Development of selective real-time PCR (SPCR) asays for the detection of K103N resistance mutation in minor HIV-1 populations

Thesis (MScMedSc)--Stellenbosch University, 2011.ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%.AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%.Poliomyelitis Research Foundation (PRF)National Research Fund (NRF)National Health Laboratory Service Research Trust (NHLS RT

The role of influenza, RSV and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high HIV sero-prevalence, South Africa, 2012-2015

BACKGROUND : Viruses detected in patients with acute respiratory infections may be the cause of illness or colonizers. METHODS : We compared the prevalence of 10 common respiratory viruses (influenza A and B viruses, parainfluenza virus 1, 2, and 3; respiratory syncytial virus (RSV); adenovirus, rhinovirus, human metapneumovirus (hMPV) and enterovirus) in patients hospitalized with severe acute respiratory illness (SARI), outpatients with influenza-like illness (ILI), and control subjects who did not report any febrile, respiratory or gastrointestinal illness during 2012-2015 in South Africa. We estimated the attributable fraction (AF) and the detection rate attributable to illness for each of the different respiratory viruses. RESULTS : We enrolled 1959 SARI, 3784 ILI and 1793 controls. Influenza virus (AF: 86.3%; 95%CI: 77.7%-91.6%), hMPV (AF: 85.6%%; 95%CI: 72.0%-92.6%), and RSV (AF: 83.7%; 95%CI: 77.5%-88.2%) infections were highly associated with severe disease, while rhinovirus (AF: 46.9%; 95%CI: 37.6%-56.5%) and adenovirus (AF: 36.4%; 95%CI: 20.6%-49.0%) were only moderately associated. The estimated detection rate associated with severe disease was: 20.2% for rhinovirus, 16.7% for RSV, 7.0% for adenovirus, 4.9% for influenza virus and 3.8% for hMPV. Similar patterns were observed for patients with ILI. CONCLUSIONS : Influenza, RSV and hMPV can be considered likely pathogens if detected in patients with ILI and SARI while rhinovirus and adenovirus were commonly identified also among controls suggesting that they may cause only a proportion of clinical disease observed in positive patients. Nonetheless, given their high estimated detection rate attributable to illness, they may be important contributors to disease.Co-operative agreement 5U51/IP000155 with the Centers for Disease Control and Prevention, Atlanta, Georgia, USA.http://www.elsevier.com/locate/jcv2017-02-28hb2016Medical Virolog

Epidemiology of influenza B/Yamagata and B/Victoria lineages in South Africa, 2005-2014

BACKGROUND : Studies describing the epidemiology of influenza B lineages in South Africa are lacking. METHODS : We conducted a prospective study to describe the circulation of influenza B/Victoria and B/ Yamagata lineages among patients of all ages enrolled in South Africa through three respiratory illness surveillance systems between 2005 and 2014: (i) the Viral Watch (VW) program enrolled outpatients with influenza-like illness (ILI) from private healthcare facilities during 2005±2014; (ii) the influenza-like illnesses program enrolled outpatients in public healthcare clinics (ILI/PHC) during 2012±2014; and (iii) the severe acute respiratory illnesses (SARI) program enrolled inpatients from public hospitals during 2009±2014. Influenza B viruses were detected by virus isolation during 2005 to 2009 and by real-time reverse transcription polymerase chain reaction from 2009±2014. Clinical and epidemiological characteristics of patients hospitalized with SARI and infected with different influenza B lineages were also compared using unconditional logistic regression. RESULTS : Influenza viruses were detected in 22% (8,706/39,804) of specimens from patients with ILI or SARI during 2005±2014, of which 24% (2,087) were positive for influenza B. Influenza B viruses predominated in all three surveillance systems in 2010. B/Victoria predominated prior to 2011 (except 2008) whereas B/Yamagata predominated thereafter (except 2012). B lineages co-circulated in all seasons, except in 2013 and 2014 for SARI and ILI/PHC surveillance. Among influenza B-positive SARI cases, the detection of influenza B/Yamagata compared to influenza B/Victoria was significantly higher in individuals aged 45±64 years (adjusted odds ratio [aOR]: 4.2; 95% confidence interval [CI]: 1.1±16.5) and 65 years (aOR: 12.2; 95% CI: 2.3±64.4) compared to children aged 0±4 years, but was significantly lower in HIV-infected patients (aOR: 0.4; 95% CI: 0.2±0.9). CONCLUSION : B lineages co-circulated in most seasons except in 2013 and 2014. Hospitalized SARI cases display differential susceptibility for the two influenza B lineages, with B/Victoria being more prevalent among children and HIV-infected persons.The National Institute for Communicable Diseases (NICD) (http://www.nicd.ac.za/) and the US Centers for Disease Control and Prevention (https://www.cdc. gov/) grant number 5U51/IP000155.http://www.plosone.orgam2017Medical Virolog

