Masters Degrees (Medical Microbiology)

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Open Access
Beta-lactam resistance profiles in urinary tract infection among Escherichia coli isolates in Bloemfontein
(University of the Free State, 2005-05) Maqutu, Lennox Makhelane; De Kock, M. J.
English: This study was designed to elucidate the epidemiology, nature and extent of β-lactam resistance in urinary tract infections caused by Escherichia coli isolates in Bloemfontein hospitals. To reach this goal it was necessary to phenotypically characterise and re-identify the E. coli isolates by the Mastascan identification system. Pure cultures were obtained by streaking single colonies onto MacConkey agar containing 50 µg / ml ampicillin. Single colonies were then picked off and inoculated into Mueller-Hinton broth, grown overnight at 37°C and re-streaked onto MacConkey agar containing 50 µg / ml ampicillin. Three colonies were then picked and stored at -20°C in a freeze mixture of 10 % proteose and 10 % glycerol. E. coli isolates were mated to a universal recipient strain (162) in pre-warmed Mueller- Hinton broth. Transconjugants were selected on MacConkey agar supplemented with 50 µg / ml ampicillin and 50 µg / ml nalidixic acid. Lactose-negative colonies resistant to nalidixic acid and ampicillin were inoculated into Mueller-Hinton broth, incubated for six hours and restreaked onto MacConkey agar containing ampicillin. Lactose-negative colonies were picked and considered to be transconjugants. It was found that fifty four (45 %) out of 120 ampicillin-resistant isolates transferred ampicillin-resistance determinants to an E. coli recipient (162) by conjugation. Seventy-five percent of ampicillin-resistant isolates were from female patients, indicating that urinary tract infections are more prevalent in females than in males. The National Committee for Clinical Laboratory Standards* agar dilution method was used to determine minimum inhibitory concentration (MIC) distributions of 12 β-lactam antimicrobial agents (ampicillin, amoxycillin, piperacillin, augmentin, cefoxitin, cefotaxime, cefepime, ceftazidime, cephazolin, ceftriaxone, cefuroxime and imipenem). Strains found to be resistant had MICs that overlapped the range where susceptibility was normally assumed. This was due to inducible β-lactamase producer strains, which go undetected by the Kirby- Bauer disk diffusion method but can be identified by using the Jarlier double-disk method. MIC frequency distributions for the penicillins showed that elevated doses should be administered in order to maximise antibiotic concentration at the site of infection or that a second antibiotic agent or inhibitor should be used in combination therapy. Beta-laetam susceptibility profiles were determined by the Kirby-Bauer disk diffusion method. This method was also used to determine the correlation of MIC values with the inhibition zone diameters in order to predict treatment outcomes from inhibition zone diameters. The Jarlier double-disk technique was used to detect extended-spectrum β-lactamaseproducing organisms and the frequencies at which they occurred. There was no difference between the ratios of ESBL-producers in hospitalised and non-hospitalised patients, although the absolute numbers were different. This was probably due to the 48 hour cut-off point used to define hospitalisation. Samples taken before 48 hours were considered to be nonhospitalised. There were many more female than male patients with urinary tract infections in the Bloemfontein hospitals during the period of the study. The extent of joint resistance to β-lactam antibiotics among E. coli isolates was assessed by comparing two agents at a time. The observed incidence of joint resistance was compared to the rate of double resistance expected if it had been acquired as two independent events. It was found that even amongst two antibiotics that are biologically cross-resistant (ampicillin and augmentin) a close correlation exists between the concentrations of the two agents required to inhibit individual E. coli strains.
Open Access
Characterization of T cell responses to the non-structural proteins of the M segment in survivors of Crimean-Congo haemorrhagic fever
(University of the Free State, 2019) Maotoana, Makgotso Golda; Goedhals, Dominique; Burt, Felicity Jane
Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is one of the most widely distributed arboviruses globally. The disease caused by the virus, Crimean- Congo haemorrhagic fever (CCHF), continues to emerge and re-emerge across the globe. There are currently various vaccines under development for CCHF prevention. The non-structural M protein (NSM), GP38, highly variable mucin-like domain and N-terminus of GC regions in CCHFV have proved to be immunogenic in vaccine studies. Furthermore, both arms of the immune system have been found to be fundamental for protection in mice. However, there is limited information about immunity in patients following natural infection. The aim of the study was to characterize T cell immune responses against the NSM, GP38 and the highly variable mucin-like domain of CCHFV in survivors of CCHF. This was achieved by first identifying immunogenic peptides in the regions of interest and determining the amino acid conservation of the identified peptides. An overlapping peptide library spanning the NSM, GP38 and highly variable mucin-like domain was designed using the South African CCHFV isolate SPU 103/87. The secretion of interferon-gamma (IFN-γ) by peripheral blood mononuclear cells isolated from 12 participants was screened using 24 peptide pools in an IFN-γ enzyme linked immunospot (ELISpot) assay. IFN-γ secretion was detected in eight of the twelve participants. Two participants showed no detectable IFN-γ responses to any of the peptide pools, and another two were excluded from the analysis due to a high background in the negative controls indicating non-specific reactivity. Nine peptides were identified with the IFN-γ ELISpot, including five peptides in the GP38 region and four in the NSM region, thus confirming the immunogenic potential of these regions during natural infection. No immunogenic peptides were identified in the highly variable mucin-like domain, which is possibly because of the high genetic diversity in the region. The identified immunogenic peptides were used to stimulate T cells of participants and a flow cytometry assay was performed to characterize the immune responses, with the focus on detecting the presence of the T cell memory subsets, the expression of CD107a, which is a cytotoxic marker, and the secretion of IFN-γ and tumour necrosis factor-alpha (TNF-α), which are antiviral cytokines. Cytotoxic CD8+ T cells were detected in six participants in response to nine peptides. IFN-γ and TNF-α secretion within the CD4 and CD8 populations were comparable; thus, highlighting the ability of the CD8+ T cell population to secrete antiviral cytokines, even though the population is known to be predominantly cytotoxic. The secretion of IFN-γ was more frequent than TNF-α secretion in both the CD4 and CD8 populations. Polyfunctional T cells were detected with the phenotypes IFN- γ+CD107a+ and IFN-γ+ TNF-α+, in both the CD4 and CD8 populations. Therefore, the results indicate the possibility of efficient antiviral responses upon stimulation with viral epitopes in survivors of infection. Heterogeneous functionality of the T cell memory subsets was observed, however the terminally differentiated (TEMRA) subset was the most dominant and abundant, followed by the naïve (TN), effector memory (TEM), with the least abundant being the central memory (TCM) T cell memory subset. The IFN-γ secretion detected with the IFN-γ ELISpot and the flow cytometry assay was used as basis for comparing the sensitivity of the two techniques. The IFN-γ ELISpot proved to be comparable to the flow cytometry assay. The ELISpot is suited for screening purposes, while the flow cytometry allowed further characterization of the T cell responses. Therefore, it is recommended that these complementary assays be used in combination. In conclusion, T cell epitopes were identified in the NSM and GP38 regions of CCHFV. Polyfunctional T cells were found in both the CD4 and CD8 populations, thus suggesting the presence of effective long-term memory T cells responses in survivors of CCHF.
