Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

Background: The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. Methods: The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Results: Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

The Immunosuppressive Effect of TNFR2 Expression in the Colorectal Cancer Microenvironment.

Colorectal cancer (CRC) represents one of the most common causes of death among cancers worldwide. Its incidence has been increasing among the young population. Many risk factors contribute to the development and progression of CRC and about 70% of them are sporadic. The CRC microenvironment is highly heterogeneous and represents a very complex immunosuppressive platform. Many cytokines and their receptors are vital participants in this immunosuppressive microenvironment. Tumor necrosis factors (TNFs) and TNF receptor 2 (TNFR2) are critical players in the development of CRC. TNFR2 was observed to have increased the immunosuppressive activity of CRC cells via regulatory T cells (T regs) and myeloid-derived suppressor cells (MDSC) in the CRC microenvironment. However, the exact mechanism of TNFR2 in regulating the CRC prognosis remains elusive. Here, we discuss the role of TNFR2 in immune escape mechanism of CRC in the immunosuppressive cells, including Tregs and MDSCs, and the complex signaling pathways that facilitate the development of CRC. It is suggested that extensive studies on TNFR2 downstream signaling must be done, since TNFR2 has a high potential to be developed into a therapeutic agent and cancer biomarker in the future.

Putative Pathogenic Genes of Leptospira interrogans and Leptospira weilii Isolated from Patients with Acute Febrile Illness.

Leptospirosis is an important worldwide tropical disease caused by pathogenic Leptospira spp. The determination of virulence genes is important, as it influences patients' clinical manifestations and clinical outcomes. This case report focused on detecting the pathogenic genes of Leptospira in association with the clinical manifestations of patients at the Hospital Universiti Sains Malaysia, Malaysia, who presented with acute febrile illness. Two cases were found and, to the best of our knowledge, these were the first two cases in Malaysia in which patients presented with febrile illness were associated with successful Leptospira isolation from clinical samples. Both clinical isolates were identified by 16S rRNA sequencing as Leptospira weilii and Leptospira interrogans, respectively, and they were classified as pathogenic Leptospira by the presence of different pathogenic genes, based on a polymerase chain reaction (PCR) amplification of targeted genes. This report emphasizes that different infecting Leptospira species and the presence of different virulence factors cause a slight difference in clinical manifestations and laboratory findings of leptospirosis. Genomic sequencing and annotation revealed the detection of classical leptospiral virulence factor genes that were otherwise missed using PCR for detection of Leptospira weilii genome B208.

Prognostic Epstein-Barr Virus (EBV) miRNA biomarkers for survival outcome in EBV-associated epithelial malignancies: Systematic review and meta-analysis.

BACKGROUND: The EBV-associated epithelial tumours consist 80% of all EBV-associated cancer, where the nasopharyngeal cancer (NPC) and EBV-associated gastric carcinoma (EBVaGC) are considered as the most frequent EBV-associated epithelial tumours. It has been shown that the BART-encoded miRNAs are abundantly expressed in EBV-associated epithelial tumours, hence, these miRNAs may serve as diagnostic and prognostic biomarkers for EBV-associated epithelial tumours. Therefore, the purpose of this systematic review and meta-analysis is to assess these EBV miRNAs as prognostic biomarkers for NPC and GC. METHOD: This systematic review was developed based on PRISMA guidelines and utilizing PubMed, Web of Science, Scopus, Cochrane, and Google scholar databases. The retrieved articles were thoroughly screened in accordance with the selection criteria. The hazard ratio (HR) and 95% confidence interval (CI) for patient survival outcomes were used to evaluate EBV miRNA expression levels. To assess the risk of bias, funnel plot symmetry and Egger's bias test were employed. RESULT: Eleven studies met the selection criteria for inclusion, and four were included in the meta-analysis. Most of the articles considered in this study were from China, with one study from South Korea. The overall pooled effect size estimation (HR) for upregulated EBV miRNAs was 3.168 (95% CI: 2.020-4.969), demonstrating that upregulated EBV miRNA expression enhanced the mortality risk in NPC and GC patients by three times. CONCLUSION: To the best of our knowledge, this is the first meta-analysis that investigates the significance of EBV miRNAs as prognostic biomarkers in NPC and GC patients. The pooled effect estimates of HR of the various studies revealed that higher EBV miRNA expression in NPC and GC may result in a worse survival outcome. To assess the clinical significance of EBV miRNAs as prognostic biomarkers, larger-scale prospective studies are needed.

