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

Correlates of multi-drug non-susceptibility in enteric bacteria isolated from Kenyan children with acute diarrhea.

Reduced antimicrobial susceptibility threatens treatment efficacy in sub-Saharan Africa, where data on the burden and correlates of antibiotic resistance among enteric pathogens are limited.Fecal samples from children aged 6 mos-15 yrs presenting with acute diarrhea in western Kenya were cultured for bacterial pathogens. HIV-uninfected children with identified Shigella or Salmonella species or pathogenic Escherichia coli (EPEC, ETEC, EAEC or EIEC) were included in this cross-sectional sub-study. Non-susceptibility to ampicillin, ceftriaxone, ciprofloxacin, cotrimoxazole, and tetracycline was determined using MicroScan Walkaway40 Plus. Multivariable log-binomial regression was used to identify correlates of multi-drug non-susceptibility (MDNS, non-susceptibility to ≥ 3 of these antibiotics).Of 292 included children, median age was 22.5 mos. MDNS was identified in 62.5% of 318 isolates. Non-susceptibility to cotrimoxazole (92.8%), ampicillin (81.3%), and tetracycline (75.0%) was common. Young age (6-24 mos vs. 24-59 mos adjusted prevalence ratio [aPR] = 1.519 [95% confidence interval: 1.19, 1.91]), maternal HIV (aPR = 1.29 [1.01, 1.66]); and acute malnutrition (aPR = 1.28 [1.06, 1.55]) were associated with higher prevalence of MDNS, as were open defecation (aPR = 2.25 [1.13, 4.50]), household crowding (aPR = 1.29 [1.08, 1.53]) and infrequent caregiver hand-washing (aPR = 1.50 [1.15, 1.95]).Young age, HIV exposure, acute malnutrition and poor sanitation may increase risk of antibiotic non-susceptible enteric pathogen infections among children in Kenya

Non-susceptibility patterns of <i>Shigella</i> spp., <i>Salmonella</i> spp., and select <i>E</i>. <i>Coli</i> strains isolated from study participants all Salmonella spp., Shigella spp., EPEC, ETEC, and EIEC isolates were susceptible to ceftriaxone.

<p>All Salmonella spp., Shigella spp., and ETEC isolates were susceptible to ciprofloxacin.</p

Prevalence of multi-drug non-susceptibility (MDNS) by age group, stratified by pathogen.

<p>Prevalence of multi-drug non-susceptibility (MDNS) by age group, stratified by pathogen.</p

Correlates of multi-drug non-susceptibility (MDNS) in enteropathogens in Kenyan children 6 mos–15 yrs with acute diarrhea.

<p>Correlates of multi-drug non-susceptibility (MDNS) in enteropathogens in Kenyan children 6 mos–15 yrs with acute diarrhea.</p

Characteristics of children with and without multi-drug non-susceptible enteric infections.

<p>Characteristics of children with and without multi-drug non-susceptible enteric infections.</p

Specific pathogens identified in the 26 children who had more than one pathogen of interest identified in the same stool sample.

<p>Specific pathogens identified in the 26 children who had more than one pathogen of interest identified in the same stool sample.</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

Summary of AMR genes in the tested population.

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