Open Access
Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
(University of the Free State, 2021) Dimaculangan, Micah; Burt, Felicity Jane
Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, taking into account the transcontinental transmission of the virus between South Africa and northern Europe by migratory birds. The conventional nested RT-PCR assay may be able to detect most alphaviruses due to minimal mismatches (0 – 1) detected between the primers and the partial nsP4 sequences of the alphavirus isolates, while the RT-HDA assay may not be well suited to detect other alphaviruses due to the many mismatches (>4) detected between the primers and the partial nsP4 sequences of the alphavirus isolates. Nevertheless, this shows that the RT-HDA is theoretically more specific that the conventional nested RT-PCR assay. The RT-HDA however failed to detect any alphaviruses in the 42 mosquito pools tested, which was not unexpected as the assay could only detect up to 4.8 x 105 copies of RNA. In contrast, the conventional nested RT-PCR assay was able to detect alphaviral RNA in five out of the 42 mosquito pools tested, and the nucleotide sequences were determined to identify the alphavirus species. SINV RNA was detected in three mosquito pools and Middelburg virus (MIDV) was detected in two pools. Phylogenetic analysis was subsequently performed to determine the genetic relationship of these isolates from the Free State with previously published/ reported SINV and MIDV isolates in South Africa, Africa, and around the world. The conventional nested RT-PCR assay developed in this study has shown to be a useful surveillance tool for the detection of mosquito-borne alphavirus infections. Given the low sensitivity determined for the RT-HDA assay, improvements, or alternative rapid and fieldable NAATs should be considered in the future for alphavirus surveillance applications in low resource settings.
Open Access
The development of a method for the detection and estimation of CCHF virus RNA in tick species
(University of the Free State, 2000-05) Du Preez, Patrick Hendrik; Pretorius, G. H. J.; Janse van Rensburg, M. N.
Crimean Congo haemorrhagic fever (CCHF), caused by a RNA virus, is a tick-borne viral zoonosis occurring in Europe, Asia and Africa. The fatality rate is ±30%. Rapid and accurate diagnosis is essential. The aim of this study was to develop a reverse transcription-polymerase chain reaction (RT-PCR) with internal control for the detection of CCHF RNA. Primers were selected for a region in the nucleocapsid - gene of the S segment. The internal control was constructed by ligating this PCR product into a pGEMEX-1 vector. Sequencing of the PCR product (381 bp) revealed two unique restriction sites, BIn I and BstE II which were used to delete a fragment of 59 bp. The shortened PCR-product was re-inserted into E. coli. T3 RNA polymerase produced plasmid derived RNA (322 bp) was used to spike specimens. Standard RT-PCR was then performed. The minimum concentration of target RNA the RTPCR can detect was estimated to be 4 x 10-5 pmol RNA, giving more or less the same sensitivity as the PCR alone. The size difference of 59 bp is enough to distinguish between the full-length and the deletion variant inserts when visualised and therefore provides an internal control. RT-PCR on fifty Hyalomma ticks was negative. The CCHF virus was probably not present or at concentrations below detection level, as RT-PCR of control CCHF virus RNA confirmed the accuracy of the method. RT-PCR allows rapid detection of CCHF virus RNA. The constructed internal control precludes the use of Dugbe virus, an antigenically related nairovirus.
Open Access
Development of detection assays for sindbus virus and investigating in vitro infection of mammalian cells
(University of the Free State, 2013-08) Hanekom, Hermanus Albertus; Burt, F. J.
Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virus has a positive sense RNA genome of 11700 bases which encodes for both structural and non structural proteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria. Laboratory confirmation is essential as SINV infections must be distinguished from various conditions that share similar clinical manifestations. The most frequently used methods for identification are haemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization tests as well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are not readily available commercially and due to the non-specific symptoms caused by SINV infection the number of infections per annum may be under diagnosed. The purpose of this study was to develop serological assays such as ELISA and a novel neutralization assay that could be used in serological surveys for the detection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determine the level of viral replication in mammalian cells for characterizing infection in mammalian cells as well as investigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINV infection. An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitro neutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISA and the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-house ELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity of the in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical density values to differentiate positive and negative samples. A forward and reverse primer for the amplification of a conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with a TaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammalian cells, human macrophages and HeLa cells, was determined by measuring viral loads with a real time quantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strain from Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viral replication but were considered insufficient to warrant further investigation and insufficient to investigate occurrence of antibody dependent enhancement of disease in macrophages. The mechanism possibly interfering with replication of virus in the human macrophages was investigated. Supernatant fluid samples from macrophage infections were tested for the release of interferon gamma which could inhibit viral replication. There were nine to fifteen fold differences in the concentration of 2 interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells were treated with similar concentrations of human interferon gamma at different time intervals. Pretreatment and concurrent treatment with infection showed reduced levels of viral load compared with no treatment or delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viral replication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNA fragments separated through agarose gel electrophoreses. There were multiple bands visible in the infected samples whereas the negative control did not show multiple bands, only one large band of genomic DNA. The presence of multiple DNA fragments in infected cells and absence of those fragments from uninfected cells were suggestive of virus induced apoptosis.
Open Access
Development of molecular and serological assays for diagnosis and surveillance of Crimean-Congo haemorrhagic fever virus
(University of the Free State, 2015-05) Pieters, Danelle; Burt, F. J.; Jansen van Vuren, P.
