Biomedical subjects

Henk D F H Schallig

Publications and source records attributed to Henk D F H Schallig.

10 recordsLinked to original sources

PubMed2006

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification.

BACKGROUND: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/mul) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/microl. METHODS: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. RESULTS: The lower detection limit of the assay is 100-1000 molecules in vitro RNA for all species, which corresponds to 0.01-0.1 parasite per diagnostic sample (i.e. 50 microl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. CONCLUSION: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 microl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies.

Animals↗

PubMed2005

Application of direct agglutination test (DAT) and fast agglutination screening test (FAST) for sero-diagnosis of visceral leishmaniasis in endemic area of Minas Gerais, Brazil.

BACKGROUND: The direct agglutination test (DAT) has proved to be a very important sero-diagnostic tool combining high levels of intrinsic validity and ease of performance. Otherwise, fast agglutination screening test (FAST) utilises only one serum dilution making the test very suitable for the screening of large populations. RESULTS: We have tested FAST and DAT for the detection anti-Leishmania antibodies in serum samples from patients with American visceral (AVL) and cutaneous leishmaniases (ACL) in Minas Gerais State, Brazil. The DAT on serum and blood samples of confirmed AVL patients found all samples positive at a serum dilution of > or = 1:800. This dilution was subsequently used as cut off value in the study. The blood and serum samples of these confirmed patients could also be clearly read in FAST using a 1:100 dilution with the same high sensitivity. DAT and FAST were not able to detect significant amounts of antibodies in samples from ACL patients and are not suitable for the diagnosis of this manifestation of the disease. CONCLUSION: We suggest that both DAT and FAST are very practical diagnostic tools for the sero-diagnosis of AVL under rural conditions as both serological tests do not require sophisticated equipment, a cold chain and are very simple to perform.

Journal Article↗

PubMed2005

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples.

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.

Adolescent↗

PubMed2004

Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijó (Alto Douro, Portugal).

Visceral leishmaniosis caused by Leishmania infantum is a zoonotic disease in the Mediterranean basin. We report an epidemiological survey carried out in dogs from the municipality of Alijó in the endemic region of Alto Douro (north Portugal). Performance of the direct agglutination test (DAT) was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; k = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from other dogs of the same municipality. The detected sero-prevalence was 18.7% (288/1540), with values ranging from 0.0 to 81.1% in each of the 19 parishes of Alijó. Three distinct geographical zones of mean sero-prevalence could be defined: northwestern (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) sero-prevalences (P = 0.560). Dogs of 9-11 years of age showed the highest sero-prevalence (28.4%), but all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values (15.0-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine Leishmania infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of canine leishmaniosis and the overall prevalence of disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high sero-prevalence.

Age Factors↗

PubMed2004

Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST).

Leishmaniasis caused by Leishmania infantum is a prevalent disease in dogs and humans. A serological survey of Leishmania infection in dogs was carried out in the endemic region of Alto Douro (north Portugal). Two hundred and ninety-four dogs from the municipality of Peso da Régua were examined for clinical signs of canine leishmaniasis (CanL), and sera samples were evaluated by the direct agglutination test (DAT) and the fast agglutination screening test (FAST). The sero-prevalence of infection was 20.4%, after screening the study population by FAST and subsequent confirmation by DAT. The overall prevalence of disease was 3.1%. Only 15.0% of the sero-positive dogs had clinical signs of CanL. A high degree of agreement (88.4%; kappa value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test for canine Leishmania infection in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of CanL.

Agglutination Tests↗

PubMed2004

Evaluation of the direct agglutination test based on freeze-dried Leishmania donovani promastigotes for the serodiagnosis of visceral leishmaniasis in Sudanese patients.

The direct agglutination test (DAT) based on freeze-dried (FD) Leishmania donovani antigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%; r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%; r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL.

Agglutination Tests↗

PubMed2004

Development of a dipstick assay for detection of Leishmania-specific canine antibodies.

A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; kappa value, 0.86), between the dipstick test and FAST (91.8%; kappa value, 0.82), and between the DAT and FAST (95.2%; kappa value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys.

Agglutination Tests↗

PubMed2003

PCR diagnosis and characterization of Leishmania in local and imported clinical samples.

Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides.

Animals↗

PubMed2002

Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification.

Molecular biology is increasingly relevant to the diagnosis and control of infectious diseases. Information on DNA sequences has been extensively exploited for the development of polymerase chain reaction-based assays for the diagnosis of leishmaniasis and the identification of parasite species. It has also led to the use of cloned antigen for serodiagnosis. It is expected that the sequencing of the Leishmania major genome and the genomes of other Leishmania species will enable important progress in further improving diagnosis and control. The ability to use genome data to clone and sequence genes, which, when expressed, provide antigens for vaccine development, will increase the possibilities for rational vaccine development. Moreover, DNA on its own will provide the basis for the development of DNA vaccines that may overcome some of the problems encountered with protein-based vaccines. One of the greatest threats to parasite control is the development of drug resistance in parasites. Knowing the molecular basis of drug resistance and the ability to monitor its development with sensitive and specific DNA-based assays for 'resistance alleles' may aid maintaining the effectiveness of available anti-Leishmania drugs. Finally, techniques such as microarrays and nucleic acid sequence-based amplification will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies.

Animals↗

PubMed2002

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis.

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.

Agglutination Tests↗