Replacement of neuraminidase inhibitor-susceptible influenza A(H1N1) with resistant phenotype in 2008 and circulation of susceptible influenza A and B viruses during 2009-2013, South Africa

BACKGROUND : Data on the susceptibility of influenza viruses from South Africa to neuraminidase inhibitors (NAIs) are scarce, and no extensive analysis was done. OBJECTIVES : We aimed to determine oseltamivir and zanamivir susceptibility of influenza A and B virus neuraminidases (NAs), 2007-2013, South Africa. PATIENTS/METHODS : We enrolled participants through national influenza-like illness surveillance, 2007-2013. Influenza diagnosis was by virus isolation and quantitative polymerase chain reaction (qPCR). Drug susceptibility was determined by chemiluminescence-based NA-STAR/ NA-XTD assay. Sanger sequencing was used to determine molecular markers of NAI resistance. RESULTS : Forty percent (6341/15 985) of participants were positive for influenza viruses using virus isolation (2007-2009) and qPCR (2009-2013) methods. A total of 1236/6341 (19.5%) virus isolates were generated of which 307/1236 (25%) were tested for drug susceptibility. During 2007-2008, the median 50% inhibitory concentration (IC50) of oseltamivir for seasonal influenza A(H1N1) increased from of 0.08 nmol/L (range 0.01-3.60) in 2007 to 73 nmol/L (range 1.56-305 nmol/L) in 2008. Influenza A isolates from 2009 to 2013 were susceptible to oseltamivir [A(H3N2) median IC50 = 0.05 nmol/L (range 0.01-0.08); A(H1N1)pdm09 = 0.11 nmol/L (range 0.01-0.78)] and zanamivir [A(H3N2) median IC50 = 0.56 nmol/L (range 0.47-0.66); A(H1N1)pdm09 = 0.35 nmol/L (range 0.27-0.533)]. Influenza B viruses were susceptible to both NAIs. NAI resistance-associated substitutions H275Y, E119V, and R150K (N1 numbering) were not detected in influenza A viruses that circulated in 2009-2013. CONCLUSIONS : We confirm replacement of NAI susceptible by resistant phenotype influenza A(H1N1) in 2008. Influenza A and B viruses (2009-2013) remained susceptible to NAIs; therefore, these drugs are useful for treating influenza-infected patients.This work was supported by the National Institute for Communicable Diseases of the National Health Laboratory Service and the US Centers for Disease Control and Prevention (cooperative agreement number: 1U51IP000528).The National Institute for Communicable Diseases of the National Health Laboratory Service and the US Centers for Disease Control and Prevention (cooperative agreement number: 1U51IP000528).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1750-2659http://www.wileyonlinelibrary.com/journal/irvam2019Medical Virolog

Epidemiology of influenza B/Yamagata and B/Victoria lineages in South Africa, 2005-2014.

BACKGROUND:Studies describing the epidemiology of influenza B lineages in South Africa are lacking. METHODS:We conducted a prospective study to describe the circulation of influenza B/Victoria and B/Yamagata lineages among patients of all ages enrolled in South Africa through three respiratory illness surveillance systems between 2005 and 2014: (i) the Viral Watch (VW) program enrolled outpatients with influenza-like illness (ILI) from private healthcare facilities during 2005-2014; (ii) the influenza-like illnesses program enrolled outpatients in public healthcare clinics (ILI/PHC) during 2012-2014; and (iii) the severe acute respiratory illnesses (SARI) program enrolled inpatients from public hospitals during 2009-2014. Influenza B viruses were detected by virus isolation during 2005 to 2009 and by real-time reverse transcription polymerase chain reaction from 2009-2014. Clinical and epidemiological characteristics of patients hospitalized with SARI and infected with different influenza B lineages were also compared using unconditional logistic regression. RESULTS:Influenza viruses were detected in 22% (8,706/39,804) of specimens from patients with ILI or SARI during 2005-2014, of which 24% (2,087) were positive for influenza B. Influenza B viruses predominated in all three surveillance systems in 2010. B/Victoria predominated prior to 2011 (except 2008) whereas B/Yamagata predominated thereafter (except 2012). B lineages co-circulated in all seasons, except in 2013 and 2014 for SARI and ILI/PHC surveillance. Among influenza B-positive SARI cases, the detection of influenza B/Yamagata compared to influenza B/Victoria was significantly higher in individuals aged 45-64 years (adjusted odds ratio [aOR]: 4.2; 95% confidence interval [CI]: 1.1-16.5) and ≥65 years (aOR: 12.2; 95% CI: 2.3-64.4) compared to children aged 0-4 years, but was significantly lower in HIV-infected patients (aOR: 0.4; 95% CI: 0.2-0.9). CONCLUSION:B lineages co-circulated in most seasons except in 2013 and 2014. Hospitalized SARI cases display differential susceptibility for the two influenza B lineages, with B/Victoria being more prevalent among children and HIV-infected persons

Risk factors for influenza-associated severe acute respiratory illness hospitalization in South Africa, 2012–2015

BACKGROUND : Data on risk factors for influenza-associated hospitalizations in low- and middle-income countries are limited. METHODS : We conducted active syndromic surveillance for hospitalized severe acute respiratory illness (SARI) and outpatient influenza-like illness (ILI) in 2 provinces of South Africa during 2012–2015. We compared the characteristics of influenza-positive patients with SARI to those with ILI to identify factors associated with severe disease requiring hospitalization, using unconditional logistic regression. RESULTS : During the study period, influenza virus was detected in 5.9% (110 of 1861) and 15.8% (577 of 3652) of SARI and ILI cases, respectively. On multivariable analysis factors significantly associated with increased risk of influenza-associated SARI hospitalization were as follows: younger and older age (<6 months [adjusted odds ratio {aOR}, 37.6], 6–11 months [aOR, 31.9], 12–23 months [aOR, 22.1], 24–59 months [aOR, 7.1], and ≥65 years [aOR, 40.7] compared with 5–24 years of age), underlying medical conditions (aOR, 4.5), human immunodeficiency virus infection (aOR, 4.3), and Streptococcus pneumoniae colonization density ≥1000 deoxyribonucleic acid copies/mL (aOR, 4.8). Underlying medical conditions in children aged <5 years included asthma (aOR, 22.7), malnutrition (aOR, 2.4), and prematurity (aOR, 4.8); in persons aged ≥5 years, conditions included asthma (aOR, 3.6), diabetes (aOR, 7.1), chronic lung diseases (aOR, 10.7), chronic heart diseases (aOR, 9.6), and obesity (aOR, 21.3). Mine workers (aOR, 13.8) and pregnant women (aOR, 12.5) were also at increased risk for influenza-associated hospitalization. CONCLUSIONS : The risk groups identified in this study may benefit most from annual influenza immunization, and children <6 months of age may be protected through vaccination of their mothers during pregnancyThe National Institute for Communicable Diseases (National Health Laboratory Service) and the US Centers for Disease Control and Prevention (cooperative agreement number 5U51IP000155).https://academic.oup.com/ofidam2018Medical Virolog

Detection rates for influenza viruses in the three surveillance program, Viral Watch (VW), Illnesses-like influenza surveillance in public health clinics (ILI/PHC) and severe acute respiratory illnesses (SARI) in South Africa, 2005–2014.

<p>Detection rates for influenza viruses in the three surveillance program, Viral Watch (VW), Illnesses-like influenza surveillance in public health clinics (ILI/PHC) and severe acute respiratory illnesses (SARI) in South Africa, 2005–2014.</p

Proportion of typed samples by influenza B Ct-value among patients with SARI.

<p>Proportion of typed samples by influenza B Ct-value among patients with SARI.</p

Monthly number of influenza-positive specimens in South Africa.

<p>(<b>A</b>) Viral Watch program (2005–2014), (<b>B</b>) Severe acute respiratory illness surveillance program (2009–2014), and (<b>C</b>) Influenza-like illness surveillance program in public health clinics (2012–2014).</p