Performance Validation of COVID-19 Self-Conduct Buccal and Nasal Swabs RTK-Antigen Diagnostic Kit.

The antigen rapid diagnostic test (Ag-RDT) is an immunodiagnostic test that detects the presence of viral proteins (antigens) expressed by the COVID-19 virus in a sample from a patient's respiratory tract. This study focused on evaluating the performance of self-conduct buccal and nasal swabs RTK-antigen test compared to nasopharyngeal swab RTK-based COVID-19 diagnostic assays, Panbio™ COVID-19 Ag Rapid Test Device (Nasopharyngeal) (Abbott Rapid Diagnostics Jena GmbH, Jena, Germany) used in hospitals for first-line screening. The sensitivity and specificity of the paired RTK-Ag test in detecting the an-tigen were calculated at 96.4% and 100%, respectively. Fisher exact tests showed the association between nasopharyngeal swabs RTK-Ag assay and buccal-nasal swabs RTK-Ag from ProdetectTM is significant (p-values < 0.001). The result showed that a self-conducted buccal and nasal RTK-antigen rapid test by the patients is comparable to the results obtained from a rapid test device conducted by trained medical personnel using a nasopharyngeal swab.

A Global Mutational Profile of SARS-CoV-2: A Systematic Review and Meta-Analysis of 368,316 COVID-19 Patients.

Since its first detection in December 2019, more than 232 million cases of COVID-19, including 4.7 million deaths, have been reported by the WHO. The SARS-CoV-2 viral genomes have evolved rapidly worldwide, causing the emergence of new variants. This systematic review and meta-analysis was conducted to provide a global mutational profile of SARS-CoV-2 from December 2019 to October 2020. The review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA), and a study protocol was lodged with PROSPERO. Data from 62 eligible studies involving 368,316 SARS-CoV-2 genomes were analyzed. The mutational data analyzed showed most studies detected mutations in the Spike protein (n = 50), Nucleocapsid phosphoprotein (n = 34), ORF1ab gene (n = 29), 5'-UTR (n = 28) and ORF3a (n = 25). Under the random-effects model, pooled prevalence of SARS-CoV-2 variants was estimated at 95.1% (95% CI; 93.3-96.4%; I2 = 98.952%; p = 0.000) while subgroup meta-analysis by country showed majority of the studies were conducted 'Worldwide' (n = 10), followed by 'Multiple countries' (n = 6) and the USA (n = 5). The estimated prevalence indicated a need to continuously monitor the prevalence of new mutations due to their potential influence on disease severity, transmissibility and vaccine effectiveness.

Linezolid-resistant Enterococcus casseliflavus and Enterococcus gallinarum isolated from poultry farms in Kelantan, Malaysia

Aims@#Linezolid has become a decisive therapy in treating infections with vancomycin-resistant Enterococcus (VRE). Currently, the emergence of linezolid-resistant Enterococcus further complicates the therapeutic options and leads to global health threat not only in hospital setting but in the community. The study aimed at antimicrobial pattern of Enterococcus isolated from 6 poultry farms in Kelantan, Malaysia.@*Methodology and results@#Between February and December 2019, 300 broiler cloacal swab sample (Gallus gallus domesticus) were collected and screened for linezolid-resistant enterococci (LRE) using a standard biochemical and antimicrobial susceptibility tests. Among all the samples, 32.3% (n=97/300) grew Enterococcus, 71.1% (n=69/97) of it were identified Enterococcus casseliflavus by molecular identification, whilst remaining isolates 28.9% (n=28/97) were further identified as Enterococcus gallinarum by 16S rRNA sequencing. None of the isolates were found to exhibit high-level resistance to vancomycin. However, 3/97 (3.1%) were exhibit resistance to high-level gentamicin based on Kirby-Bauer disk diffusion test. Whereas 48/97 (49.5%) of isolates were observed to be resistant to ampicillin, 28/97 (28.9%) were resistant to penicillin. Surprisingly, among the two strains isolated, 18.6% (n=18/97) of it were resistant to linezolid. Isolates showed resistance to linezolid by disk diffusion test were verified by VITEK-2 automated system (bioMérieux, USA) with MIC ≥8 µg/mL. All antimicrobial susceptibility test and minimal inhibitory concentration (MIC) results were interpreted according to Clinical and Laboratory Standard Institute (CLSI). @*Conclusion, significance and impact of study@#In conclusion, this study has reported the prevalence of linezolid resistant Enterococcus (LRE) in highly intrinsic antibiotic resistant of E. casseliflavus and E. gallinarum in Malaysia poultry farms, alongside with the truancy of vanA strains. The emergence of LRE strains is an alarming problem to the animal husbandry and healthcare setting worldwide. This could lead to potentially untreatable and life-threatening enterococcal infections. Even more worrying is the spread of LRE to geographical regions where these strains were previously unreported, which may pose a global health threat. Antimicrobial surveillance in poultry husbandry is thus, dimly necessary to prevent wide spread of multidrug-resistant bacteria.