Crimean-Congo haemorrhagic fever virus (CCHFV) an arthropod-borne virus associated with haemorrhagic disease in humans. The global distribution of CCHFV correlates with that of ticks from the Hyalomma genus. CCHFV infection is diagnosed by detection of viral nucleic acid using reverse-transcription polymerase-chain-reaction (RT-PCR) or other molecular assays, by virus isolation from infected cell culture or suckling mouse brain or by detection of anti-CCHFV antibodies using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA). High biocontainment facilities are required for virus isolation and preparation of whole virus native antigen for use in serological assays. Currently, treatment is limited to supportive therapy. CCHFV is currently emerging and re-emerging in many regions, which emphasize the requirement for safe, reliable and inexpensive assays to increase diagnostic capacity and monitor emergence of the virus. A nucleic acid sequence-based amplification (NASBA) molecular assay for detection of CCHFV ribonucleic acid (RNA) was developed. The assay can be performed without the requirement for sophisticated laboratory equipment. A commercially available enzyme mixture and buffer were compared with a more cost effective and easier to obtain in-house enzyme mixture and amplification buffer. Specificity of the NASBA assays were determined by testing viral RNA extracted from Vero cell culture infected with genetically diverse southern African CCHFV strains. A total of 41/48 samples tested were positive. Sensitivity of the NASBA assays was determined using dilutions of viral RNA and transcribed RNA to detect minimal copy number that could be amplified. The NASBA assay was able to detect at least 3.7 RNA copies. Diagnostic application of the NASBA assays was investigated by amplifying RNA extracted from clinical samples and the results compared with two commercial real-time RT-PCR assays. A total of 20/22 samples tested positive using the NASBA whereas the commercially available assays were able to amplify 22/22 samples. Subsequently, the inhibitory effect of sera on the amplification of CCHFV RNA using the NASBA assay was investigated using sera spiked with transcribed RNA. Two expression systems were investigated for the expression of recombinant CCHFV nucleocapsid protein (NP) for use in serological assays. The baculovirus expression system was initially investigated. The open reading frame of the S segment of a CCHFV strain was codon optimized for expression in insect cells. A pFastBac HT B transfer vector containing the optimized CCHFV NP gene was prepared and used to transform DH10Bac™ Escherichia coli cells to transpose the optimized CCHFV NP gene to a bacmid. The recombinant bacmid was utilized to transfect Spodoptera frugiperda 9 cells. The cell lysates were analysed, however, no expression of the CCHFV NP could be confirmed. A mammalian expression system was subsequently investigated. A pcDNATM 3.1D/V5-His-TOPO.CCHFV.NP construct was used to transfect baby hamster kidney-21 cells. Expression of CCHFV NP was detected in transiently transfected cells using IFA and serum collected from a convalescent CCHFV patient. To profile the immune response against CCHF viral proteins, 15 sera collected from convalescent patients at various times after onset of illness were tested for antibody against CCHFV NP and glycoproteins (GP) using commercially available slides. The antigen slides were prepared from transfected cells expressing recombinant CCHFV NP and GP. Antibody against CCHFV GP and NP were detected in all samples. End point titers of anti-CCHFV NP and GP were determined for two serum samples. Commercially available slides are expensive and therefore have limited application for testing large numbers. Application of in-house antigen slides prepared from transfected cells expressing CCHFV NP were tested using IFA and 14 sera collected from convalescent CCHFV patients. All sera tested positive, suggesting that preparation of a stable cell line expressing CCHFV NP is warranted for application in detection of antibody against CCHFV.
Open Access
Die voorkoms van giste tydens hoenderproduksie
(University of the Free State, 2000-12) Laubscher, Willem Diederik Froneman; Viljoen, B. C.
Afrikaans: Die verhoging in die plasing van die aantal hoenders per oppervlakte verhouding in moderne pluimvee boerdery praktyke, skep In klimaat waar mikrobiese groei (insluitend patogeniese mikroorganismes) gestimuleer word. Die stimulering van mikroorganismes word veralopgemerk in respiratories geassosieerde vrektes in groeihokke. Op grond van die verhoging in vrektes van jong kuikens is daar gekyk na die mikrobiese populasies teenwoordig in die tracheas van groeihok kuikens met spesiale karakterisering van die gis populasies teenwoordig. Die data het aangetoon dat die tracheas van jong braaikuikens hoofsaaklik besmet word deur die teenwoordigheid van gis species. Ten spyte van lae populasies in die eerste weke na uitbroeiing, ontwikkel die gispopulasies vinnig tot essensieel 'n klimaks populasie tydens die vierde week in die groeihokke. Die karakterisering van die gispopulasies het daarop gedui dat die belangrikste species blyk as volg te wees: Debaryomyces hansenii, Candida b/ankii, Toru/aspora de/brueckii en Trichosporon beige/ii. Omdat giste ook 'n belangrike bydrae maak tot die bederf van hoenderkarkasse, en deel uitmaak van die normale mikrobiologiese populasie teenwoordig op die eetbare hoenderkarkasse en hoenderstukke, is die voorkoms van hierdie flora verder deur die prosesseringsaanleg gevolg. Alhoewel giste proporsioneel in kleiner getalle voorkom as bakterieë, kan die giste as sekondêre populasie ontwikkel wanneer die bakteriese populasie geinhibeer word soos gevind nadat die karkasse onderdompel is in chloorbevattende immersie-wentelverkoelings baddens tydens prosessering. Cryptococcus en Candida species domineer voor verkoeling van die karkasse tydens ontvering en ontweiding terwyl Debaryomyces species oorheers na verkoeling en tydens bevriessing. Die data dui verder daarop dat die implementasie van chloor as ontsmettingsmiddel tydens verkoeling geen invloed gehad het op die voorkoms van die gisspecies nie. Die bronne van giskontaminasie in die onmiddelike omgewing van die slag pale het dieselfde tipes van giste opgelewer as gevind op die karkasse. Weens die abnormale hoë getalle giste gevind op die karkasse, en die beperkte invloed van die ontsmettingsmiddels tydens verkoeling op die getalle van giste is verskillende bruikbare ontsmettingsmiddels in die immersie-wentelverkoelings bad ondersoek. Goeie inhiberende en dodings resultate is verkry met behulp van meeste van die produkte. Slegs chloor dioksied het egter In genoegsame dodingseffek gehad op Salmonella species en In betekenisvolle inhibering van gisgetalle.
Open Access
The distribution, patient characteristics, therapy and patient outcome in culture positive invasive mold infections in a tertiary hospital in the Free State province, South Africa
(University of the Free State, 2019) Van der Westhuizen, B.; Coovadia, Y.; Potgieter, S.; Abrahams, M. S.
