Rapid and sensitive detection of Mycobacterium ulcerans by use of a loop-mediated isothermal amplification test.

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B.

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25min at 63 degrees C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83bp and 89bp sized bands and the LAMP product melt curves showed consistent melting temperature (T(m)) of approximately 89 degrees C. The assay analytical sensitivity is approximately 0.1tryps/ml while that of classical PCR test targeting the same gene is approximately 10tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.

The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta, Canada, and observations on some cohabiting parasites.

Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5-25% of the two groups of animals studied. Giardia (12.5-21.7%) and Cryptosporidium (0-17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.

Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea.

Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers). Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

Loop-Mediated Isothermal Amplification Test for Trypanosoma gambiense Group 1 with Stem Primers: A Molecular Xenomonitoring Test for Sleeping Sickness.

The World Health Organization has targeted Human African Trypanosomiasis (HAT) for elimination by 2020 with zero incidence by 2030. To achieve and sustain this goal, accurate and easy-to-deploy diagnostic tests for Gambian trypanosomiasis which accounts for over 98% of reported cases will play a crucial role. Most needed will be tools for surveillance of pathogen in vectors (xenomonitoring) since population screening tests are readily available. The development of new tests is expensive and takes a long time while incremental improvement of existing technologies that have potential for xenomonitoring may offer a shorter pathway to tools for HAT surveillance. We have investigated the effect of including a second set of reaction accelerating primers (stem primers) to the standard T. brucei gambiense LAMP test format. The new test format was analyzed with and without outer primers. Amplification was carried out using Rotorgene 6000 and the portable ESE Quant amplification unit capable of real-time data output. The stem LAMP formats indicated shorter time to results (~8 min), were 10-100-fold more sensitive, and indicated higher diagnostic sensitivity and accuracy compared to the standard LAMP test. It was possible to confirm the predicted product using ESE melt curves demonstrating the potential of combining LAMP and real-time technologies as possible tool for HAT molecular xenomonitoring.

Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

BACKGROUND: Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. METHODS: In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. RESULTS: The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10-7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10-5 (~3 ng/ml) and 10-4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. CONCLUSION: The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

Detection of trypanosomes in small ruminants and pigs in western Kenya: important reservoirs in the epidemiology of sleeping sickness?

BACKGROUND: Trypanosomosis is a major impediment to livestock farming in sub-Saharan Africa and limits the full potential of agricultural development in the 36 countries where it is endemic. In man, sleeping sickness is fatal if untreated and causes severe morbidity. This study was undertaken in western Kenya, an area that is endemic for both human and livestock trypanosomosis. While trypanosomosis in livestock is present at high levels of endemicity, sleeping sickness occurs at low levels over long periods, interspersed with epidemics, underscoring the complexity of the disease epidemiology. In this study, we sought to investigate the prevalence of trypanosomes in small ruminants and pigs, and the potential of these livestock as reservoirs of potentially human-infective trypanosomes. The study was undertaken in 5 villages, to address two key questions: i) are small ruminants and pigs important in the transmission dynamics of trypanosomosis? and ii), do they harbour potentially human infective trypanosomes? Answers to these questions are important in developing strategies for the control of both livestock and human trypanosomosis. RESULTS: Eighty-six animals, representing 21.3% of the 402 sampled in the 5 villages, were detected as positive by PCR using a panel of primers that identify trypanosomes to the level of the species and sub-species. These were categorised as 23 (5.7%) infections of T. vivax, 22 (5.5%) of T. simiae, 21 (5.2%) of the T. congolense clade and 20 (5.0%) of T. brucei ssp. The sheep was more susceptible to trypanosome infection as compared to goats and pigs. The 20 T. brucei positive samples were evaluated by PCR for the presence of the Serum Resistance Associated (SRA) gene, which has been linked to human infectivity in T. b. rhodesiense. Three samples (one pig, one sheep and one goat) were found to have the SRA gene. These results suggest that sheep, goats and pigs, which are kept alongside cattle, may harbour human-infective trypanosomes. CONCLUSION: We conclude that all livestock kept in this T. b. rhodesiense endemic area acquire natural infections of trypanosomes, and are therefore important in the transmission cycle. Sheep, goats and pigs harbour trypanosomes that are potentially infective to man. Hence, the control of trypanosomosis in these livestock is essential to the success of any strategy to control the disease in man and livestock.

The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR.

The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.

Towards Point-of-Care Diagnostic and Staging Tools for Human African Trypanosomiaisis.

Human African trypanosomiasis is a debilitating disease prevalent in rural sub-Saharan Africa. Control of this disease almost exclusively relies on chemotherapy that should be driven by accurate diagnosis, given the unacceptable toxicity of the few available drugs. Unfortunately, the available diagnostics are characterised by low sensitivities due to the inherent low parasitaemia in natural infections. Demonstration of the trypanosomes in body fluids, which is a prerequisite before treatment, often follows complex algorithms. In this paper, we review the available diagnostics and explore recent advances towards development of novel point-of-care diagnostic tests.

Rapid and sensitive detection of human African trypanosomiasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick.

A combined loop-mediated isothermal amplification lateral flow dipstick (LAMP-LFD) format was evaluated in the detection of human infective trypanosome DNA from clinical samples. The LAMP-LFD showed analytical sensitivity equivalent to 0.01 tryps/mL, levels that were identical to using gel electrophoresis and SYBR Green I dye. The LAMP-LFD showed superior specificity to SYBR Green I when supernatant prepared from boiled human biological samples was used as template. These results indicate that the use of nonspecific DNA intercalators may produce false positives when partially processed templates are used. The LAMP-LFD format presented here is simple, rapid, and has future potential use in diagnosis of sleeping sickness.

Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense.

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.