Suharni Mohamad

Development and Evaluation of a Sensitive and Specific Assay for Diagnosis of Human Toxocariasis by Use of Three Recombinant Antigens (TES-26, TES-30USM, and TES-120)

ABSTRACT Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.

Assessing subtypes and drug resistance mutations among HIV-1 infected children who failed antiretroviral therapy in Kelantan, Malaysia

Antiretroviral (ARV) therapy has dramatically reduced morbidity and mortality in human immunodeficiency virus 1 (HIV-1) infected children. However, development of ARV resistance in these children is a major public health problem due to lack of availability of and access to new drugs. This study was conducted in order to identify circulating HIV subtypes and recombinant forms and evaluate the drug resistance mutation patterns in 18 HIV-1 infected children failing ARV treatment in Kelantan, Malaysia. Genotyping for codon 1-99 of protease (PR) and 1-250 of reverse transcriptase (RT) were performed by polymerase chain reaction (PCR) amplification and DNA sequencing. Subsequently, these were phylogenetically analyzed to determine the subtypes. CRF33_01B (44.4%) was found to be the predominant HIV subtype, followed by B (27.8%), CRF15_01B (16.7%) and CRF01_AE (11.1%) subtypes. The most prevalent RT mutations were T215F/V/Y (66.7%), D67G/N (55.6%), K219Q/E/R (44.4%), M184V/I (38.9%), K70R/E (27.8%) and M41L (27.8%), associated with nucleoside reverse transcriptase inhibitors (NRTI) resistance; and K103N (55.6%), G190A (33.3%), and K101P/E/H (27.8%) associated with non-nucleoside reverse transcriptase inhibitors (NNRTI) resistance. The results showed a possible emergence of CRF33_01B as current predominant subtypes/circulating recombinant forms (CRFs), and a high frequency of primary mutations among HIV-1 infected children after failure of ARV therapy in Kelantan, Malaysia.

Antiretroviral drug resistance and HIV-1 subtypes among treatment-naive prisoners in Kelantan, Malaysia

INTRODUCTION The widespread use of highly active antiretroviral therapy (HAART) and continuous reports of HIV-1 strains developing resistance to these drugs is rather alarming, as transmission of resistant viruses to newly infected persons is possible. This study aimed to determine HIV-1 subtypes and the prevalence of primary mutations associated with antiretroviral (ARV) resistance among treatment-naive prisoners on the east coast of Malaysia. METHODOLOGY Viral RNA was extracted from plasma samples of 21 treatment-naive prisoners. Protease (PR) and reverse transcriptase (RT) regions were amplified and sequenced. Stanford HIV database algorithms were used for interpretation of resistance, and phylogenetic analysis was performed for subtype assignment. RESULTS In the PR gene, no antiviral resistance-associated mutation was detected. For RT-associated mutations, K103N was the most prevalent in sequenced samples (14.3%). Genetic subtyping on the pol gene revealed that the majority of the prisoners were infected with subtype CRF33_01B (52.4%). CONCLUSION Continuous surveillance of newly infected individuals is required to help strategize the best antiviral treatment for these patients.

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.

In-vitro antimicrobial effectiveness of herbal-based mouthrinses against oral microorganisms

ABSTRACT Objective To evaluate the in vitro antimicrobial effectiveness of commercial herbal-based mouthrinses against oral microorganisms. Methods A total of three mouthrinses (OX, Pesona and Watsons) were tested for their antimicrobial activity against six oral organisms, Streptococcus mutans ( S. mutans ), Streptococcus sobrinus ( S. sobrinus ), Lactobacillus salivarius ( L. salivarius ), Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P. aeruginosa ) and Candida albicans ( C. albicans ) by standard agar-disk diffusion assay. Oradex mouthrinse containing 0.12% chlorhexidine gluconate and sterile distilled water was served as positive and negative controls, respectively. Results All mouthrinse formulations were effective in inhibiting the growth of S. mutans, S. sobrinus, L. salivarius and C. albicans . Among the tested mouthrinses, Pesona was the only effective mouthrinse against S. aureus and P. aeruginosa , similar to Oradex mouthrinse. Pesona mouthrinse formulation appears to be as effective as Oradex mouthrinse formulation to kill S. aureus and P. aeruginosa . Statistical analysis showed no significant difference among the tested formulations regarding their antimicrobial activities ( P > 0.05). Conclusions Pesona was not the only herbal mouthrinse effective in inhibiting the growth of S. mutans, S. sobrinus, L. salivarius and C. albicans in vitro . All tested formulations were effective against those strains. Our findings may serve as a guide for selecting a kind of herbal mouthrinses as well as providing information to the dental professionals about the efficacy of these products.