Browsing by Author "Smuts, Heidi"

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    Open Access
    A Molecular Epidemiological Study of Human Parainfluenza 4 in the Western Cape, South Africa
    (2021) Parsons, Jane; Hardie, Diana; Smuts, Heidi
    Background Human parainfluenza 4 (HPIV 4) is a recognised cause of acute respiratory infection (ARI). However, there is no published data on the epidemiology of this virus in South Africa. This thesis describes the molecular epidemiology of HPIV 4 over a 4-year period (2014-2017). Respiratory samples from infants, children and adults presenting with respiratory illness in the Western Cape, South Africa were studied. Method A retrospective 4-year study using routine diagnostic samples from patients with ARI was conducted in Western Cape, South Africa. A database search of positive HPIV 4 samples detected by the Seegene Anyplex RV 16 diagnostic assay was extracted. Epidemiological information was recorded to determine age, gender, hospital ward (used as a proxy for disease severity), specimen type (upper or lower respiratory tract) and collection date (to indicate seasonality). To determine genetic evolution, novel primers targeting the haemagglutinin-neuraminidase (HN) in both HPIV 4 subtypes were designed to amplify a 733 bp and 738 bp sequence for HPIV 4A and HPIV 4B respectively. This product was then sequenced and aligned with known reference sequences from GenBank, using BioEdit. These aligned sequences were analysed using the phylogenetic analysis tool, MEGA 6, and Highlighter plots to determine sequence divergence events and evolution. A real-time PCR assay, targeting the phosphoprotein, was developed to rapidly distinguish subtype A and B viruses. Results HPIVs were the 6th most common respiratory viruses detected in diagnostic samples. In all, there were 312/7456 (4.2 %) HPIV 4 positive samples in patients with a median age of 12 months. Males had a higher infection rate. HPIV 4 was the most prevalent of the HPIVs accounting for 47% of all HPIVs. Respiratory infections due to HPIV 4 were seasonal, peaking in autumn and mid-winter (March to August). The overall prevalence of HPIV 4 increased over the study period. Of the HPIV 4-positive samples that were subtyped, 59 were subtype A and 26 subtype B. Both subtypes co-circulated during each season. 71 % of patients who were positive for HPIV 4 were co-infected with one or more additional respiratory virus with Adenovirus (27 %), Human Rhinovirus (23 %) and Bocavirus (19 %) as the most common. HPIV 1 and HPIV 3 were both able to co-infect patients with HPIV 4, but no co-infections with HPIV 2 were detected. Phylogenetic trees constructed using neighbour joining (NJ) method showed that most of the South African HPIV 4 subtypes did not group with the closest significant reference sequences from GenBank. The phylogenetic tree for HPIV 4A revealed 4 genetic groupings. There were many nucleotide changes increasing with time as well as a non-synonymous change in HPIV 4A, at location N161D. HPIV 4B had an amino acid change in location G198R in the HN protein sequenced. Conclusion HPIV 4 with an overall prevalence of 4 % over the study period was identified as a significant cause of ARI in the Western Cape, South Africa. Mono-infection with HPIV4 was associated with severe disease. In hospitalized infants who were HPIV 4 positive, between ¼ to 1/3 were from patients in ICU. Of these almost half (46 %) had HPIV 4 as a single infection. Further studies are needed to fully understand the molecular epidemiology of this infection.
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    Analysis of cytomegalovirus UL97 drug resistance mutations in patients receiving Ganciclovir
    (2018) Nkosi, Nokwazi Pearl; Hsiao, Nei-Yuan; Korsman, Stephen; Smuts, Heidi
    Introduction: Cytomegalovirus (CMV) drug resistance mutations, because of the widespread use of ganciclovir, have been widely reported in international literature, particularly in the post-transplant setting. However, a genotypic assay to detect CMV drug resistance is not available in South Africa and the prevalence of these mutations is therefore unknown. We aimed to document the prevalence and types of CMV UL97 mutations following exposure to ganciclovir in adult and paediatric oncology patients, transplant recipients and HIV-infected patients in the local tertiary level hospitals: Red Cross War Memorial Children's Hospital, Groote Schuur Hospital and Tygerberg Hospital. Methods: The study had two components, the first component being a retrospective cross-sectional study using stored extracted DNA from patients with serially elevated CMV viral load levels. Thirty-three samples were tested for this component. The second component was a prospective case series on patients who were referred by clinicians for genotypic testing in whom CMV drug resistance was suspected. Eight samples were tested for this component. The CMV UL97 gene was amplified by conventional nested polymerase chain reaction (PCR) and Sanger sequencing performed. Results: CMV UL97 mutations were identified in five of thirty-three (15%) retrospectively screened samples while the prospective testing of eight patient samples identified drug resistance mutations in three patients (38%). Overall 8/41 (20%) patients had CMV UL97 mutations. A trend of higher risk for development of drug resistance mutations among haematological oncology patients 7/23 (30%) compared to solid organ transplant recipients 1/10 (10%) was observed, however, this difference was not statistically significant (P=0.306). Conclusion: This study, the first of its nature in South Africa, identified the presence of CMV UL97 mutations conferring resistance to ganciclovir in the haematological oncology, primary immunodeficiency and solid organ transplant patients in the Western Cape. The assay successfully detected CMV UL97 drug resistance mutations in whole blood and cerebrospinal fluid clinical samples. Ongoing viral replication in the background of intensive immunosuppression and prolonged antiviral therapy selects for the emergence of CMV UL97 drug resistance mutations.
