Browsing by Author "Hardie, Diana"

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    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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    Cytomegalovirus viraemia in immunocompromised children in Cape Town
    (2009) Hsiao, Nei-Yuan; Hardie, Diana
    Includes bibliographical references (leaves 54-62).
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    Evaluation of two SARS-CoV-2 immunoassays
    (2022) Naidoo, Michelle; Hardie, Diana
    Aim: The purpose of this study is to verify the performance of the Roche Elecsys ® antinucleocapsid (qualitative) and anti-spike (quantitative) SARS-CoV-2 immunoassays to determine whether the performance of the assays is acceptable for diagnostic use in the Groote Schuur Hospital virology/chemistry laboratory, as well as other National Health Laboratory Service (NHLS) laboratories in South Africa. Methods: We performed a verification study using de-identified remnant serum or plasma samples. Standard verification experiments including sensitivity, specificity and precision were performed. Pre-pandemic samples were used to assess specificity. Samples with a linked positive SARS-CoV-2 polymerase chain reaction (PCR) result on a respiratory sample >10 days before the serum/plasma collection date were used to assess sensitivity. Additionally, postvaccine humoral response and other parameters was assessed in a cohort of laboratory staff. Results: For the anti-nucleocapsid antibody assay, specificity was 99.7% based on 316 samples and sensitivity 91.3% based on 404 samples. For the anti-spike antibody assay, the specificity based on 194 samples was 100%, and the sensitivity based on 384 samples was 93.8%. Both assays demonstrated acceptable precision. Furthermore, the anti-spike antibody assay sensitivity was >92% during the first three waves in South Africa, dominated by different SARS-CoV-2 variants. Post-vaccine seroconversion in 115 staff with no evidence of prior natural infection was 99% and hybrid immunity produced higher anti-spike antibody titres compared to vaccine-only participants. Conclusion: Both immunoassays met our acceptance criteria. Both assays can be used for seroprevalence studies. The anti-nucleocapsid immunoassay assay is valuable in confirming past natural infection in patients with previous asymptomatic infection, previous symptomatic infection where no PCR was done or PCR-negative patients who present to hospital with COVID-19 during the second week of illness or later. Most importantly, the antispike immunoassay can be used as a reliable, cheap, and easily accessible surrogate marker of post-vaccine humoral immune response and we recommend using this to confirm and monitor humoral immune response in patients with risk factors for non-seroconversion following vaccination and increased risk for morbidity and mortality following infection with SARS-CoV-2.
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    The failure of routine rapid HIV testing: a case study of improving low sensitivity in the field
    (BioMed Central Ltd, 2010) Wolpaw, Benjamin; Mathews, Catherine; Chopra, Mickey; Hardie, Diana; de Azevedo, Virginia; Jennings, Karen; Lurie, Mark
    BACKGROUND:The rapid HIV antibody test is the diagnostic tool of choice in low and middle-income countries. Previous evidence suggests that rapid HIV diagnostic tests may underperform in the field, failing to detect a substantial number of infections. A research study inadvertently discovered that a clinic rapid HIV testing process was failing to detect cases of established (high antibody titer) infection, exhibiting an estimated 68.7% sensitivity (95% CI [41.3%-89.0%]) over the course of the first three weeks of observation. The setting is a public service clinic that provides STI diagnosis and treatment in an impoverished, peri-urban community outside of Cape Town, South Africa. METHODS: The researchers and local health administrators collaborated to investigate the cause of the poor test performance and make necessary corrections. The clinic changed the brand of rapid test being used and later introduced quality improvement measures. Observations were made of the clinic staff as they administered rapid HIV tests to real patients. Estimated testing sensitivity was calculated as the number of rapid HIV test positive individuals detected by the clinic divided by this number plus the number of PCR positive, highly reactive 3rd generation ELISA patients identified among those who were rapid test negative at the clinic. RESULTS: In the period of five months after the clinic made the switch of rapid HIV tests, estimated sensitivity improved to 93.5% (95% CI [86.5%-97.6%]), during which time observations of counselors administering tests at the clinic found poor adherence to the recommended testing protocol. Quality improvement measures were implemented and estimated sensitivity rose to 95.1% (95% CI [83.5%-99.4%]) during the final two months of full observation. CONCLUSIONS: Poor testing procedure in the field can lead to exceedingly low levels of rapid HIV test sensitivity, making it imperative that stringent quality control measures are implemented where they do not already exist. Certain brands of rapid-testing kits may perform better than others when faced with sub-optimal use.
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    Hepatitis A seroprevalence in South Africa: Are we in epidemiological transition?
    (2018) Enoch, Annabel; Kagina, Benjamin M; Hussey, Gregory D; Andersson, Monique; Hardie, Diana
    Hepatitis A virus (HAV) is the most common cause of viral hepatitis worldwide. Infection with HAV is vaccine preventable, however, a vaccine against HAV is not included in the Expanded Programme on Immunization in South Africa (SA). South Africa was considered to be a high endemic country for hepatitis A in the past, hence there was no need for routine immunization against the virus. Our hypothesis is that SA is changing from high to intermediate endemic setting for hepatitis A. To test our hypothesis, we conducted a cross-sectional seroprevalence study in the 1-7 year age group in the Western Cape Province. Our samples for this study were from specimens, collected between August and October 2015, sent for routine diagnosis to referral hospitals in the Western Cape Province. We tested remaining serum of 482 samples sent for routine tests. A Siemens enzyme immunoassay was used to test for hepatitis A antibodies. We also analysed hepatitis A test results from the National Health Laboratory Services (NHLS) Disa*Lab database at Groote Schuur hospital from 2009-2014, as well as hepatitis A surveillance data from the National Institute for Communicable Diseases (NICD) from 2009-2014, to look at the past hepatitis A prevalence trend. Our cross-sectional study showed the seroprevalence to be 44.1% in the 1-7 year age group. The NHLS data showed a seroprevalence of <90% up to age 10 years, indicating intermediate endemicity. The NICD data showed that a substantial number of symptomatic hepatitis A infections occurred in the 7-40 year age group, suggesting an increasing proportion of a susceptible population to HAV infection. Taken together, these results indicate the need for further studies designed to aid the development of vaccination policies against HAV infection in South Africa.