Introduction Fungi, including molds, are increasingly recognized as important pathogens carrying a high morbidity and mortality in critically ill and immune compromised patients and our understanding of these diseases remain incomplete, largely due to the lack of surveillance data. This study aimed to better quantify the distribution, patient characteristics, risk factors, therapy and treatment outcome in culture positive invasive mold infections at Universitas Academic Hospital in the Free State province, South Africa. Methods All culture positive mold isolates cultured from sterile specimens were identified retrospectively from 1 July 2014 to 30 June 2017. Laboratory and clinical data were reviewed for those that met the inclusion criteria. Results A total of 48 isolates were included in this study. There was a similar distribution between males and females and the mean age was 40.5 years. Aspergillus species were the most commonly isolated mold. The most common risk factors identified were HIV infection with a median CD4 of 88.5 cells/μl followed by hematological conditions. The treatment strategies in our study group were heterogeneous with 73.1% (19/26) of patients treated with antifungal therapy alone, 19.2% (5/26) with surgery alone and 7.7% (2/26) with a combined medical and surgical approach. Many patients received no treatment 45.8% (22/48). The overall mortality was 25% (12/48). Conclusions The diagnosis of invasive mold infections remains a challenge. In the current study, molds were found to cause serious infections, especially in at risk patients. Despite treatment with appropriate antifungal agents, the associated mortality rate was still high. This study contributes to the growing knowledge on the distribution, patient characteristics and outcomes of invasive mold infections, particularly in patients in the Free State, and lays the foundation for further research in the field of invasive mold infections.
Open Access
Evaluating the role of efflux pumps in bacterial disinfectant resistance
(University of the Free State, 2022) Staats, Gunther Johann; Bragg, R. R.
The global rise of antibiotic resistance could lead to the advent of a post-antibiotic era, where disinfectants and biosecurity will be vital parts to control microbial proliferation. The SARS-CoV-2 pandemic has shown how important proper biosecurity and disinfection protocols are to control disease outbreaks. Disinfectant resistance has the potential to alter every aspect of disease control, as this phenomenon impacts human life from food security to healthcare systems. Antimicrobial resistance at its core originates from the presence and regulation of specific genes within the genome of a microorganism able to combat/resist specific action of an antimicrobial. This study focuses on investigating whether specific resistance determinants are responsible for the insusceptibility of a highly resistant isolate, Serratia sp. HRI. This isolate has high levels of disinfectant resistance; therefore, it provides an opportune chance to study if a specific mechanism is responsible. To achieve this the genomes of the Serratia sp. HRI and its closest related type strain were investigated for efflux pump genes. The efflux pump genes were predicted using an automatic annotation pipeline. The predictions revealed a plethora of resistance efflux genes mostly harbouring multidrug functioning. Additionally, disinfectant-specific efflux pump genes were identified (emrE, sugE, qacA, qacE, and ssmE). Susceptibility testing using three disinfectants revealed how the resistance levels between the two Serratia isolates differed. Further investigation using efflux pump inhibitors (EPIs) showed how specific families of efflux pumps confer resistance in both isolates. Time-kill analyses over an extended period showed how Serratia sp. HRI tolerates long-term disinfection. Using EPI reserpine, the efflux pump activity was determined during long-term disinfection. The results showed that concentration-dependent recruitment of efflux pumps was seen by Serratia sp. HRI. At low disinfectant concentrations, another mechanism was responsible for the survivability of the bacteria. Using liquid chromatography with tandem mass spectrometry it was established that disinfectant levels introduced with Serratia sp. HRI decreased over time. Suggesting that the HRI isolate has some mechanism to alter the structure of the disinfectant, such as metabolism or degradation pathways. This work highlights the role of efflux pumps in disinfectant resistance and the potential of other mechanisms to be involved. Future research will include gene deletion and expression studies to fully determine the efflux pump reliance for disinfectant resistance. The work completed in this thesis added to the knowledge of efflux-mediated disinfectant resistance. This work also highlighted the potential role of metabolism/degradation in resistance to low-level disinfection.
Open Access
The evaluation of a continual disinfection program on a commercial broiler chicken farm
(University of the Free State, 2022) Beauzec, Deon; Bragg, R.; Carlie, S.
The rise of antibiotic resistance as a global health threat has been linked to overuse of antibiotics, in particular, in the agricultural sector. A post-antibiotic world is fast approaching where antibiotics may be completely ineffective. To delay and prepare for this reality, alternative solutions to the use of antibiotics in animal production are required. Biosecurity describes some of the oldest techniques related to animal health, including infectious agent destruction and exclusion. Novel solutions to biosecurity must be investigated, however the possible loss of production related to halting antibiotic use is a major risk to food security. This study focused on assessing a continuous disinfection program using Virukill®, a modified- didecyldimethylammonium chloride-based disinfectant, for post-placement cleaning and disinfection, direct spray, and administration in water supply, at a commercial scale. This work provides a proof of concept for industrial application of a continuous disinfection program as a biosecurity measure in poultry production. Through bacterial and viral evaluation methods it was established that the continuous disinfection program was equal or better than standard aldehyde disinfectants in reducing viral loads. Organisms that survived the cleaning and disinfection process were also isolated and identified with molecular methods. The possibility of disinfectant resistance was also investigated but no evidence of resistance was observed. It was established that Virukill® was more effective than DDAC and BC in inhibiting the growth of organism isolated. Through growth performance studies this work establishes for the first time that the Virukill® continuous disinfection program improves performance of broilers, and a correlation was established between cleaning efficacy and performance. This work provides a viable alternative biosecurity-based alternative to the use of antibiotics in broiler production.
