Oriel M. M. Thekisoe

Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of Cryptosporidium oocysts in fecal and water samples

ABSTRACT A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.

Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing

Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/microL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.

Comparative Diagnosis of Malaria Infections by Microscopy, Nested PCR, and LAMP in Northern Thailand

Three methods, microscopy, nested polymerase chain reaction (nPCR), and loop-mediated isothermal amplification (LAMP) have been applied for malaria diagnosis in 105 human blood samples collected in Northern Thailand. Only Plasmodium falciparum and Plasmodium vivax infections were detected. A total number of 57 positives (54%) could be detected for P. falciparum and 25 (24%) for P. vivax when all samples that were positive in any of the three methods are counted together. The nPCR was used as a reference standard for comparison with the other methods, microscopy and LAMP. The sensitivity of LAMP for P. falciparum was 100%. All nPCR-negative samples for P. falciparum were also negative by both microscopy and LAMP (specificity, 100%). For diagnosis of P. vivax, microscopy detected 15 of 23 nPCR-positive samples (sensitivity, 65%). LAMP detected 22 of 23 nPCR-positives (sensitivity, 96%). Among the 82 nPCR-negative samples microscopy detected two samples (specificity, 98%). All 82 nPCR-negative were also negative by the LAMP method (specificity, 100%). Both Plasmodium genus- and species-specific LAMP primer sets yielded the same results in all samples. There were no significant differences in the prevalence detected by each method. We assume that LAMP was as reliable as nPCR and more reliable than microscopy in the detection of Plasmodium DNA tested in the examined Thai field blood samples. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of malaria cases and together with nPCR can also be used as supplementary methods for clinical and epidemiological use.

Prevalence of Trypanosoma sp. in cattle from Tanzania estimated by conventional PCR and loop-mediated isothermal amplification (LAMP)

This study compared the prevalence of trypanosome infections estimated by PFR-loop-mediated isothermal amplification (LAMP) with conventional polymerase chain reaction (PCR) tests. One hundred forty eight cattle blood samples were collected from Robanda village, Mara region, Tanzania in April 2008. In conventional PCR, four sets of primers, specific for the detection of Trypanosoma sp., Trypanosoma brucei rhodesiense, Trypanosoma vivax, and Trypanozoon, as well as a modified LAMP were used. Conventional PCR detected no infection or up to 8, 1, and 3 infections with Trypanosoma congolense savannah, Trypanozoon, and T. vivax, respectively, whereas LAMP detected additional 44 Trypanozoon positive cases. Our results clearly indicate that the prevalence of Trypanozoon spp. in cattle in Robanda village estimated by PFR-LAMP (30.4%) was significantly higher than the estimates by PCR assays (0.6–2%). As such, future studies should target epidemiological surveys of Trypanozoon and T. brucei rhodesiense infections in possible reservoir animals by LAMP to further elucidate the actual prevalence of these parasites.

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.

Detection of Trypanosoma cruzi and T. rangeli infections from Rhodnius pallescens bugs by loop-mediated isothermal amplification (LAMP).

We have developed two loop-mediated isothermal amplification (LAMP) assays for specific detection of Trypanosoma cruzi and Trypanosoma rangeli based on the 18S ribosomal RNA (rRNA) and the small nucleolar RNA (snoRNA) genes, respectively. The detection limit of the assays is 100 fg and 1 pg for T. cruzi and T. rangeli, respectively, with reactions conducted in 60 minutes. The two LAMP assays were used in detection of T. cruzi and T. rangeli infections in comparison with polymerase chain reaction (PCR) for DNA samples extracted from Rhodnius pallescens bugs collected from palm trees in Panama. Out of a total of 52 DNA samples from R. pallescens bugs 17 (33%) and 14 (27%) were T. cruzi-positive by LAMP and PCR, respectively, while, 7 (13%) and 4 (8%) were T. rangeli-positive by LAMP and PCR, respectively. Further evaluation of these LAMP assays is needed, especially with specimens collected from human patients as well as blood kept for transfusion purposes.

Epidemiology and evolution of the genetic variability of Anaplasma marginale in South Africa

Bovine anaplasmosis caused by infection of cattle with Anaplasma marginale has been considered to be endemic in South Africa, an assumption based primarily on the distribution of the tick vectors of A. marginale and serological studies on the prevalence of anaplasmosis in Limpopo, Free State, and North West. However, molecular evidence of the distribution of anaplasmosis has only been reported in the Free State province. In order to establish effective control measures for anaplasmosis, epidemiological surveys are needed to define the prevalence and distribution of A. marginale in South Africa. In addition, a proposed control strategy for anaplasmosis is the development of an A. marginale major surface protein 1a (MSP1a)-based vaccine. Nevertheless, regional variations of this gene would need to be characterized prior to vaccine development for South Africa. The objectives of the present study were therefore to conduct a national survey of the prevalence of A. marginale in South Africa, followed by an evaluation of the diversity and evolution of msp1a in South African strains of A. marginale. To accomplish these objectives, species-specific PCR was used to test 250 blood samples from cattle collected from all South African provinces (including 26 districts and municipalities), except the Free State province where similar studies were reported previously. The prevalence of A. marginale ranged from 65% to 100%, except in Northern Cape province where A. marginale was not detected. A correlation was found between the prevalence and genetic diversity of A. marginale MSP1a. Additionally, the genetic diversity of the A. marginale MSP1a was found to evolve under negative and positive selection, and 23 new tandem repeats in South Africa were shown to have evolved from the extant tandem repeat 4. Despite the MSP1a genetic variability, some types of tandem repeats were found to be conserved among the A. marginale strains, and low-variable peptides in MSP1a tandem repeats were subsequently identified. The results of this research confirmed that anaplasmosis is endemic in South Africa. The results of the molecular characterization of the MSP1a can then be used as the basis for development of new and novel vaccines for anaplasmosis control in South Africa.

A trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks in Kagoshima Prefecture, Japan

Common arthropod vectors for trypanosomes are flies, fleas and bugs. This study reports on an unknown trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks, hereby, referred to as Trypanosoma KG1 isolate. The parasite has been successfully cultured in vitro with L929 or HEK 293T cell line as feeder cells. This trypanosome cannot survive in vitro without feeder cells. Following experimental infections of ticks, the trypomastigote-like and the epimastigote-like forms of this trypanosome could be detected by Giemsa-stained smears of the midgut and salivary glands of Ornithodoros moubata ticks which were made to feed on a culturing medium containing Trypanosoma KG1 isolate through an artificial membrane. Trypanosoma KG1 isolate could also be detected from Giemsa-stained smears of the haemolymph up to 30 days post-inoculation into the O. moubata haemocoel. Trypanosoma KG1 isolate cannot be propagated in laboratory animals including mice, rats, rabbits and sheep. A phylogenetic tree constructed with the 18S rRNA gene indicates that Trypanosoma KG1 is a member of the stercorarian trypanosomes.