Rahmah Noordin

A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths.

Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs.

Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial

The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs.

Recombinant proteins in the diagnosis of toxoplasmosis

Kotresha D, Rahmah N. Recombinant proteins in the diagnosis of toxoplasmosis. APMIS 2010; 118: 529–42.

Transmission assessment surveys (TAS) to define endpoints for lymphatic filariasis mass drug administration: a multicenter evaluation.

Background Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings. Methodology The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6–7 year olds or 1st–2nd graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs. Principal Findings/Conclusions In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation.

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.

Application of Real-Time Polymerase Chain Reaction in Detection of Entamoeba histolytica in Pus Aspirates of Liver Abscess Patients

Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.

In vitro Anti-Toxoplasma gondii Activity of Root Extract/Fractions of Eurycoma longifolia Jack

BackgroundToxoplasma gondii infection causes toxoplasmosis, an infectious disease with worldwide prevalence. The limited efficiency of drugs against this infection, their side effects and the potential appearance of resistant strains make the search of novel drugs an essential need. We examined Eurycoma longifolia root extract and fractions as potential sources of new compounds with high activity and low toxicity. The main goal of this study was to investigate the anti-T. gondii activity of crude extract (TACME) and four fractions (TAF 273, TAF 355, TAF 191 and TAF 401) from E. longifolia, with clindamycin as the positive control.MethodsIn vitro toxoplasmacidal evaluation was performed using Vero cells as host for T. gondii. Light microscopy technique was used to study in situ antiparasitic activity.ResultsSignificant anti-T. gondii activity was observed with clindamycin (EC50 = 0.016 μg/ml), follow by TAF 355 (EC50 = 0.369 μg/ml) and TAF 401 (EC50 = 0.882 μg/ml). Light microscopy revealed that most Vero cells were infected after 3 h of exposure to T. gondii. After 36 h of exposure to the E. longifolia fraction, the host Vero cells showed no visible intracellular parasite and no remarkable morphological changes.ConclusionsOur study demonstrated that TAF 355 and TAF401 fractions may be the sources of new anti-T. gondii compounds.

Impact of Six Rounds of Mass Drug Administration on Brugian Filariasis and Soil-Transmitted Helminth Infections in Eastern Indonesia

Background The lymphatic filarial parasite Brugia timori occurs only in eastern Indonesia where it causes high morbidity. The absence of an animal reservoir, the inefficient transmission by Anopheles mosquitoes and the high sensitivity to DEC/albendazole treatment make this species a prime candidate for elimination by mass drug administration (MDA). Methodology/Principal Findings We evaluated the effect of MDA using DEC and albendazole on B. timori and soil transmitted helminths (STH) in a cross-sectional study of a sentinel village on Alor Island annually over a period of 10 years. Pre-MDA the microfilaria (MF) prevalence was 26% and 80% of the residents had filaria-specific IgG4 antibodies. In 2010, 34 months after the 6th round of MDA, MF and antibody rates were only 0.17% and 6.4%, respectively. The MDA campaign had also a beneficial effect on STH. Baseline prevalence rates for Ascaris, hookworm and Trichuris were 34%, 28%, and 11%, respectively; these rates were reduced to 27%, 4%, and 2% one year after the 5th round of MDA. Unfortunately, STH rates rebounded 34 months after cessation of MDA and approached pre-MDA rates. However, the intensity of STH infection in 2009 was still reduced, and no heavy infections were detected. Conclusions/Significance MDA with DEC/albendazole has had a major impact on B. timori MF and IgG4 antibody rates, providing a proof of principle that elimination is feasible. We also documented the value of annual DEC/albendazole as a mass de-worming intervention and the importance of continuing some form of STH control after cessation of MDA for filariasis.

Pan LF-ELISA using BmR1 and BmSXP recombinant antigens for detection of lymphatic filariasis

BACKGROUND Anti-filarial IgG4 antibody has been shown to be a good marker for detection of lymphatic filaria infection. Previous studies demonstrated that anti-filarial IgG4 assay using BmR1 recombinant antigen was highly specific and sensitive for detection of brugian filariasis. For bancroftian filariasis, an equivalent assay employing recombinant antigen expressed from the ORF of SXP1 gene has been reported. In order to detect infections by all species of lymphatic filarial, BmR1 and BmSXP recombinant antigens were employed in the development of a pan LF-ELISA. METHODS BmR1 was previously produced while BmSXP recombinant antigen was produced by cloning the ORF of SXP1 gene from a Brugia malayi cDNA library, followed by expression in a bacterial expression system. Subsequently, each of the purified recombinant antigens (BmR1 and BmSXP) and mixture of different ratios of the two antigens (1:1, 2:1 and 1:2) were tested using IgG4-ELISA with various categories of infection and normal human serum samples. RESULTS The results showed that both recombinant antigens were highly specific (99%-100%). For detection of brugian filariasis, BmR1 antigen alone and the mixture of BmR1 with BmSXP (1:1) gave 98% sensitivity; while BmSXP antigen alone showed 84% sensitivity. For detection of bancroftian filariasis, BmSXP antigen was more sensitive (95%) than assays using either BmR1 or mixtures of the two recombinant antigens. CONCLUSION A sensitive and specific pan LF-ELISA for detection of lymphatic filariasis was successfully developed using two adjacent wells, each separately coated with BmR1 and BmSXP.

The effects of size and synthesis methods of gold nanoparticle-conjugated MαHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis

This study describes the properties of colloidal gold nanoparticles (AuNPs) with sizes of 20, 30 and 40 nm, which were synthesized using citrate reduction or seeding-growth methods. Likewise, the conjugation of these AuNPs to mouse anti-human IgG(4) (MαHIgG(4)) was evaluated for an immunochromatographic (ICG) strip test to detect brugian filariasis. The morphology of the AuNPs was studied based on the degree of ellipticity (G) of the transmission electron microscopy images. The AuNPs produced using the seeding-growth method showed lower ellipticity (G ≤ 1.11) as compared with the AuNPs synthesized using the citrate reduction method (G ≤ 1.18). Zetasizer analysis showed that the AuNPs that were synthesized using the seeding-growth method were almost monodispersed with a lower polydispersity index (PDI; PDI≤0.079), as compared with the AuNPs synthesized using the citrate reduction method (PDI≤0.177). UV-visible spectroscopic analysis showed a red-shift of the absorbance spectra after the reaction with MαHIgG(4), which indicated that the AuNPs were successfully conjugated. The optimum concentration of the BmR1 recombinant antigen that was immobilized on the surface of the ICG strip on the test line was 1.0 mg ml(-1). When used with the ICG test strip assay and brugian filariasis serum samples, the conjugated AuNPs-MαHIgG(4) synthesized using the seeding-growth method had faster detection times, as compared with the AuNPs synthesized using the citrate reduction method. The 30 nm AuNPs-MαHIgG(4), with an optical density of 4 from the seeding-growth method, demonstrated the best performance for labelling ICG strips because it displayed the best sensitivity and the highest specificity when tested with serum samples from brugian filariasis patients and controls.