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.
Isolation and culture of Burkholderia pseudomallei remains the main stay in the diagnosis of melioidosis. Thus, the search for selective and differential media for B. pseudomallei has been ongoing. A number of such media have been reported with varying efficacy. Ashdown medium is the most established selective medium for the isolation of B. pseudomallei. There are no reports of differential media differentiating B. pseudomallei from Burkholderia cepacia. This report documents such a selective and differentiating medium for B. pseudomallei. Of a total of 1042 clinical specimens containing mixed flora and gram-negative isolates that were tested on this medium, 16 of the specimens yielded B. pseudomallei. The isolation rate was found to be 1.5%. This medium was found to be simple and inexpensive, can be made by small laboratories, and called as Francis medium. Based on the colony morphology and color, a preliminary report can be made within 18-24 h for the presence of B. pseudomallei. Our study showed that this medium had an overall sensitivity of 78.4% with a specificity of 92.2%. The use of this medium as an early diagnostic tool will help to reduce mortality and morbidity of melioidosis patients.
Hydroxyapatite is a calcium phosphate bioceramic that has been shown by many authors to be biocompatible with bioactive properties. It is widely accepted as the best synthetic material available for surgical use as a bone graft substitute. HA granules produced by AMREC-SIRIM from local materials underwent 5 types of sterilisation techniques with different ageing periods. Samples were tested for chemical and phase composition and microbial contamination before and after being sterilised. From the microbiological tests done, none of the unsterilised positive control yielded a positive culture. Results from X-Ray diffraction studies found that all the sterilisation techniques did not chemically degrade or structurally change the HA granules significantly.
The haemolysins produced by Aeromonas species were detected and compared by two assay methods--a modified blood agar plate assay and the rabbit erythrocyte haemolysin method. Both assays showed a high level of agreement (86%). The titres of the rabbit erythrocyte haemolysin assay correlated with the haemolytic zone diameter of the ox blood agar assay. In addition the agar haemolysin assay had simple media requirements, was easy to perform and results were well defined.
OBJECTIVE: To systematically review Indian literature on delays in tuberculosis (TB) diagnosis and treatment.
METHODS: We searched multiple sources for studies on delays in patients with pulmonary TB and those with chest symptoms. Studies were included if numeric data on any delay were reported. Patient delay was defined as the interval between onset of symptoms and the patient's first contact with a health care provider. Diagnostic delay was defined as the interval between the first consultation with a health care provider and diagnosis. Treatment delay was defined as the interval between diagnosis and initiation of anti-tuberculosis treatment. Total delay was defined as time interval from the onset of symptoms until treatment initiation.
RESULTS: Among 541 potential citations identified, 23 studies met the inclusion criteria. Included studies used a variety of definitions for onset of symptoms and delays. Median estimates of patient, diagnostic and treatment delay were respectively 18.4 (IQR 14.3-27.0), 31.0 (IQR 24.5-35.4) and 2.5 days (IQR 1.9-3.6) for patients with TB and those with chest symptoms combined. The median total delay was 55.3 days (IQR 46.5-61.5). About 48% of all patients first consulted private providers; an average of 2.7 health care providers were consulted before diagnosis. Number and type of provider first consulted were the most important risk factors for delay.
CONCLUSIONS: These findings underscore the need to develop novel strategies for reducing patient and diagnostic delays and engaging first-contact health care providers.
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.
The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.
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.
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.
This study was carried out to compare the performance of BACTEC MGIT 960 with the BACTEC 460 TB for growth and detection of Mycobacteria from human clinical specimens. The BACTEC MGIT 960 instrument is a fully automated system that utilizes MGIT tubes containing an oxygen sensor embedded in silicon at the bottom and filled with 7 mL of modified Middlebrook 7H9 broth. Identical samples were inoculated into the two automated systems and incubated for six weeks. Over a period of three months, 279 specimens were decontaminated and processed according to the standard CDC NALC/NaOH method, using the commercial MycoPrep kit. Forty-two specimens (15%) yielded Mycobacterium tuberculosis; 37 (88%) were detected by the fluorescent BACTEC MGIT 960 and 35 (83%) detected by the radiometric BACTEC 460 TB. Fifteen specimens (5%) yielded Mycobacterium Other Than Tuberculosis (MOTT); 10 (66%) were detected by BACTEC MGIT 960 and 11 (73%) detected by BACTEC 460 TB. The average time to detection and contamination rates and the average time to obtain results of antimicrobial susceptibility tests between the two systems were compared. The performance of the BACTEC MGIT 960 was comparable to the BACTEC 460 TB system which has been the "Gold Standard" for automated detection of TB. The former was more rapid, as sensitive and less labour intensive than the BACTEC 460. Our data demonstrates that the BACTEC MGIT 960 system is an accurate, automated and a non-radioactive alternative to the BACTEC 460 TB for the culture and susceptibility testing of M. tuberculosis.
A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp.
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.
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, 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.
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.
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.
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.
Introduction.Burkholderia pseudomallei (melioidosis) is an important cause of community-acquired pneumonia (CAP) in the tropics. Selective medium is recommended for laboratory diagnosis with non-sterile respiratory samples, while PCR is not routinely used due to variable reported performance. The effectiveness of these diagnostic modalities varies by site.Aim. To compare selective media and real-time PCR (qPCR) with routine media in detecting B. pseudomallei in CAP respiratory samples in a low-incidence setting in Kuala Lumpur, Malaysia.Methodology. Respiratory samples were routinely cultured on blood, chocolate and MacConkey agar (RESP-ROUTINE), and compared to culture on selective Ashdown medium (RESP-SELECTIVE) and qPCR. The gold standard was routine culture of B. pseudomallei from any site (ALL-ROUTINE).Results.B. pseudomallei was detected in 8/204 (3.9 %) samples. Overall sensitivity rates differed (P=0.03) for qPCR (100%), RESP-SELECTIVE (87.5%) and RESP-ROUTINE (50%). There was a trend towards lower median days to positive culture for RESP-SELECTIVE (1 day) compared to RESP-ROUTINE (2 days, P=0.08) and ALL-ROUTINE (2 days, P=0.06). Reagent costs for each additional detection were USD59 for RESP-SELECTIVE and USD354 for PCR.Conclusions. In a low-incidence setting, selective culture of respiratory samples on Ashdown was more sensitive and allowed quicker identification than routine media, at reasonable cost. Blood cultures are critical, confirming four cases missed by routine respiratory culture. Selective medium is useful in early pneumonia (pre-sepsis) and resource-limited settings where blood cultures are infrequently done. Real-time PCR is costly, but highly sensitive and useful for high-risk patients with diabetes, cancer or immunosuppressants, or requiring ventilation or intensive care.