Open Access
Genetic analysis of human papillomavirus type 11 isolates from patients with recurrent respiratory papillomatosis treated at Universitas Academic Hospital
(University of the Free State, 2021-07) Thuynsma, Corne; Burt, Felicity Jane; Seedat, Riaz
Human papillomavirus type 11 (HPV11) is a causative agent of recurrent respiratory papillomatosis (RRP), a common benign laryngeal neoplasm that presents mainly in children. The genome comprises three regions: the early region (E1, E2, E4, E5a/b, E6 and E7), the late region (L1 and L2), and the upper regulatory region (URR). A sequence-based classification system is primarily used to genotype HPV. The L1 is used for HPV type discrimination, and in combination with the URR, can be used to differentiate between various lineages. However, optimal sub-lineage classification requires whole genome sequencing (WGS). A recent study investigating the genomic diversity of globally circulating HPV11 isolates identified a novel lineage and two novel sub-lineages. It has been proposed that phylogenetic tree topologies using the sequences of concatenated E5a/b- L1-URR genes, a 208bp segment of the E2 gene, and the complete genome generates similar tree topologies. Also, there is currently no published data on the HPV11 intratypic variants circulating in the Free State region. Hence, this study investigated HPV11 intratypic variants circulating in patients with RRP at the Universitas Academic Hospital, and aimed to identify novel (sub)lineages through phylogenetic investigations. The study population included patients diagnosed with RRP caused by HPV11, and sequence data for geographically distinct HPV11 (sub)lineage representatives. The genetic variation of HPV11 isolated from patients with RRP was determined by sequencing the E5a/b, L1, URR and a segment of E2 genes. Four isolates of interest were selected for whole-genome sequencing and phylogenetically analysed to determine the presence of potentially novel isolates. Many nucleic heterogeneities and non-synonymous substitutions were identified in isolates characterised in this study. Phylogenetic analysis of the concatenated L1-URR and E5a/b-L1-URR resolved into lineages A and B; however, sub-lineage classification was unclear. Analysis of the complete genome determined the presence of a lineage B isolate and two isolates of interest. Comparative analysis of genetic variability determined that the concatenated E5a/b-L1-URR could not reliably classify isolates. A segment of the E2 gene could reliably distinguish between all lineages and sub-lineages, suggesting that this gene segment contains stable sub-lineage specific single nucleotide polymorphisms (SNPs) and may serve in sub-lineage identification. In conclusion this study provides the most comprehensive data on the genomic diversity of HPV11 in the Free State to date. Results obtained in the current study support WGS for HPV11 classification below lineage level as a standard, as it generates more information regarding genetic variants.
Open Access
HBV viral load and drug resistance among HIV-HBV co-infected patients: a cross-sectional study in central South Africa
(University of the Free State, 2021-12) Kotze, Jacobus Charles; Goedhals, Dominique
In South Africa, human immunodeficiency virus (HIV) infected individuals co-infected with hepatitis B virus (HBV) do not routinely undergo HBV viral load (VL) testing when on antiretroviral therapy in the public sector treatment programme. We set out to explore whether HIV VL can be used as a proxy for HBV treatment response, since HIV VL testing is routinely performed in HIV/HBV co-infected patients. The clinical utility of HIV VL testing in this context may be impacted by the slower rate of viral decay which has been described for HBV as compared to HIV. In total, 224 patient samples were tested for HBV VL to determine the relatedness between HIV VL and HBV VL results. Samples with detectable HBV VL were sequenced to identify HBV associated drug mutations, hepatitis B surface antigen (HBsAg) mutations and genotype. Chi-square test for independence (χ2) was used to determine the relatedness between the viral loads, which indicated that the two viral loads are related (p-value<0.0001). However, in samples with an undetectable HIV VL, 29.27% (36/123) had a detectable HBV viral load, with 7.32% having an HBV VL >2000 IU/mL which has previously been linked to an increased risk of HBV related complications. Sequencing results showed that 10 samples had lamivudine resistance, however, no tenofovir resistance was detected. Three samples had immune escape mutations, two caused by the HBsAg mutations E164D and I195M and one by the immune-associated escape mutations N131T and D144A. The results from the study show that patients with HIV/HBV co-infection need to be monitored more closely in South Africa regarding HBV treatment response. The extensive use of lamivudine for HIV treatment in South Africa can be a driver of immune escape and further research needs to be done to determine the possible public health impact.
Open Access
Identification of antigen-specific serological cross-reactivity among survivors of Crimean-Congo Haemorrhagic fever
(University of the Free State, 2013) Rangunwala, Azeeza; Burt, F. J.
Abstract not available
Open Access
Identification of antigenic regions and linear B cell epitopes on yellow fever virus
(University of the Free State, 2013-02) Smouse, Shannon Lucrecia; Burt, F. J.
English: Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.
Open Access
Immunogenicity of Sindbis based replicons for Crimean-Congo hemorrhagic fever virus
(University of the Free State, 2019-02) Tipih, Thomas; Burt, Felicity
Introduction and Aim: Crimean-Congo hemorrhagic fever virus (CCHFV) infrequently causes hemorrhagic fever in humans with a case fatality rate of 30%. Currently, there is neither an internationally approved antiviral drug nor vaccine against the virus. In a move aimed at averting future epidemics, the World Health Organization has added the virus to the list of priority infectious organisms. The aim of the study was to investigate mechanisms of immunogenicity of Sindbis replicons encoding CCHFV glycoproteins and nucleoproteins for future development of an efficacious vaccine. Methodology: Genes encoding the complete open reading frames of the CCHFV nucleoprotein and glycoprotein precursor proteins of South African strains were amplified by the reverse transcription polymerase chain reaction technique and cloned into a Sindbis virus replicon vector. Sanger sequencing and next-generation sequencing were carried out to confirm gene sequences. Nucleoprotein and glycoprotein expression were demonstrated by transfecting baby hamster kidney cells and human embryonic kidney cells. Vaccine construct self-replication rates were assessed by transfecting BHK-21 cells and assaying for CCHFV RNA using gene-specific primers. Apoptosis induction in transfected BHK-21 cells was determined by measuring the enrichment of nucleosomes in the cytoplasm using an ELISA. Groups of three NIH mice were immunized with 100 μg of vaccine constructs three times intramuscularly three weeks apart with plasmid constructs pSinCCHF-31S, pSinCCHF-52S and pSinCCHF-52M. To augment cytokine responses the adjuvant poly (I:C) was co-inoculated with pSinCCHF-52S and pSinCCHF-52M separately. In addition, the constructs pSinCCHF-52M and pSinCCHF-52S were co-immunised with and without poly(I:C) to induce a response against both proteins simultaneously. Two weeks after receiving the third dose mice were sacrificed and blood was collected for determination of humoral immune responses while harvested splenocytes were stimulated with a CCHFV antigen for cytokine responses. Results: Two vaccine constructs (pSinCCHF-31S and pSinCCHF-52S) expressing CCHFV nucleoprotein and a construct (pSinCCHF-52M) expressing CCHFV glycoprotein were prepared. Recombinant protein expression was demonstrated by immunofluorescence assays targeting the histidine tag fused to the CCHFV proteins. Further confirmation of protein expression was performed by immunofluorescence assays using serum from CCHF survivors. All prepared vaccine constructs transcribed CCHFV RNA, as demonstrated by detection of protein using immunofluorescent antibody assays, and induced apoptosis in transfected cells. Immunized mice responded with the production of high titers of CCHFV IgG NP specific antibodies and higher levels of IgG2a in comparison to IgG1 responses were observed in responders suggesting a predominant Th1 antibody response. CCHFV IgG GP specific antibodies were not induced in vaccinated mice. Vaccine construct pSinCCHF-52S resulted in higher secretion of IL-2, (p = 0.0495) IFN-γ (p = 0.0369) and TNF-α (p = 0.0495) relative to immunisation with pSinGFP. An enhanced secretion of IFN-γ and IL-2 (p = 0.0463) was observed from splenocytes from mice co-immunised with pSinCCHF-52S and pSinCCHF-52M while vaccinating with pSinCCHF-52M increased IL-2 secretion (p = 0.0463). Co-administration of pSinCCHF-52M and pSinCCHF-52S constructs augmented IFN-γ (p = 0.0463) secretion. Co-inoculation of vaccine constructs with adjuvant poly (I:C) did not enhance cytokine secretion. Conclusion: The study demonstrated the expression of CCHFV nucleoproteins and glycoproteins by a Sindbis virus vector in mammalian cells. Vaccination of mice with construct pSinCCHF-52S induced type 1 immunity. Immunoglobulin G subtyping demonstrated IgG2a/IgG1 >1 as well as significantly higher IL-2, IFN- γ and TNF- α. Immunisation with pSinCCHF-31S and pSinCCHF-52M did not elicit specific antibody production and cytokines responses were weak. Further studies in CCHFV susceptible animals are necessary to determine whether the immune responses generated by vaccinating with pSinCCHF-52S are protective. However, this study shows the utility of Sindbis replicons in vaccine development against CCHFV.
