6 research outputs found
Resistance sniffer : an online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data
The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct
treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the
rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the
problems that are associated with current phenotypic drug susceptibility testing. However, the application of
WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the
technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic
application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms
associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs
for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic
polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.
ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial
genome datasets in different file formats including raw Illumina fastq read files. The program was validated on
sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB
Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for
probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.The South African National Research Foundation (NRF)https://www.elsevier.com/locate/ijmmam2020BiochemistryGeneticsMicrobiology and Plant Patholog
Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle
Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and
Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas
difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested
polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase
chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in
South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by
the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of
A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay
detected more A. marginale- and A. centrale-positive samples than the other assays, either as
single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement
for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale
using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region
of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field
samples. The results indicated sequence variation in the internal forward primer (AM100) area
amongst the South African A. marginale msp1β sequences, resulting in false negatives. We
propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the
benefits of quantification and multiplex detection of both Anaplasma spp.The
National Research Foundation (NRF) of South Africa
(grant number 81840 awarded to Dr Nicola Collins) and
Technology Innovation Agency (TIA), Tshwane Animal
Health Cluster (grant TAHC12-00037 awarded to Professor
Marinda Oosthuizen).http://www.ojvr.org/am2017GeneticsVeterinary Tropical Disease
Recommended from our members
In-vitro bioactivity of fractions from a local medicinal plant on HIV-1 replication, and selected fungal and bacterial pathogens
MSc (Microbiology)Department of MicrobiologySee the attached abstract below
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.This research was funded by National Research Foundation.Peer Reviewe
Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity
BACKGROUND : Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in
South Africa, and no studies have comprehensively examined these species across the whole country. To undertake
this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but
found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field
samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced
from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α
genotype to gauge strain diversity.
RESULTS : Although the A. marginale msp1β gene is variable, the qPCR functions at an acceptable efficiency. The A.
centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay,
57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the
cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous
studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can
be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two
South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa.
CONCLUSIONS : Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and
absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent
in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A.
centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse
population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across
the country, and others appearing only once in one province. This diversity should be taken into account in future
vaccine development studies.The National Research Foundation (NRF) (Nicola Collins, grant number 81840); Technology Innovation Agency, Tshwane Animal Health Cluster (TIA, TAHC) (Marinda Oosthuizen, grant number TAHC12-00037).http://www.parasitesandvectors.comam2018Forestry and Agricultural Biotechnology Institute (FABI)Veterinary Tropical Disease
Comparison of three nucleic acid-based tests for detecting <i>Anaplasma marginale</i> and <i>Anaplasma centrale</i> in cattle
Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp