scholarly journals Improved performance of saliva for the detection of the B.1.351 variant in South Africa

2021 ◽  
Author(s):  
Chun Yat Chu ◽  
Gert Marais ◽  
Christoffel Opperman ◽  
Deelan Doolabh ◽  
Arash Iranzadeh ◽  
...  
Keyword(s):  
South Africa ◽  
Marked Improvement ◽  
Molecular Detection ◽  
Cape Town ◽  
The Novel ◽  
Wild Type ◽  
Rt Pcr ◽  
Diagnostic Laboratory ◽  
Percent Agreement ◽  
Improved Performance

Assessment of the unknown performance of saliva for the detection of the novel SARS-CoV-2 variant of concern (VOC) B.1.351 (501Y.V2) lineage is essential as saliva has been shown to be an equivalent, less invasive and a less costly alternative to nasopharyngeal swabs for the molecular detection of SARS-CoV-2 infection in pre-variant studies. Between 1st August 2020 and 16th January 2021, we enrolled 410 eligible ambulatory participants who presented to Groote Schuur Hospital (GSH) in Cape Town, South Africa for SARS-CoV-2 testing. Of these, 300 were enrolled prior to, and 110 after, the initial detection and replacement of wild-type by the B.1.351 variant. All participants provided a supervised self-collected mid-turbinate (MT) and saliva (SA) swab, in addition to the standard HCW collected NP swab which were all tested by RT-PCR in an accredited diagnostic laboratory. Positive percent agreement to NP swab for SA swabs pre- and post-variant were 51.5% and 72.5% respectively while these values for MT swabs were 75.8% and 77.5%. The negative percent agreement for all swab types during all periods was >98%. The basis for this marked improvement of SA swabs as a diagnostic sample for B.1.351 virus is still being investigated.

scholarly journals The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

2020 ◽  
Author(s):  
Gert Johannes Kruger Marais ◽  
Michelle Naidoo ◽  
Nei-yuan Hsiao ◽  
Ziyaad Valley-Omar ◽  
Heidi Smuts ◽  
...  
Keyword(s):  
South Africa ◽  
Nucleic Acid ◽  
Sample Preparation ◽  
Preparation Method ◽  
Preparation Technique ◽  
Rt Pcr ◽  
Sample Preparation Method ◽  
Diagnostic Laboratory ◽  
Percent Agreement ◽  
Nucleic Acid Purification

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 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. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

Characterization of a Novel Aberrant Splice Site, 79bp Downstream of Exon 5 in the Human Factor 7 Gene Detected in Patients with Severe Congenital Factor VII Deficiency.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3481-3481
Author(s):  
Karin Wulff ◽  
Jan Astermark ◽  
Falko F H Herrmann ◽  
Günther Auerswald ◽  
Winnie Schröder
Keyword(s):  
Splice Site ◽  
Donor Splice Site ◽  
Aberrant Splice ◽  
The Novel ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Donor Splice ◽  
Naturally Occurring ◽  
Fvii Deficiency

Abstract Abstract 3481 Poster Board III-418 Hereditary FVII deficiency (FVIID) is a rare congenital bleeding disorder with an estimated prevalence of symptomatic individuals of 1:500,000. In the “Greifswald Registry FVII Deficiency” molecular defects of more than 1000 FVII deficient patients were described. By direct sequencing of the F7 genes in congenital FVIID revealed 146 different F7 gene mutations including 25 different mutations (18% of all) in the naturally-occurring acceptor or donor splice sites (Tab.1) were identified. In seven FVIID patients from Sweden and Germany the novel lesion g.IVS5+78G>A - downstream of the naturally-occurring donor splice sites of exon 5 - was identified. This variation was detected heterozygous in FVIID patients with FVII: C levels of 15%, 27%, 31%, 40% and 65%, and FVII: Ag levels between 25-50%. In two compound heterozygous patients with FVII: C levels of 1% und FVII: Ag levels of 2% and 3% respectively, one well-known causative FVII mutation is combined with the novel lesion g.IVS5+78G>A. The influence of this novel F7 gene variation on splicing was investigated by RT-PCR analysis and in vitro expression studies using exon-trap vector constructs. The total RNA was isolated from peripheral leucocytes and analyzed by one step RT-PCR and sequencing. Fragments of exon 5 and a part of the flanking intron 5 region (g.7679 –g.8073) were amplified of patients' DNA and cloned into the exon trap-vector pET01. Different vector constructs containing minigenes of the wild type (g.IVS5+78G) or mutant form (g.IVS5+78A) and the corresponding minigenes with an “optimized” naturally-occurring donor splice site in position +5 respectively were transfected into HEK293 cells. The expressed RNA was isolated and characterized. Consensus Values (CV) for all donor splice sites were calculated using a splice site detection tool according Shapiro and Senapathy (1987). The RT-PCR analysis in patients indicate that the novel variation g.IVS5+78G>A in intron 5 created an aberrant splice site in position 79bp downstream of exon 5 even though the naturally-occurring donor-splice-site of exon 5 is not abolished. An insertion of 79bp of intron 5 into the mRNA leads to a frame shift and predicts a premature termination 11 codons past the last unaltered codon. Minigenes include the naturally-occurring splice site and the variation g.IVS5+78A used exclusively the aberrant splice position 79bp downstream of exon 5 whereas wild type minigenes with the naturally-occurring splice site and the wild type form g.IVS5+78G produced normally spliced mRNA. In a following experiment the “naturally-occurring splice site” of exon 5 was optimized by the additional substitution g.IVS5+5C>G which increased their CV from 76.6 to 90.9 compared to the CV of the novel mutant g.IVS5+78A of 80.3. In presence of both mutations (g.IVS5+5G and g.IVS5+78A) only normal spliced mRNA was expressed of this minigene. In this construct the mutation g.IVS5+78G>A was without importance for the mRNA splicing. The results of the in vitro experiments demonstrated, that the Consensus Values (CV) seems to be an important factor for the selection of donor splice sites in the F7 gene. In the “Greifswald Registry FVII Deficiency” 26 different splice site variations in F7 gene were identified (Tab. 1). The atypical splice site variation g.IVS5+78G>A, +78bp downstream of exon 5 was present in 7 FVIID patients from Sweden and Germany in different genotypes. This novel F7 gene mutation g.IVS5+78G>A creates an aberrant splice site in position +79 of intron 5 and predicts premature termination. RNA analysis and expression studies demonstrated, that this novel F7 gene lesion is a type I mutation with low FVII:C and FVII: Ag levels and is the basis defect in 7 FVIID patients of the “Greifswald Registry FVII Deficiency”. Tab. 1 26 different intronic F7 gene mutations analysed in FVII deficiency patients of the “Greifswald Registry FVII Deficiency” Intron Acceptor splice site Intron Donor splice site 1b g.IVS1b-11G>A 1a g.IVS1a+5G>A 1b *g.IVS1b8del14bp 1a *g.IVS1a+6T>G 1b *g.IVS1b-3C>G 1a *g.IVS1a+8C>T 2 *g.IVS2-3C>G 2 g.IVS2+1G>A 3 g.IVS3-1G>A 2 *g.IVS2+1G>T 3 *g-IVS3-1G>T 2 *g.IVS2+1G>C 4 *g.IVS4-7T>G 2 *g.IVS2+1delG 7 *g.IVS7-10T>C 2 g.IVS2+5G>T 7 *g.IVS7-3C>G 3 *g.IVS3+1G>T 7 *g.IVS7-1G>A 4 g.IVS4+1G>A 5 *g.IVS5+78G>A 6 *g.IVS6+1G>A 6 g.IVS6+1G>T 6 *g.IVS6+3A>G 7 *g.IVS7+1G>A 7 g.IVS7+3_6 del4bp * novel mutations (HGMD Factor 7 Database, 2009 /http://www.hgmd.org) Disclosures: No relevant conflicts of interest to declare.

Ultra Rapid, One Step Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Amplification Reaction.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2515-2515
Author(s):  
Elena D'agostini ◽  
Giulia Minnucci ◽  
Giulia Amicarelli ◽  
Cinzia Pultrone ◽  
Veronica Tettamanzi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Detection ◽  
Lymphoblastic Leukemia ◽  
Internal Quality ◽  
Clinical Samples ◽  
Wild Type ◽  
Rt Pcr ◽  
Automatic Instrument ◽  
Fusion Transcripts ◽  
One Step

Abstract Abstract 2515 Background: The molecular identification of the BCRABL transcripts in clinical samples is actually based on a conventional Reverse Transcription Polymerase Chain reaction (RT-PCR). Here we present a novel molecular method, based on Loop Mediated Amplification assay that, starting from RNA (RT-LAMP) in one single tube reaction ensures a rapid and simultaneous detection of either the BCR-ABL p190 or p210 fusion transcript as well as the housekeeping gene used as internal quality control. Methods: The BCR-ABL RT-LAMP is a multiplex, isothermal method for retro-transcription, amplification and detection of the Minor (p190) and Major (p210) t(9;22) transcripts and the endogenous Gusb RNA, as internal control for validation of negative results. The employment of fluorescent specific probes allows real-time monitoring of the reaction, so that the test result is obtained in a single, homogeneous step. RT-LAMP is carried out on the Liaison IAM, a 8-wells manageable instrument suitable for isothermal reactions. Liaison IAM incubates at constant temperature, monitors the fluorescent signals and the data produced can be analyzed, upon connection to up to 6 other instruments, for a throughput of 8 to 48 samples. Thanks to the three channels of fluorescence, it can monitor multiplex assays, providing elaborated final objective results with no need of further data analysis by the operator. Results: The level of sensitivity of the triplex BCR-ABL RT-LAMP has been analytically evaluated directly on serial dilutions of RNA extracted respectively from t(9;22) positive cell lines (TOM-1 for p190 or K-562 for p210) into wild type RNA from HL-60 cell line (30 replicates). The p190 and p210 transcripts have been detected and distinguished down to 10−4 and 10−5respectively within 50 minutes. The assay demonstrated 100% specificity since 70 replicates of wild type RNA from 7 cell lines resulted BCR-ABL negative and GUSb positive (internal amplification control). This assay was validated on 60 clinical samples (30 white blood cells RNA from Chronic Myeloid Leukemia, 30 mononuclear cells RNA from B-lineage Acute Lymphoblastic Leukemia). All these samples were obtained at diagnosis and were previously analyzed by conventional RT-PCR. RT-LAMP detected and identified the BCR-ABL fusion transcripts correctly in all cases with a 100% concordance with the reference method. Fully concordant results were obtained also on 30 RNA samples from patients affected by t(9;22) negative hematologic malignancies and on 30 RNA samples obtained from healthy donors in which the RT-LAMP amplified exclusively the housekeeping GUSb transcript. Conclusions: The triplex p190-p210-GUSb RT-LAMP is a one-step procedure for specific, highly sensitive and rapid molecular detection of the BCR-ABL fusion transcripts. The semi-automatic instrument Liaison IAM, simplifies the entire procedure, reduces the contamination risks deriving from the conventional, multi step RT-PCR and significantly improve the diagnostic lab routine. Disclosures: D'agostini: DIASORIN S.p.A: Employment. Minnucci:Diasorin S.p.A.: Employment. Amicarelli:DIASPRIN S.p.A.: Employment. Pultrone:DIASORIN S.p.A.: Employment. Tettamanzi:DIASORIN S.p.A.: Employment. Salmoiraghi:DIASORIN S.p.A.: Consultancy. Spinelli:DIASORIN S.p.A: Consultancy. Colotta:DIASORIN S.p.A: Employment. Rambaldi:DIASORIN S.p.A: Consultancy.

scholarly journals Molecular detection of airborne Emergomyces africanus, a thermally dimorphic fungal pathogen, in Cape Town, South Africa

2018 ◽  
Vol 12 (1) ◽  
pp. e0006174 ◽  
Author(s):  
Ilan S. Schwartz ◽  
Josh D. McLoud ◽  
Dilys Berman ◽  
Alfred Botha ◽  
Barbra Lerm ◽  
...  
Keyword(s):  
South Africa ◽  
Fungal Pathogen ◽  
Molecular Detection ◽  
Cape Town

Time and Latitude in South Africa

1972 ◽  
Vol 1 ◽  
pp. 27-38
Author(s):  
J. Hers
Keyword(s):  
South Africa ◽  
Cape Town ◽  
Astronomical Observations ◽  
Royal Observatory ◽  
The Republic ◽  
Astronomical Determination ◽  
Limited Accuracy ◽  
Better Than

In South Africa the modern outlook towards time may be said to have started in 1948. Both the two major observatories, The Royal Observatory in Cape Town and the Union Observatory (now known as the Republic Observatory) in Johannesburg had, of course, been involved in the astronomical determination of time almost from their inception, and the Johannesburg Observatory has been responsible for the official time of South Africa since 1908. However the pendulum clocks then in use could not be relied on to provide an accuracy better than about 1/10 second, which was of the same order as that of the astronomical observations. It is doubtful if much use was made of even this limited accuracy outside the two observatories, and although there may – occasionally have been a demand for more accurate time, it was certainly not voiced.

Knowledge about Newborn Discharge Follow-up in Cape Town, South Africa

PEDIATRICS ◽  
2016 ◽  
Vol 137 (Supplement 3) ◽  
pp. 393A-393A
Author(s):  
KaWing Cho ◽  
Jean P Milambo ◽  
Leonidas Ndayisaba ◽  
Charles Okwundu ◽  
Abiola Olowoyeye ◽  
...  
Keyword(s):  
South Africa ◽  
Cape Town

Experiences of Hearing Parents Following the Diagnosis of Their Child's Hearing Loss

2018 ◽  
Vol 30 (2) ◽  
Author(s):  
Ronel Sanet Davids ◽  
Mariana De Jager
Keyword(s):  
South Africa ◽  
Hearing Loss ◽  
Thematic Analysis ◽  
Qualitative Methodology ◽  
Cape Town ◽  
Best Interest ◽  
Ethical Considerations ◽  
Hearing Parents ◽  
Phenomenological Design ◽  
Depth Interviews

An estimated 90 per cent of children with a hearing loss are born to hearing parents. Most parents are unprepared for the diagnosis, leaving them shocked, confused, sad and bewildered. This article reports on a study aimed at exploring and describing the experiences of hearing parents regarding their child’s hearing loss. The study was conducted in Cape Town, South Africa. The study applied a qualitative methodology with a phenomenological design. Purposive sampling was implemented and data were collected by means of unstructured in-depth interviews. Data were analysed using thematic analysis. Ethical considerations were adhered to. The main findings of the study indicated that hearing parents experience a myriad of emotions when their child is diagnosed with a hearing loss. This study advocates for various stakeholders in the helping profession to collaborate in the best interest of hearing parents and a child with hearing loss. Furthermore, these findings serve as guidelines for professionals working with these families.

The Perpetuation of Gender Norms and White Hegemonic Patriarchy in the South African Defence Force as Represented in André van der Merwe’s Moffie (2006)

2020 ◽  
Vol 8 (2) ◽  
Author(s):  
Bernard Nolen Fortuin
Keyword(s):  
South Africa ◽  
Military Service ◽  
Well Being ◽  
The Novel ◽  
White Family ◽  
White Children ◽  
White Masculinity ◽  
Party Government ◽  
Compulsory Military Service ◽  
The Military

With the institution of compulsory military service in South Africa in 1948 the National Party government effected a tool well shaped for the construction of hegemonic masculinities. Through this, and other structures like schools and families, white children were shaped into submissive abiding citizens. Due to the brutal nature of a militarised society, gender roles become strictly defined and perpetuated. As such, white men’s time served on the border also “toughened” them up and shaped them into hegemonic copies of each other, ready to enforce patriarchal and racist ideologies. In this article, I look at how the novel Moffie by André Carl van der Merwe (2006) illustrates hegemonic white masculinity in South Africa and how it has long been strictly regulated to perpetuate the well-being of the white family as representative of the capitalist state. I discuss the novel by looking at the ways in which the narrator is marked by service in the military, which functions as a socialising agent, but as importantly by the looming threat of the application of the term “moffie” to himself, by self or others.  

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