Open Access
Molecular epidemiology of Mycobacterium tuberculosis strains from the Free State and Northern Cape provinces, South Africa
(University of the Free State, 2004-05) Mokhethi, Sehloho Zacharia; Van der Spoel van Dijk, Anneke
Background. Tuberculosis is increasing in the Free State and Northern Cape provinces of South Africa, but it is not clear how much of the disease is caused by reactivated latent infection and how much is attributed to interpersonal transmission. The discovery of the transposable DNA insertion sequence, IS6110, provided the desired polymorphism among different strains to track routes of transmission, study the degree of inter-person transmission versus reactivation, to detect laboratory contamination and disease outbreaks. Alternative methods include spoligotyping and the mycobacterial intergenic repetitive units or variable number of tandem repeats (MIRU-VNTR). Sustained studies performed on a small area in the Western Cape Province and some mines in the Gauteng Province of South Africa have found person-to-person transmission of tuberculosis to be high in these populations. In addition, resistance determinants to key antituberculosis drugs have remained unknown among tuberculosis causative organisms circulating in the Free State and Northern Cape. Thus, extensive DNA fingerprinting and gene mutation studies are needed to address these problems. Methods. An area in the Free State suitable for long-term surveillance studies was defined using available information from the governmental database, the 1996 census statistics, and tuberculosis (TB) case loads and transfer data obtained from the National Tuberculosis Database. Each clinic’s catchment information was provided by clinic managers and the population movement data from a 2002 student project. Sputum samples were collected and Mycobacterium tuberculosis isolated from tuberculosis positive patients from the defined area (Gamadi). Isoniazid resistant isolates received from a representative sample from the Free State and a few strains from the Northern Cape Province were also included in the study. IS6110-directed restriction-fragment-length polymorphism (RFLP) analysis was performed on all isolates and drug susceptibility testing (indirect proportion method) done on the Gamadi isolates. Subtyping of identical strains (RFLP clusters) and some of the isolates with less than six IS6110 bands was done using spoligotyping and the MIRU-VNTR typing. DNA sequencing analysis of the katG and rpoB genes was done in resistant isolates and a rapid PCR-based restriction enzyme katG gene mutation detecting method evaluated. Results. An area characterised by extreme poverty (unemployment rate 69.0%), a relatively young population (69.0% below 35 years) of 61534 and with high incidence of tuberculosis (840/100 000) suitable for long -term surveillance studies was identified in the Free State. The area is served by three clinics and a hospital and is situated near the rural town of Thaba Nchu in the Free State province. Eighty eight M. tuberculosis isolates and a mycobacterium-other-than-tuberculosis (MOTT) were isolated from the 286 sputum specimens collected from the Gamadi area. Only two M. tuberculosis isolates tested isoniazid (INH) resistant and no rifampicin (RIF) resistant isolates were found. The MOTT was resistant to INH (0.2, 1 and 5 µg/ml) and to RIF. Standard IS6110-based DNA fingerprinting of 84 of 88 (96.5%) isolates from the defined area was performed. Four of the isolates were cultured from duplicate sputum specimens provided by four patients. Two of these had identical fingerprint patterns to the first isolate of the patient and two had a different profile. The latter pair could be attributed to laboratory error. IS6110 sequences were not detected in six isolates. Fourteen isolates had less than six IS 6110 hybridisation bands and four strains were in clusters. The remaining 57 (88.9%) strains had distinct RFLP profiles with more than six bands. The number of IS6110 copies varied from seven to 21. A total of five strains was distributed in two clusters, one with two and the other with three members. Thirteen family groups, clustered at 65.0% on the similarity dendogram, each with two to eight strains, but no dominant groups were evident. A cluster of three isolates with five identical IS 6110 bands each was confirmed as one strain by MIRU-VNTR typing while two further isolates (both had three bands of different sizes) were confirmed as different strains by MIRU typing. A total of 37 isoniazid-resistant M. tuberculosis was analysed. DNA fingerprint profiles showed nine isolates with less than six insertions (24.3%). Six of these isolates were from the Free State and three from the Northern Cape Province. Three of these isolates were multidrug resistant. The remaining 28 isolates (75.7%) contained between 9 and 18 copies of the IS6110 insertion sequence. Twenty-six different IS 6110 RFLP types were identified. Only two clusters with two isolates, respectively, were found in each province. Eight clonally related groups (65.0% similarity) with two to four strains were present. Three clusters of two isolates (each with more than six bands) also exhibited identical spoligotype patterns. Spoligotyping of two of three isolates from a fourth cluster (4 RFLP bands each) showed two different banding patterns and all were shown to be different by MIRU-VNTR typing. The fifth cluster (2 bands) was made up of one isolate from each province. Spoligotyping of these strains was identical, but the MIRU was different. One isolate from Bloemfontein had identical IS 6110-RFLP and spoligotyping patterns to a susceptible isolate from Gamadi. Isoniazid resistance in 22/37 isolates was sequence linked to altered nucleotides of codon 315 of the katG gene. Twenty harboured the ACC variant at the codon. One strain carried the AAC mutation at this codon and the other GGC. The remaining 15 carried the wild type (AGC) genotype at this site. Two of the strains harbouring the AGC315ACC mutation belonged to the same IS6110 cluster. Two mutations were found at codon 463 (CGG ® CTG; CGG ® CCG). Thirteen MDR strains were investigated for rpoB gene alterations. Four of these isolates carried no mutations within the 157-bp amplified fragment while the others had various mutations. Analysis of an 808bp fragment of the katG gene from INH-resistant M. tuberculosis isolates after restriction with Msp I agreed with results obtained by sequencing. Thirteen isolates carried a pattern consisting of 228, 153, 146, 109, 79, 65 base pairs with the 153 bp fragment indicating the presence of the wild type AGC at codon 315 of the katG gene. Seventeen isolates demonstrated the 228, 146, 132, 109, 79, 65, 21 profile with the 132 bp fragments indicating the presence of an ACC mutation. Three isolates contained a mixed genotype and were digested into the fragments 228 bp, 153 bp, 146 bp, 132 bp, 109 bp, 79 bp, and 65 bp. Fragments with 146 bp and 65 bp are seen in strains with no mutation (bases CGG) at codon 463, while a 211 bp fragment shows a mutation at this spot. Four strains had the fragments 228, 211, 153, 109, and 79 bp. One strain was digested into six fragments of 228 bp, 211 bp, 132 bp, 109 bp 79 bp and 21 bp containing both a 315 (ACC) and 463 (CTG) codon mutation. Discussion and conclusions. An area consisting of ten villages and characterised by a high incidence of tuberculosis was defined for long-term surveillance studies. Resistance in the area appears to be low and compares favourably to the situation in the Free State. Strains received from this area were highly diverse, but the presence of a cluster of five isolates indicated the need for continuous investigation. Recent transmission of INH resistance in the Free State province is not a significant factor, but since the isolates from the Northern Cape were not representative, no deduction could be made for this province. Resistance to INH is mostly associated with mutation AGC to ACC at codon 315 of the katG gene. The absence of alterations in a proportion of isolates is in agreement with published data implicating the involvement of more genes in causing INH resistance. Resistance to RIF was associated with various point mutations in the 81-bp core region of the rpoB gene. The high proportion of the ACC allele found among INH-resistant strains, cost effectiveness, ease to perform and rapid results, make PCR-RFLP an attractive option for detection of resistance especially in resource-poor countries.
