Evaluation of the performance of a multiplex reverse transcription polymerase chain reaction kit as a potential diagnostic and surveillance kit for rotavirus in Kenya

Abstract Background Diarrhea is a serious concern worldwide, especially in developing countries. Rotavirus is implicated in approximately 400,000 infant deaths annually. It is highly contagious elevating the risk of outbreaks especially in enclosed settings such as daycare centers, hospitals, and boarding schools. Reliable testing methods are critical for early detection of infections, better clinical management, pathogen surveillance and evaluation of interventions such as vaccines. Enzyme immunoassays have proved to be reliable and practical in most settings; however, newer multiplex reverse transcription polymerase assays have been introduced in the Kenya market but have not been evaluated locally. Methods Stool samples collected from an ongoing Surveillance of Enteric Pathogens Causing diarrheal illness in Kenya (EPS) study were used to compare an established enzyme immunoassay, Premier™ Rotaclone® (Meridian Bioscience, Cincinnati, Ohio, U.S.A.), that can only detect group A rotavirus against a novel multiplex reverse transcription polymerase chain reaction kit, Seeplex® Diarrhea-V ACE Detection (Seegene, Seoul, Republic of Korea), that can detect rotavirus, astrovirus, adenovirus, and norovirus genogroups I and II. Detection frequency, sensitivity, specificity, turnaround time, and cost were compared to determine the suitability of each assay for clinical work in austere settings versus public health work in well-funded institutes in Kenya. Results The Premier™ Rotaclone® kit had a detection frequency of 11.2%, sensitivity of 77.8%, specificity of 100%, turnaround time of 93 min and an average cost per sample of 13.33 United States dollars (USD). The Seeplex® Diarrhea-V ACE Detection kit had a detection frequency of 16.0%, sensitivity of 100%, specificity of 98.1%, turnaround time of 359 min and an average cost per samples 32.74 United States dollars respectively. The detection frequency sensitivity and specificity of the Seeplex® Diarrhea-V ACE Detection kit mentioned above are for rotavirus only. Conclusions The higher sensitivity and multiplex nature of the Seeplex® Diarrhea-V ACE Detection kit make it suitable for surveillance of enteric viruses circulating in Kenya. However, its higher cost, longer turnaround time and complexity favor well-resourced clinical labs and research applications. The Premier™ Rotaclone®, on the other hand, had a higher specificity, shorter turnaround time, and lower cost making it more attractive for clinical work in low complexity labs in austere regions of the country. It is important to continuously evaluate assay platforms’ performance, operational cost, turnaround time, and usability in different settings so as to ensure quality results that are useful to the patients and public health practitioners.https://deepblue.lib.umich.edu/bitstream/2027.42/152177/1/40794_2019_Article_87.pd

Identification of a novel late domain in human parainfluenza virus type 3 matrix protein

Human parainfluenza virus type 3 (HPIV3) is a major cause of bronchiolitis and pneumonia in infants under 6 months of age. Like other enveloped RNA viruses, HPIV3 encodes a matrix (M) protein involved in the final assembly and budding steps of the viral life cycle. Illustrating the important role of M protein is its ability to induce its own budding from cells in the form of enveloped virus-like particles (VLPs). For related viruses, the feature of viral M proteins that allows this independent budding to occur is a critical amino acid sequence, called a late (L) domain, which interacts with the host cell vesicle-forming machinery. To identify the HPIV3 L domains, we selected four HPIV3 M protein sequences (PPKH, YLDV, KPEL, and YPNI) based on their sequence similarity to established L domains and made alanine-substitution mutants of each potential L domain sequence. When these mutant M proteins were expressed in cells, we found that disrupting the YLDV sequence caused a severe budding defect. To confirm these results, we then inserted these sequences into poorly budding L domain-deficient mutants of the Ebola virus matrix protein. Consistent with our previous findings, the YLDV-containing VP40-delta N13 mutant was able to restore budding efficiency to levels on par with wild-type VP40. These findings provide strong evidence to support the likelihood that the YLDV sequence of HPIV3 M protein functions as an L domain

Reverse genetics systems as tools to overcome the genetic diversity of Lassa virus