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    The formulation and refinement of a polymerase chain reaction (PCR) assay for early diagnosis of paediatric HIV infection and genetic analysis of variants involved in vertical transmission of HIV-1
    (1996) Nolte, Jeanine Lucasta; Smuts, Heidi; Stannard, Linda
    Paediatric human immunodeficiency virus (HIV) infection has become a major socio-economic health problem in recent years as the number of HIV-1 infected children steadily increases. The majority of these infants are infected through mother-to-child transmission, with the frequency of vertical transmission varying between 12,9% and 65%. In order to implement appropriate management and possible treatment of these infected neonates, it is essential to have reliable laboratory tests for the early diagnosis of an HIV infection. At the time that this study was initiated, the diagnosis of HIV-1 infection in the Groote Schuur Hospital Virology Laboratory depended almost exclusively on serological assays. Such assays are of limited value for infants under 18 months of age, as maternal lgG antibody to HIV-1 is transferred via the placenta and may persist in the baby for up to 18 months. Available lgG antibody tests do not distinguish reliably between passively acquired maternal antibody and that produced by the infant itself. A valuable method of establishing the presence of true infection is provided by the polymerase chain reaction (PCR) technique which allows the identification, and subsequent exponential amplification of low levels of specific viral nucleic acid using specific oligonucleotide primers. A major aim of this study was to develop and instigate a (PCR) assay for the early diagnosis of HIV infection in infected infants. This was successfully achieved by the adaptation and optimization of an existing standard PCR protocol to suit the specific needs of a routine diagnostic service. Preliminary requirements involved the selection of primers and probes and establishing optimal parameters for: ionic strength, Taq DNA polymerase concentration, primer concentration, deoxynucleotide triphosphate concentration, and hybridization conditions for most efficient functioning of the test. The devised method entailed the extraction of proviral DNA from peripheral blood mononuclear cells, amplification of HIV-1 specific sequences by PCR, and identification by Southern blot hybridization with digoxigenin (DIG)-labelled probes. Thereafter the efficacy of the assay was tested on 45 infants (under 15 months of age) all born to seropositive mothers and therefore at risk for HIV infection. Forty-two of these infants had antibodies to HIV-1 and the remaining 3 were seronegative. The latter 3 also tested negative for HIV proviral DNA when PCR was performed, using at least 2 different HIV-1 primer pairs and their respective DIG-labelled probes. However, 27 (64%) of the 42 seropositive infants were also HIV-PCR positive and the remaining 15 (36%) seropositive infants were negative for HIV proviral DNA. Positive PCR tests correlated well with clinical data indicative of active HIV-1 infection for the majority of infants in the neonatal period, although it could not provide proof of infection in newborn babies (less than 1 week of age). The development of an in-house PCR protocol specific for HIV-1 has not only provided a valuable diagnostic assay for neonatal infection, but has also given insight into the parameters required for high sensitivity and the stringent precautionary measures that need to be applied to avoid contamination problems. The second part of this study was devoted to DNA sequence analysis of cloned HIV isolates from an infected mother and her 3-month-old infant. Nucleotide sequence variation between isolates of HIV-1 has been well documented. Examination of the third variable region (particularly the V3- loop) in the env gene of HIV-1 of our mother-infant pair confirmed this variation and provided the first genetic epidemiological data of this nature in the local community. Proviral DNA from both mother and baby was amplified using V3-specific degenerate primers and cloned. Clones containing the insert DNA were 2 identified by colony-blot hybridization. Their nucleotide and amino acid sequences were analyzed by using various computer programs. The degree of similarity between variants from the mother and infant in this study differed to a large extent from previous studies. The virus population harboured by the mother displayed highly homogeneous V3 sequences (1,04% variation) compared to the isolates from her 3-month-old infant, which showed a higher degree (1,8%) of heterogeneity. Phylogenetic analysis of the different isolates from mother and infant demonstrated that an HIV-1 subtype C virus was the infectious agent. This classification was confirmed by the characteristic amino-acid sequence of the tetrapeptide motif of the V3 loop present in the isolates from both mother and infant as well as the absence of a potential N-linked glycosylation site proximal to the first cysteine of the V3 loop, which is characteristic of subtype C viruses. Based on the amino acids present at positions 306 and 320 of the V3 loop, it could also be concluded that isolates from both the mother and her baby were consistent with the non-syncytium inducing (NSI) phenotype of HIV-1, thus indicating that, contrary to popular belief, NSI variants can be responsible for initiating infection. Data obtained from these genetic investigations of variants involved in vertical transmission of HIV-1 can form a useful basis for future comparative studies.