Open Access
Preparation and immunogenicity of a candidate replicon based yellow fever vaccine
(University of the Free State, 2014-12) Viljoen, Natalie; Burt, Felicity Jane
English: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development. The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional cloning. The nucleotide sequence was confirmed by bidirectional sequencing at the site of insertion and subsequently protein expression was confirmed in selected mammalian cell lines. Protein expression was confirmed by detection of the C-terminal histidine tag by immunofluorescence using anti-His6 mouse monoclonal antibody. Thereafter, the expressed protein was characterised by immunofluorescence using mouse immune sera previously shown to contain anti-YFV ED-lll antibody. Reactivity of the expressed protein with anti-YFV ED-lll antibody substantiated the use of the pSinED-lll replicon in a mouse immunisation study. Mice were immunised intramuscularly according to pre-approved immunisation regimes consisting of DNA only, as well as mixed modal immunisation regimes. Serum samples were collected and spleens were harvested two weeks after the administration of the final immunisations. Serum samples were screened for anti-YFV antibodies by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to determine the induction of a humoral immune response. Five of the twenty mice in groups immunised with the DNA-launched pSinED-lll replicon developed low level antibody responses illustrating the potential of the pSinED-lll replicon to induce a humoral immune response. Splenocytes were stimulated in cell culture with concanavalin A, YFV ED-lll protein, or no stimulant to facilitate the detection of cytokine release using ELISA’s. A predominantly T-helper 1 cell-mediated immune response characterised by high level release of interferon-γ post-stimulation with the YFV ED-lll protein in vitro was elicited. The release of interleukin-10 post-stimulation may be associated with the prevention of immunologically mediated damage to the host by preventing an overzealous inflammatory immune response. Antibody-mediated anti-vector immunity should not negatively impact the immunogenicity of the DNA-launched pSinED-lll replicon as antibody directed against the encoded Sindbis virus non-structural proteins was not detected in mouse sera. In conclusion, the pSinED-lll replicon was shown to have the potential to induce both a cell-mediated and a humoral immune response post-immunisation. However, optimisation of the humoral immune response will be required.
Open Access
Preparation of multiplex immunofluorescent platform for serology
(University of the Free State, 2023) Maswanganyi, Nyiko Given; Burt, Felicity Jane; Makoah, Nigel Aminake
Arboviruses are transmitted by blood-feeding insects such as mosquitoes and ticks, and pose significant global health challenges. In South Africa there are arboviruses that are known to occur causing outbreaks annually after heavy rainfall and there are arboviruses that have been detected historically but their medical and veterinary importance is unknown. Due to the unavailability of commercial assays for many arboviruses, the development of in-house assays becomes crucial. This study aimed to develop a cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides for simultaneous screening of IgG antibodies against multiple arboviruses. The nucleoprotein (NP), envelope domain III (EDIII), and capsid (C) genes for Crimean-Congo hemorrhagic fever virus (CCHFV), West Nile virus (WNV), and Sindbis virus (SINV) respectively were codon optimized and cloned into pcDNA 3.1+ plasmids. The plasmids were transfected into human embryonic kidney (HEK) 293 mammalian cells, and the expression of these genes was confirmed through immunofluorescent assay (IFA). The expressed proteins were further validated by SDS and Western blot analyses. In-house enzyme-linked immunosorbent assays (ELISAs) were developed and optimized to screen human and cattle serum samples for IgG antibodies against SINV and CCHFV. A total of 386 human serum samples and 97 cattle serum samples were screened. Polyvalent IFA slides were developed and used to screen all positive serum samples, some negative and borderline from inhouse ELISAs. The results demonstrated successful expression of CCHFV NP, confirmed through positive IFA reactions with anti-CCHF IgG. SDS and Western blot analyses further validated the size of the expressed CCHFV. WNV showed low IFA reactivity with antihis antibody and SINV showed no expression. Hence it was decided to continue using SINV-infected cells to replace SINV transfected cells, confirmed using IFA with antiSINV sera. WNV was omitted going forward due to low yield. Among the samples positive for SINV IgG ELISA, 39 exhibited concordant positive results with the polyvalent slides. Similarly, three samples positive for CCHFV IgG antibodies with CCHFV ELISA were concordant with polyvalent slides, while eight negative serum samples tested negative using the ELISAs and IFA. Three ELISA borderline samples for SINV ELISA tested positive for SINV suggesting increased sensitivity for IFA. Furthermore, specificity testing of IFA revealed an accuracy rate of 83.3%. Multiplex assays offer a low-cost and fast method for simultaneous detection of IgG antibodies against multiple arboviruses on a single platform. The IFA results were concordant with the ELISA, suggesting the reliability of the developed technique. The positive reactors among the borderline samples for SINV antibody ELISA indicate that additional screening of negative samples could improve the cut-off value. Importantly, all samples tested negative with ELISAs were also negative with the multiplex IFA. This approach enables efficient serosurveillance, particularly in resource-limited regions. The accuracy and sensitivity of the multiplex assay were evident through the confirmation of IgG presence by monovalent IFA. In conclusion, the developed cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides provides a valuable tool for simultaneous screening of IgG antibodies against multiple arboviruses. The accuracy and sensitivity of the multiplex assay were validated through monovalent IFA confirmation. Subsequent research and refinement of this technique hold the potential to broaden its platform, enabling the screening of a more extensive array of viruses.