Lassa virus is endemic in a large area of sub-Saharan Africa, and exhibits a large amount of genetic diversity. Of the four currently recognized lineages, lineages I–III circulate in Nigeria, and lineage IV circulates in Sierra Leone, Guinea, and Liberia. However, several newly detected lineages have been proposed. LASV genetic diversity may result in differences in pathogenicity or response to medical countermeasures, necessitating the testing of multiple lineages during the development of countermeasures and diagnostics. Logistical and biosafety concerns can make it difficult to obtain representative collections of divergent LASV clades for comparison studies. For example, lack of a cold chain in remote areas, or shipping restrictions on live viruses can prevent the dissemination of natural virus isolates to researchers. Reverse genetics systems that have been developed for LASV can facilitate acquisition of hard-to-obtain LASV strains and enable comprehensive development of medical countermeasures

Summary of AMR genes in the tested population.

<p>Summary of AMR genes in the tested population.</p

Antimicrobial resistance of <i>Klebsiella pneumoniae</i> stool isolates circulating in Kenya

<div><p>We sought to determine the genetic and phenotypic antimicrobial resistance (AMR) profiles of commensal <i>Klebsiella</i> spp. circulating in Kenya by testing human stool isolates of 87 <i>K</i>. <i>pneumoniae</i> and three <i>K</i>. <i>oxytoca</i> collected at eight locations. Over one-third of the isolates were resistant to ≥3 categories of antimicrobials and were considered multidrug-resistant (MDR). We then compared the resistance phenotype to the presence/absence of 238 AMR genes determined by a broad-spectrum microarray and PCR. Forty-six genes/gene families were identified conferring resistance to β-lactams (<i>ampC</i>/<i>bla</i>_DHA, <i>bla</i>_CMY/LAT, <i>bla</i>_LEN-1, <i>bla</i>_OKP-A/OKP-B1, <i>bla</i>_OXA-1-like family, <i>bla</i>_OXY-1, <i>bla</i>_SHV, <i>bla</i>_TEM, <i>bla</i>_CTX-M-1 and <i>bla</i>_CTX-M-2 families), aminoglycosides (<i>aac(3)-III</i>, <i>aac(6)-Ib</i>, <i>aad</i>(A1/A2), <i>aad</i>(A4), <i>aph</i>(AI), <i>aph3/str</i>(A), <i>aph6/str</i>(B), and <i>rmtB</i>), macrolides (<i>mac</i>(A), <i>mac</i>(B), <i>mph</i>(A)<i>/mph</i>(K)), tetracyclines (<i>tet</i>(A), <i>tet</i>(B), <i>tet</i>(D), <i>tet</i>(G)), ansamycins (<i>arr</i>), phenicols (<i>catA1/cat4</i>, <i>floR</i>, <i>cmlA</i>, <i>cmr</i>), fluoroquinolones (<i>qnrS</i>), quaternary amines (<i>qacE</i>Δ<i>1</i>), streptothricin (<i>sat2</i>), sulfonamides (<i>sul1</i>, <i>sul2</i>, <i>sul3</i>), and diaminopyrimidines (<i>dfrA1</i>, <i>dfrA5</i>, <i>dfrA7</i>, <i>dfrA8</i>, <i>dfrA12</i>, <i>dfrA13/21/22/23</i> family, <i>dfrA14</i>, <i>dfrA15</i>, <i>dfrA16</i>, <i>dfrA17</i>). This is the first profile of genes conferring resistance to multiple categories of antimicrobial agents in western and central Kenya. The large number and wide variety of resistance genes detected suggest the presence of significant selective pressure. The presence of five or more resistance determinants in almost two-thirds of the isolates points to the need for more effective, targeted public health policies and infection control/prevention measures.</p></div

Summary of antimicrobial phenotypic susceptibility for diarrheal and control isolates.

<p>Summary of antimicrobial phenotypic susceptibility for diarrheal and control isolates.</p

Genomic Surveillance of Rabies Virus in Georgian Canines

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685)

Metadata and phenotypic antimicrobial susceptibility for individual isolates.

<p>Metadata and phenotypic antimicrobial susceptibility for individual isolates.</p