Patient experiences of switching from Efavirenz- to Dolutegravir-based antiretroviral therapy: a qualitative study in Uganda.

BACKGROUND: In 2019, the World Health Organisation (WHO) recommended Dolutegravir (DTG) as the preferred first-line antiretroviral treatment (ART) for all persons with HIV. ART regimen switches may affect HIV treatment adherence. We sought to describe patient experiences switching from EFV to DTG-based ART in Kampala, Uganda. METHODS: Between July and September 2019, we purposively sampled adults living with HIV who had switched to DTG at the Infectious Diseases Institute HIV clinic. We conducted in-depth interviews with adults who switched to DTG, to explore their preparation to switch and experiences on DTG. Interviews were audio-recorded, transcribed and analysed thematically using Atlas ti version 8 software. RESULTS: We interviewed 25 adults: 18 (72%) were women, and the median age was 35 years (interquartile range [IQR] 30-40). Median length on ART before switching to DTG was 67 months (IQR 51-125). Duration on DTG after switching was 16 months (IQR 10-18). Participants reported accepting provider recommendations to switch to DTG mainly because they anticipated that swallowing a smaller pill once a day would be more convenient. While most participants initially felt uncertain about drug switching, their providers offer of frequent appointments and a toll-free number to call in the event of side effects allayed their anxiety. At the same time, participants said they felt rushed to switch to the new ART regimen considering that they had been on their previous regimen(s) for several years and the switch to DTG happened during a routine visit when they had expected their regular prescription. Some participants felt unprepared for new adverse events associated with DTG and for the abrupt change in treatment schedule. Most participants said they needed additional support from their health providers before and after switching to DTG. CONCLUSION AND RECOMMENDATIONS: Adults living with HIV stable on an EFV-based regimen but were switched to DTG in a program-wide policy change found the duration between counselling and drug switching inadequate. DTG was nonetheless largely preferred because of the small pill size, once daily dosing, and absence of EFV-like side effects. Community-engaged research is needed to devise acceptable ways to prepare participants for switching ART at scale.

Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

Detection of monkeypox virus with real-time PCR assays.

BACKGROUND: Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak. OBJECTIVES: We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak. STUDY DESIGN: A TaqMan-based assay (E9L-NVAR) targets the orthopoxvirus DNA polymerase gene and detects Eurasian orthopoxviruses other than Variola. A hybridization assay, utilizing a MGB Eclipsetrade mark (Epoch Biosciences) probe, targets an envelope protein gene (B6R) and specifically detects monkeypox virus (MPXV). Assays were validated using coded orthopoxvirus DNA samples and used to evaluate lesion samples from five confirmed US monkeypox cases. RESULTS: E9L-NVAR did not detect variola (48 strains), North American orthopoxviruses (2), or DNA derived from non-poxviral rash illnesses. The assay reproducibly identified various concentrations of 13 Eurasian orthopoxvirus strains and was sensitive to 12.5 vaccinia genomes. The B6R assay recognized 15 different MPXV strains, while other orthopoxvirus (9) and bacteria (15) strains did not cross-react. Of the 13 human samples tested from confirmed cases, both assays identified 100% as containing MPXV DNA. CONCLUSIONS: E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections.

Genome sequence diversity and clues to the evolution of variola (smallpox) virus.

Comparative genomics of 45 epidemiologically varied variola virus isolates from the past 30 years of the smallpox era indicate low sequence diversity, suggesting that there is probably little difference in the isolates' functional gene content. Phylogenetic clustering inferred three clades coincident with their geographical origin and case-fatality rate; the latter implicated putative proteins that mediate viral virulence differences. Analysis of the viral linear DNA genome suggests that its evolution involved direct descent and DNA end-region recombination events. Knowing the sequences will help understand the viral proteome and improve diagnostic test precision, therapeutics, and systems for their assessment.

Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus.

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus ORTHOPOXVIRUS: A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T(m)s) determined for 46 strains of variola virus (T(m)s, 55.9 to 57.8 degrees C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (T(m)s, 61.7 to 62.7 degrees C), 15 strains of monkeypox virus (T(m)s, 61.9 to 62.2 degrees C), 40 strains of cowpox virus (T(m)s, 61.3 to 63.7 degrees C), 8 strains of mousepox virus (T(m), 61.9 degrees C), and 8 strains of camelpox virus (T(m)s, 64.0 to 65.0 degrees C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.

Random amplified polymorphic DNA and amplified fragment length polymorphism analyses of Pasteurella multocida isolates from fatal fowl cholera infections.

Fowl cholera, a disease caused by Pasteurella multocida, continues to be a major problem for the poultry industry. The sources of pathogenic organisms responsible for most sporadic epidemics remain unconfirmed, although attenuated vaccines that retain a low level of virulence have occasionally been implicated in outbreaks of the disease. One of the vaccines most commonly used to prevent fowl cholera is the M-9 strain. In the present study, 61 clinical isolates from turkeys that died of fowl cholera from 1997 to 1999 on 36 Utah farms were analyzed and compared to the M-9 vaccine strain. Genetic analyses of the isolates were done by random amplified polymorphic DNA (RAPD) analysis and amplified fragment length polymorphism (AFLP) fingerprinting. The results of these genetic analyses were correlated with the vaccination status of the flock, isolate serotype, and geographic location. Although both genetic techniques effectively identified similar subtle genomic differences, RAPD analysis provided only 77% of the detail provided by AFLP analysis. While a relationship between genetic profile and serotype was evident, no significant relationship indicating geographic influence was found (P = 0.351). Interestingly, organisms isolated from vaccinated flocks were significantly closer genetically to the M-9 vaccine strain than isolates from unvaccinated birds were (P = 0.020). Statistical analyses revealed that this relationship could not have been determined by serotyping alone (P = 0.320), demonstrating the value of AFLP and RAPD analyses in the characterization of disease-causing strains.