Open Access
Preparation of recombinant antigen for serological detection of African hantaviruses
(University of the Free State, 2017-07) Damane, Deborah Rethabile; Burt, Felicity Jane
Unlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined. Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required. The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples. There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it was considered that the preparation of a recombinant African hantavirus antigen based on SANGV could have application in serological surveillance studies. The S gene segment of the SA14 strain of SANGV was modified and codon optimized for enhanced expression and detection. The construct was sequenced and aligned to the native S gene. It was used to transfect baby hamster kidney cells (BHK-21). Expression of a 50kDA protein was confirmed by SDS-PAGE and Western blot assay. Antigen slides were prepared from transfected cells fixed on 12 well chamber slides. Positive controls from the commercially available ELISA kits were used in the IFA. Four of the 176 serum samples tested gave a positive test. For the in-house ELISA, protein was harvested from T75 culture flasks. The antigen was tested using positive and negative controls from the commercial ELISA kits. A suitable differentiation between positive and negative samples was not detectable despite attempts to optimize the in house ELISA. It is likely that the protein yield was insufficient for the ELISA and further attempts, beyond the scope of this project, to increase the protein yield will be investigated using a stable cell line. Commercially available ELISA kits comprising of HNTV, Dobrava (DOBV) and Puumala (PUUV) recombinant NP antigens for Europe and Asia group and Andes virus (ANDV) and SNV for the American group were used to screen acute human sera in the laboratory. Positive reactors were identified using both kits. The significance of the results is difficult to interpret as there was lack of concordance. However, it does suggest that hantaviruses are to be found in this area and that the use of a homologous antigen for serological surveillance is essential. Results confirmed some serological cross-reactivity between heterologous Asian, American and African hantaviruses and a potential application for an African hantavirus as a tool for surveillance.
Open Access
Preparation of recombinant antigens for demonstrating antibody responses in patients with Crimean-Congo haemorrhagic fever virus infections
(University of the Free State, 2011-06) Samudzi, Rudo Ruth; Burt, F. J.
Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing numbers of cases are seen in regions of Asia and in the past ten years CCHFV has emerged in several countries in the Balkans and re-emergence in south-western regions of the Russian Federation. Diagnosis of CCHFV infections during the acute phase is based on isolation of the virus or amplification of viral RNA. Patients that survive the infection have a demonstrable IgG and IgM antibody response, usually from day 5 to 7 after onset of illness. Current serological diagnostic assays based on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for culturing the virus and therefore limits the number of laboratories that can prepare suitable reagents. Preparation of recombinant antigens would enable laboratories to perform serological diagnosis of CCHFV infections and surveillance studies. The purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a bacterial expression system, to determine if the protein was immunogenic and to determine if the protein was able to detect IgG antibodies in survivors of CCHFV infection. The complete open reading frame of the gene encoding the NP of CCHFV was amplified by RT-PCR using primers specifically designed with restriction sites engineered to the primers to facilitate cloning. The amplicon was cloned into pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that the correct gene had been amplified and cloned into the vector for downstream cloning and expression applications. Initially we aimed to express the native gene using a bacterial expression system and the NP gene was rescued from the recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1 restriction sites present in the multiple cloning site on the vector. Various attempts were made to express the CCHFV NP protein however no protein was detectable using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our gene encoding the NP was analysed using the Rare Codon Analysis Tool software and we elected to codon optimize the gene for expression in E. coli. The optimized gene was synthesized by GenScript and supplied cloned in the multiple cloning site of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF bacterial expression vector. A 106 kDa protein was expressed from the construct likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP protein and confirmed by Western blot analysis. A higher yield of the protein was present in the insoluble phase and as optimization of the growth and induction conditions did not significantly alter the insoluble to soluble ratio of the expressed protein, the protein was harvested from the insoluble phase by denaturing, purification and refolding of the protein. The biological activity of the recombinant protein was confirmed using immunoassays and by immunizing mice to determine if the antibodies induced by the recombinant protein could be detected using an antigen prepared from the whole virus. Four of five mice immunized with the recombinant NP had a detectable antibody response using an immunofluorescent assay. Serum samples from acute and convalescent patients collected at varying stages after onset of illness were reacted in a Western blot with the recombinant CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all the convalescent patient sera except two sera collected on days 14 and 15 during the acute phase. In contrast all the samples were detected using the recombinant antigen in an ELISA. Due to the potential biohazardous nature of samples only samples collected two weeks after onset of illness were tested. The results showed 100% concordance with the results obtained in an ELISA using mouse brain derived antigen. The assay was shown to be reproducible and stability studies showed that four months after preparation the protein was still active. A full validation of the protein using a large panel of serum samples from confirmed CCHF patients is now required. The results suggest that bacterially expressed proteins lacking post translational modifications and folding that occur with mammalian and baculovirus expression can be used in ELISA to detect IgG antibody against CCHFV in human sera which finds application in diagnostics, epidemiologic and surveillance studies.