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    HIV Viral Load Testing in the South African Public Health Setting in the Context of Evolving ART Guidelines and Advances in Technology, 2013 - 2022
    (Multidisciplinary Digital Publishing Institute, 2023-08-22) Hans, Lucia; Cassim, Naseem; Sarang, Somayya; Hardie, Diana; Ndlovu, Silence; Venter, W.D. Francois; Da Silva, Pedro; Stevens, Wendy
    HIV viral load (VL) testing plays a key role in the clinical management of HIV as a marker of adherence and antiretroviral efficacy. To date, national and international antiretroviral treatment recommendations have evolved to endorse routine VL testing. South Africa (SA) has recommended routine VL testing since 2004. Progressively, the centralised HIV VL program managed by its National Health Laboratory Service (NHLS) has undergone expansive growth. Retrospective de-identified VL data from 2013 to 2022 were evaluated to review program performance. Test volumes increased from 1,961,720 performed in 2013 to 45,334,864 in 2022. The median total in-laboratory turnaround time (TAT) ranged from 94 h (2015) to 51 h (2022). Implementation of two new assays improved median TATs in all laboratories. Samples of VL greater than 1000 copies/mL declined steadily. Despite initial increases, samples of fewer than 50 copies/mL stagnated at about 70% from 2019 and declined to 68% in 2022. Some variations between assays were observed. Overall, the SA VL program is successful. The scale of the VL program, the largest of its kind in the world by some margin, provides lessons for future public health programs dependent on laboratories for patient outcome and program performance monitoring.
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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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    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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    Patient experiences following acute HIV infection diagnosis and counseling in South Africa
    (Public Library of Science, 2014) Wolpaw, Benjamin J; Mathews, Catherine; Mtshizana, Yolisa; Chopra, Mickey; Hardie, Diana; Lurie, Mark N; De Azevedo, Virginia; Jennings, Karen
    Individuals in the acute stage of HIV infection (AHI) have an elevated potential to transmit HIV and play a critical role in the growth of the epidemic. Routine identification and counseling of individuals during AHI could decrease transmission behavior during this key period. However, diagnosis of AHI may present challenges distinct from those experienced through diagnosis of established HIV infection. A study was conducted in a public youth clinic outside of Cape Town, South Africa, to identify and counsel individuals with acute stage HIV infection. In-depth interviews were conducted with patients following diagnosis. After counseling, patients were accepting of the testing regimen used to diagnose AHI. They used the knowledge of having been recently infected to identify the source of their infection, but did not retain or place importance on information regarding the increased ability to transmit HIV during the acute stage. Future interventions directed at the reduction of HIV transmission following diagnosis with AHI will need to find ways of making this information more salient, possibly through more culturally meaningful educational approaches.
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    Silent casualties from the measles outbreak in South Africa
    (2011) Albertyn, Christine; Van der Plas, Helen; Hardie, Diana; Candy, Sally; Tomoka, Tamiwe; LeePan, Edward B; Heckmann, Edward B
    South Africa, home to the world’s largest population of people living with HIV (5.7 million), experienced a measles outbreak that started in late 2009.1 There was a stepped increase in cases of measles, with the highest incidence reported in March 2010.2 By September 2010, more than 17 000 new measles cases had been reported to the National Institute of Communicable Diseases since January 2009. A mass vaccination campaign from mid-April to early May 2010 resulted in a significant decline in new measles cases. The measles virus is highly contagious, and outbreaks are fuelled by overcrowding and poor vaccine coverage, making elimination status in South Africa difficult to attain. Measles may infect the central nervous system (CNS) as acute viral encephalitis, or result after 2 - 4 weeks in a post-infectious immune-mediated inflammatory disorder or acute disseminated encephalomyelitis (ADEM). There are 2 further rare and latent CNS complications resulting from a preceding measles infection: subacute sclerosing panencephalitis (SSPE) caused by years of viral persistence in a seemingly immunocompetent host,3 and subacute measles encephalitis (SME), occurring in an immunocompromised host.4 SME manifests 1 - 7 months after the acute measles infection.5 Patients present with seizures, often epilepsy partialis continua, and altered mental status.5 It carries a mortality rate of 85% and survivors often have significant psychomotor retardation.5 SME has hitherto only been described in single case reports as a rare complication of measles in the context of organ transplantation,6,7 immunosuppressive therapy or immunodeficiencies,5,8 and HIV and AIDS.5,9,10 We report 8 cases of SME in HIV-infected patients who presented to a tertiary referral hospital between July and October 2010.
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    The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa
    (2022) Marais, Gert J K; Hardie, Diana
    The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation and inactivation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. A total of 195 samples were tested as part of the validation. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.