Diagnostic and Prognostic Indications of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a disease that is highly associated with the latent infection of Epstein-Barr virus. The absence of obvious clinical signs at the early stage of the disease has made early diagnosis practically impossible, thereby promoting the establishment and progression of the disease. To enhance the stride for a reliable and less invasive tool for the diagnosis and prognosis of NPC, we synopsize biomarkers belonging to the two most implicated biological domains (oncogenes and tumor suppressors) in NPC disease. Since no single biomarker is sufficient for diagnosis and prognosis, coupled with the fact that the known established methods such as methylation-specific polymerase chain reaction (PCR), multiplex methylation-specific PCR, microarray assays, etc., can only accommodate a few biomarkers, we propose a 10-biomarker panel (KIT, LMP1, PIKC3A, miR-141, and miR-18a/b (oncogenic) and p16, RASSF1A, DAP-kinase, miR-9, and miR-26a (tumor suppressors)) based on their diagnostic and prognostic values. This marker set could be explored in a multilevel or single unified assay for the diagnosis and prognosis of NPC. If carefully harnessed and standardized, it is hoped that the proposed marker set would help transform the diagnostic and prognostic realm of NPC, and ultimately, help prevent the life-threatening late-stage NPC disease.

Prevalence and association of Panton-Valentine Leukocidin gene with the risk of sepsis in patients infected with Methicillin Resistant Staphylococcus aureus.

BACKGROUND: Panton-Valentine Leukocidin (PVL), is one of the virulence gene expressed by Methicillin Resistant Staphylococcus aureus (MRSA) and is known to be associated with severe form of community acquired MRSA infection. The aim of this study is to investigate its prevalence in our setting and patient's clinical outcome. METHODS: A cross sectional study involve retrospective record review were done involving 90 MRSA positive isolates between November 2016 and October 2017. Multiplex PCR was performed to detect femA, mecA and PVL genes. Clinical presentation and outcomes of patients were reviewed and presented as descriptive analysis. RESULTS: All of the 90 MRSA isolates included in this study were positive for femA and mecA genes following PCR. PVL gene was detected in 20% (n = 18) of the isolates of which 61.1% (n = 11) were community acquired infections and 38.8% (n = 7) were hospital acquired. Case distribution from community acquired infections include patients with skin and soft tissue infections (33.3%, n = 6), infected diabetic foot ulcers (16.7%, n = 3), and one patient each (5.5%, n = 1) for community acquired pneumonia and meningitis. Half of the PVL positive MRSA cases (50%, n = 9) were having sepsis and four of them succumbed to death due to severe infection. CONCLUSION: This study shows a high prevalence of PVL positive MRSA infection in our population. Skin and soft tissue infections accounting for the major sources. In addition, the presence of the PVL gene is associated with increased risk for developing sepsis.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample.

BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model. RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites. CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Recent Advances in Diagnostic Approaches for Epstein-Barr Virus.

Epstein-Barr virus (EBV) is the causative agent of many diseases including infectious mononucleosis (IM), and it is associated with different subtypes of lymphoma, sarcoma and carcinoma such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. With the advent of improved laboratory tests for EBV, a timelier and accurate diagnosis could be made to aid better prognosis and effective treatment. For histopathological lesions, the in situ hybridization (ISH) of EBV-encoded RNA (EBER) in biopsy tissues remains the gold standard for detecting EBV. Methods such as the heterophile antibody test, immunofluorescence assays, enzyme immunoassays, Western blot, and polymerase chain reaction (PCR) are also employed in the detection of EBV in different types of samples. The determination of EBV viral load using PCR, however, is gaining more prominence in the diagnosis of EBV-associated diseases. Given the challenge of false positive/negative results that are sometimes experienced during the detection of EBV, variability in results from different laboratories, and the impact of factors such as sample type and the immunological status of patients from whom samples are collected, the need to critically examine these present methods is invaluable. This review thus presents current advances in the detection of EBV, detailing the advantages and disadvantages of the various techniques. In addition, fundamental virological concepts are highlighted to enhance the greater understanding, the proper application, and the interpretation of EBV tests.

Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation of Burkholderia pseudomallei and Leptospira spp. DNA.

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.

Development and validation of pan-Leptospira Taqman qPCR for the detection of Leptospira spp. in clinical specimens.

BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely. METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients. RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri. CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.

Isolation of Leptospira kmetyi from residential areas of patients with leptospirosis in Kelantan, Malaysia.

BACKGROUND: Environmental sampling provides important information that enhances the understanding of the leptospiral human-environment-animal relationship. Several studies have described the distribution of Leptospira in the environment. However, more targeted sites, that is, areas surrounding leptospirosis patients' houses, remain under-explored. Therefore, this study aims to detect the presence of Leptospira spp. in the residential areas of patients with leptospirosis. METHODS: Soil and water samples near leptospirosis patients' residences were collected, processed and cultured into EMJH media. Partial 16S rRNA gene sequencing was performed to confirm the identity of Leptospira. RESULTS: EMJH culture and partial 16S rRNA gene sequencing revealed predominant growth of pathogenic Leptospira kmetyi (17%, n=7/42). All tested locations had at least one Leptospira sp., mostly from the soil samples. CONCLUSION: More than one species of Leptospira may be present in a sampling area. The most common environmental isolates were pathogenic L. kmetyi.

Antibacterial activity and toxicity of Duckweed, Lemna minor L. (Arales: Lemnaceae) from Malaysia

Aims@#New therapeutics are needed to ease the prevailing waterborne disease, and one of the alternatives is by exploring the natural compounds with antimicrobial properties. Duckweed, Lemna sp. is recorded as a medicinal herb that known to have antifungal and antibacterial activities towards several fungi and bacteria. Suitability of duckweed (Lemna minor) as an antibacterial resource against selected waterborne bacteria were evaluated in terms of its antibacterial activity and toxicity.@*Methodology and results@# Antibacterial activity of the duckweed methanolic extract was tested against 11 selected waterborne bacteria using disc diffusion, minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) assay. Brine shrimp lethality assay was used to determine the toxicity of this extract. The lethal concentrations of plant extract resulting in 50% mortality of the brine shrimp (LC50) were then determined.@*Conclusion, significance and impact of study@#Results showed that duckweed extract exhibited bacteriostatic and bactericidal against the selected bacteria activity at the concentration of MIC = 1.8-2.0 mg/mL and MBC ≥ 2.0 mg/mL. This study shows that methanolic extract of L. minor may contain bioactive compounds against bacteria and potential therapeutic effect. The crude extract is slightly toxic and may not safe to be used in high concentration but is valuable in further study as a potential antitumor agent.

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae.

Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

Molecular detection of leptospirosis and melioidosis co-infection: A case report.

Leptospirosis and melioidosis are important tropical infections caused by Leptospira and Burkholdheria pseudomallei, respectively. As both infections share similar clinical manifestations yet require different managements, complementary laboratory tests are crucial for the diagnosis. We describe a case of Leptospira and B. pseudomallei co-infection in a diabetic 40-year-old woman with history of visit to a freshwater camping site in northern Malaysia. To our knowledge, this is the first case of such double-infection, simultaneously demonstrated by molecular approach. This case highlights the possibility of leptospirosis and melioidosis co-infections and their underlying challenges in the rapid and accurate detection of the etiologic microorganism.

Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii.

The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n = 1/33, and L. licerasiae, n = 7/33) and nonpathogenic leptospire, L. meyeri (n = 22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors.

A shelf-stable fluorogenic isothermal amplification assay for the detection of Burkholderia pseudomallei.

A shelf-stable loop-mediated isothermal amplification (LAMP) reagent for Burkholderia pseudomallei detection is described. The coupling of LAMP reagents with the indirect colorimetric indicator and consequently its lyophilization enable the simple evaluation of results without the need for any advance laboratory instruments. The reagents were found to have a stable shelf life of at least 30 days with well-maintained sensitivity and specificity.

A pentaplex PCR assay for the detection and differentiation of Shigella species.

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.