeting three genes was developed. Primers were designed for the amplification of Burkholderia genus-specific groEL gene, B. pseudomallei-specific mprA gene and B. cepacia-specific zmpA gene. The specificity and sensitivity of the assay was tested with 15 negative control strains and 71 Burkholderia spp. isolates including positive controls B. pseudomallei K96243 and ATCC B. cepacia strain. All B. pseudomallei strains were positive for groEL (139 bp) and mprA (162 bp), indicating a sensitivity of 100%. All B. cepacia strains produced amplicons for detection of groEL and zmpA (147 bp). Specificity using negative strains was 100%. In this study, a PCR assay specific for the detection of Burkholderia spp. and differentiation of the genus B. pseudomallei and B. cepacia was developed. The conventional assay has to be performed separately for each species due to the similar size of the PCR products amplified. This format may therefore be recommended for use as a diagnostic tool in laboratories where real-time PCR machines are not available. However, the real-time PCR was able to detect and differentiate the genus and species in single duplex assay.
Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.
Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.
Routine use of selective media improves diagnosis of Burkholderia pseudomallei, but resources may be limited in endemic developing countries. To maximise yield in the relatively low-prevalence setting of Kuala Lumpur, Malaysia, B. pseudomallei selective agar and broth were compared with routine media for 154 respiratory specimens from patients with community-acquired disease. Selective media detected three additional culture-positive specimens and one additional melioidosis patient, at a consumables cost of US$75. Burkholderia pseudomallei was not isolated from 74 diabetic foot ulcer samples. Following careful local evaluation, focused use of selective media may be cost-effective.
In this study, PCR-RFLP analysis (PRA) targeting hsp65 and rpoB gene regions was evaluated for the identification of mycobacterial species isolated from Malaysian patients. Overall, the hsp65 PRA identified 92.2 % of 90 isolates compared to 85.6 % by the rpoB PRA. With 47 rapidly growing species, the hsp65 PRA identified fewer (89.4 %) species than the rpoB PRA (95.7 %), but with 23 slow-growing species the reverse was true (91.3 % identification by the hsp65 PRA but only 52.5 % by the rpoB PRA). There were 16 isolates with discordant PRA results, which were resolved by 16S rRNA and hsp65 gene sequence analysis. The findings in this study suggest that the hsp65 PRA is more useful than the rpoB PRA for the identification of Mycobacterium species, particularly with the slow-growing members of the genus. In addition, this study reports 5 and 12 novel restriction patterns for inclusion in the hsp65 and rpoB PRA algorithms, respectively.
The established practice of sending blood cultures in an aerobic-anaerobic pair of bottles has been questioned in recent years, and this study was conducted to evaluate the routine use of an anaerobic bottle in the BACTEC blood culture set at the University of Malaya Medical Centre, Kuala Lumpur, from January to December 2004. A total of 11,663 paired blood culture sets were received, of which 3326 were from pediatric patients and 8337 were from adult patients. The overall positive isolation rate was 15%; the positive isolation rate on excluding the anaerobic bottles was 13%. Overall, there were significantly more organisms isolated from the aerobic bottle (p<0.05); however, the best yield was obtained on using the paired aerobic-anaerobic bottles. Among the positive blood culture sets, organisms were isolated from the anaerobic bottle alone in 15.2% of the pediatric sets and in 18.1% of the adult sets. Organisms that grew more frequently in the anaerobic bottle were anaerobes and some facultative anaerobes; however, the difference was not statistically significant except for anaerobes in the adult sets. We recommend that when culturing blood, an aerobic-anaerobic pair of bottles be used rather than an aerobic-aerobic pair, to optimize the recovery of a wider spectrum of organisms, including obligatory anaerobes.
Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10(3) CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.
Peritonitis still remains a serious complication with high rate of morbidity and mortality in patients on CAPD. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early and optimal treatment. The aim of this study was to use 16S rRNA and ITS gene sequencing to identify common bacterial and fungal pathogens directly from the peritoneal fluid without culturing. Ninety one peritoneal fluids obtained from 91 different patients on CAPD suspected for peritonitis were investigated for etiological agents by 16S rRNA and ITS gene sequencing. Data obtained by molecular method was compared with the results obtained by culture method. Among the 45 patients confirmed for peritonitis based on international society of peritoneal dialysis (ISPD) guidelines, the etiological agents were identified in 37(82.2%) samples by culture method, while molecular method identified the etiological agents in 40(88.9%) samples. Despite the high potential application of the 16S rRNA and ITS gene sequencing in comparison to culture method to detect the vast majority of etiological agents directly from peritoneal fluids; it could not be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in Gene Bank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.
An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of nosocomial and community-acquired infections worldwide. Molecular diagnosis for MRSA nasal carriers is increasingly important for rapid detection and screening of MRSA colonization because the conventional methods are time consuming and labor intensive. However, conventional polymerase chain reaction (PCR) tests still require cold-chain storage as well as trained personnel, which makes them unsuitable for rapid high-throughput analysis. The aim of this study was to develop a thermostabilized PCR assay for MRSA in a ready-to-use form that requires no cold chain.
Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 10 3 CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.
A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.
Flavonoids are polyphenols that are important organic chemicals in plants. The health benefits of flavonoids that result in high commercial values make them attractive targets for large-scale production through bioengineering. Strategies such as engineering a flavonoid biosynthetic pathway in microbial hosts provide an alternative way to produce these beneficial compounds. Escherichia coli, Saccharomyces cerevisiae and Streptomyces sp. are among the expression systems used to produce recombinant products, as well as for the production of flavonoid compounds through various bioengineering approaches including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering and genetically encoded biosensors to detect flavonoid biosynthesis. In this study, we review the recent advances in engineering model microbial hosts as being the factory to produce targeted flavonoid compounds.