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    Human Parainfluenza Virus (HPIV) Detection in Hospitalized Children with Acute Respiratory Tract Infection in the Western Cape, South Africa during 2014–2022 Reveals a Shift in Dominance of HPIV 3 and 4 Infections
    (Multidisciplinary Digital Publishing Institute, 2023-08-02) Parsons, Jane; Korsman, Stephen; Smuts, Heidi; Hsiao, Nei-Yuan; Valley-Omar, Ziyaad; Gelderbloem, Tathym; Hardie, Diana
    The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2–10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study.
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    Human rhinovirus infection in young African children with acute wheezing
    (BioMed Central Ltd, 2011) Smuts, Heidi; Workman, Lesley; Zar, Heather
    BACKGROUND:Infections caused by human rhinoviruses (HRVs) are important triggers of wheezing in young children. Wheezy illness has increasingly been recognised as an important cause of morbidity in African children, but there is little information on the contribution of HRV to this. The aim of this study was to determine the role of HRV as a cause of acute wheezing in South African children. METHODS: Two hundred and twenty children presenting consecutively at a tertiary children's hospital with a wheezing illness from May 2004 to November 2005 were prospectively enrolled. A nasal swab was taken and reverse transcription PCR used to screen the samples for HRV. The presence of human metapneumovirus, human bocavirus and human coronavirus-NL63 was assessed in all samples using PCR-based assays. A general shell vial culture using a pool of monoclonal antibodies was used to detect other common respiratory viruses on 26% of samples. Phylogenetic analysis to determine circulating HRV species was performed on a portion of HRV-positive samples. Categorical characteristics were analysed using Fisher's Exact test. RESULTS: HRV was detected in 128 (58.2%) of children, most (72%) of whom were under 2 years of age. Presenting symptoms between the HRV-positive and negative groups were similar. Most illness was managed with ambulatory therapy, but 45 (35%) were hospitalized for treatment and 3 (2%) were admitted to intensive care. There were no in-hospital deaths. All 3 species of HRV were detected with HRV-C being the most common (52%) followed by HRV-A (37%) and HRV-B (11%). Infection with other respiratory viruses occurred in 20/128 (16%) of HRV-positive children and in 26/92 (28%) of HRV-negative samples. CONCLUSION: HRV may be the commonest viral infection in young South African children with acute wheezing. Infection is associated with mild or moderate clinical disease.
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    Molecular characterisation of virus in the brains of patients with measles inclusion body encephalitis (MIBE)
    (BioMed Central Ltd, 2013) Hardie, Diana; Albertyn, Christine; Heckmann, Jeannine; Smuts, Heidi
    BACKGROUND: During 2009/10 a major measles epidemic caused by genotype B3 occurred in South Africa. Measles inclusion body encephalitis (MIBE) was diagnosed in a number of highly immuno-compromised HIV patients. The diagnosis was based on typical clinical and MRI findings and positive measles virus PCR in brain or CSF.To characterize the brain virus, nucleoprotein, matrix, fusion and haemagglutinin genes from 4 cases was compared with virus from acutely infected patients. METHODS: cDNA was synthesized using random primers and viral genes were amplified by nested RT-PCR. PCR products were sequenced in the forward and reverse direction and a contig of each gene was created. Sequences were aligned with reference sequences from GenBank and other local sequences. RESULTS: Brain virus was very similar to the South African epidemic virus. Features characteristic of persistent measles virus in the brain were absent. Mutation frequency in brain virus was similar to epidemic virus and had the same substitution preference (U to C and C to U). The virus of 2 patients had the same L454W mutation in the fusion protein. CONCLUSION: The brain virus was very similar to the epidemic strain. The relatively few mutations probably reflect the short time from infection to brain disease in these highly immuno-compromised patients.
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    Phenotypic and molecular analysis of Helicobacter spp. and related micro-organisms identified in clinical & environmental specimens
    (2006) Hoosain, Nisreen; Lastovica, A J; Smuts, Heidi
    Includes bibliographical references (p. 140-168).