Multiplex real-time PCR revealed very high prevalence of soil-transmitted helminth infections among aborigines in Peninsular Malaysia

Objective: To determine the true prevalence of soil-transmitted helminth infections in the Malaysian aborigines using real-time PCR. Methods: A total of 122 aborigines from seven tribes were recruited from settlements and nearby hospitals which served the communities, located in four states in Peninsular Malaysia. The stool samples were examined for the presence of soil-transmitted helminth using real-time PCR and microscopy. The latter included the direct wet mount and formalin-ether concentration technique (FECT). The infection load in FECT-positive samples was determined by the Kato-Katz method. Rotorgene real-time analyzer detected five helminth species using two sets of assays. Results: The real-time PCR detected soil-transmitted helminth in 98.4% samples (n=122), which were 1.56 times higher than by microscopy. Ascaris lumbricoides and Trichuris trichiura were detected in more than 90% of the samples, while hookworm was detected in 46.7% (Necator americanus) and 13.9% (Ancylostoma sp.) of the samples. Comparison with previous reports on the Malaysian aborigines showed that the real-time PCR markedly improved the detection of Ascaris lumbricoides, hookworm and Strongyloides stercoralis. The real-time PCR detected poly-helminths in 92.6% of the samples compared to 28.7% by microscopy. In addition, 27 samples (22.1%) showed amplification of Strongyloides stercoralis DNA. Conclusions: The real-time PCR showed very high prevalence rates of soil-transmitted helminth infections in the aborigines and is the recommended method for epidemiological investigation of soil-transmitted helminth infections in this population.

Epidemiology and immunodiagnostics of Strongyloides stercoralis infections among migrant workers in Malaysia

To investigate the status of Strongyloides(S.) stercoralis infections among migrant workers in Malaysia for the first time and identify risk factors. Methods: Four diagnostic methods were employed for the detection of S. stercoralis including microscopy, enzyme-linked immunosorbent assay (ELISA) using a commercial kit, ELISA using the rSs1a antigen and polymerase chain reaction (PCR). Low and semi-skilled workers from five working sectors (i.e. manufacturing, food service, agriculture and plantation, construction and domestic service) were tested on a voluntary basis. Results: The overall seroprevalence of S. stercoralis from 483 workers employing the ELISA commercial kit for IgG was 35.8% (n=173; 95% CI: 31.5%-40.1%) whereas seroprevalence using the rSs1a-ELISA was 13.0% (n=63; 95% CI: 10.0%-16.0%). Cross tabulation between the ELISA commercial kit and rSs1a-ELISA showed that only 6.4% (n=31; 95% CI: 4.2%-8.6%) of the samples were positive in both tests. Microscopic examination of all 388 fecal samples were negative; however subsequent testing by a nested PCR against DNA from the same samples successfully amplified DNA from three male subjects (0.8%; 3/388). Male workers, India and Myanmar nationality, food service occupation and those living in the hostel were statistically significant with seroprevalence (P<0.005). Conclusion: This is the first report on the epidemiology of S. stercoralis infections among the migrant workers in Malaysia. Our results highlight the importance of using appropriate diagnostic tools for detection. The presence of anti-S. stercoralis antibodies in the study population calls for improvements in personal hygiene and sanitation standards among migrant workers in Malaysia through control strategies including health education campaigns and programs aimed at increasing awareness and healthy behaviors.

Detection of Strongyloides stercoralis infection among cancer patients in a major hospital in Kelantan, Malaysia

<p><b>INTRODUCTION</b>Strongyloidiasis is one of the most commonly neglected but clinically important parasitic infections worldwide, especially among immunocompromised patients. Evidence of infection among immunocompromised patients in Malaysia is, however, lacking. In this study, microscopy, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs) were used to detect Strongyloides stercoralis (S. stercoralis) infection among cancer patients in a Malaysian hospital.</p><p><b>METHODS</b>A total of 192 stool and serum samples were collected from cancer patients who were receiving chemotherapy with or without steroid treatment at a hospital in northeastern Malaysia. Stool samples were examined for S. stercoralis using parasitological methods and real-time PCR. Serology by ELISA was performed to detect parasite-specific immunoglobulin G (IgG), IgG4 and immunoglobulin E (IgE) antibodies. For comparison, IgG4- and IgG-ELISAs were also performed on the sera of 150 healthy individuals from the same area.</p><p><b>RESULTS</b>Of the 192 samples examined, 1 (0.5%) sample was positive for S. stercoralis by microscopy, 3 (1.6%) by real-time PCR, 8 (4.2%) by IgG-ELISA, 6 (3.1%) by IgG4-ELISA, and none was positive by IgE-ELISA. In comparison, healthy blood donors had significantly lower prevalence of parasite-specific IgG (2.67%, p < 0.05) and IgG4 (2.67%, p < 0.05) responses.</p><p><b>CONCLUSION</b>This study showed that laboratory testing may be considered as a diagnostic investigation for S. stercoralis among immunocompromised cancer patients.</p>

Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess

<p><b>OBJECTIVE</b>To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters.</p><p><b>METHODS</b>Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.</p><p><b>RESULTS</b>A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively.</p><p><b>CONCLUSIONS</b>This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.</p>

Entamoeba histolytica acetyl-CoA synthetase: Biomarker of acute amoebic liver abscess

Objective: To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Methods: Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. Results: A ~75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. Conclusions: This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

Proteomics Technology – A Powerful Tool for the Biomedical Scientists

“Proteomics” refers to the systematic analysis of proteins. It complements other “omics” technologies such as genomics and transcriptomics in elucidating the identity of proteins of an organism, and understanding their functions. Proteomics is used in many areas of research such as discovery of markers for diagnosis and vaccine candidates, understanding pathogenic mechanisms, in the study of expression patterns at different time points and in response to different stimuli, and in elucidating functional protein networks. Proteomics analysis involves sample preparation, protein separation and protein identification. The ‘heart’ of current proteomics is mass-spectrometry, with LC-MS/MS and MALDI-TOF/TOF being commonly used equipment. However, the high costs of the equipment, software, databases and the need for skilled personnel limit the wide utilization of this technology in the less developed countries. Therefore, there need to be sharing of facilities, better networking and collaborations among our scientists and laboratories to take advantage of this powerful technology.

Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess: comparison of three staining methods

<p><b>OBJECTIVE</b>To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.</p><p><b>METHODS</b>Amoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite.</p><p><b>RESULTS</b>The three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues.</p><p><b>CONCLUSIONS</b>It can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.</p>

Symptomatic chronic strongyloidiasis in children following treatment for solid organ malignancies : case reports and literature review

Strongyloidiasis is an infection caused by the intestinal nematode Strongyloides stercoralis. Infected healthy individuals are usually asymptomatic, however it is potentially fatal in immunocompromised hosts due to its capacity to cause an overwhelming hyperinfection. Strongyloidiasis could be missed during routine screening because of low and intermittent larval output in stool and variable manifestations of the symptoms. We present two cases of strongyloidiasis occurring in children with solid organ malignancies suspected to have the infection based on their clinical conditions and treatment history for cancer. Both patients were diagnosed by molecular and serological tests and were successfully treated. Thus, strongyloidiasis in patients undergoing intensive treatment for malignancies should be suspected, properly investigated and treated accordingly.