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Clinical Evaluation of a Multiplex PCR for the Detection of Salmonella enterica Serovars Typhi and Paratyphi A from Blood Specimens in a High-Endemic Setting.

ABSTRACT: Enteric fever is a major public health concern in endemic areas, particularly in infrastructure-limited countries where Salmonella Paratyphi A has emerged in increasing proportion of cases. We aimed to evaluate a method to detect Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A (S. Paratyphi A) in febrile patients in Bangladesh. We conducted a prospective study enrolling patients with fever > 38°C admitted to two large urban hospitals and two outpatient clinics located in Dhaka, Bangladesh. We developed and evaluated a method combining short culture with a new molecular assay to simultaneously detect and differentiate S. Typhi and S. Paratyphi A from other Salmonella directly from 2 to 4 mL of whole blood in febrile patients (n = 680). A total of 680 cases were enrolled from the four participating sites. An increase in the detection rate (+38.8%) in S. Typhi and S. Paratyphi A was observed with a multiplex polymerase chain reaction (PCR) assay, and absence of non-typhoidal Salmonella detection was reported. All 45 healthy controls were culture and PCR negative, generating an estimated 92.9% of specificity on clinical samples. When clinical performance was assessed in the absence of blood volume prioritization for testing, a latent class model estimates clinical performance ≥ 95% in sensitivity and specificity with likelihood ratio (LR) LR+ > 10 and LR- < 0.1 for the multiplex PCR assay. The alternative method to blood culture we developed may be useful alone or in combination with culture or serological tests for epidemiological studies in high disease burden settings and should be considered as secondary endpoint test for future vaccine trials.

SUBMITTER: Pouzol S

PROVIDER: S-EPMC6726943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Of the > 2000 serovars of Salmonella enterica subspecies I, most cause self-limiting gastrointestinal disease in a wide range of mammalian hosts. However, S. enterica serovars Typhi and Paratyphi A are restricted to the human host and cause the similar systemic diseases typhoid and paratyphoid fever. Genome sequence similarity between Paratyphi A and Typhi has been attributed to convergent evolution via relatively recent recombination of a quarter of their genomes. The accumulation of pseudogenes is a key feature of these and other host-adapted pathogens, and overlapping pseudogene complements are evident in Paratyphi A and Typhi. RESULTS:We report the 4.5 Mbp genome of a clinical isolate of Paratyphi A, strain AKU_12601, completely sequenced using capillary techniques and subsequently checked using Illumina/Solexa resequencing. Comparison with the published genome of Paratyphi A ATCC9150 revealed the two are collinear and highly similar, with 188 single nucleotide polymorphisms and 39 insertions/deletions. A comparative analysis of pseudogene complements of these and two finished Typhi genomes (CT18, Ty2) identified several pseudogenes that had been overlooked in prior genome annotations of one or both serovars, and identified 66 pseudogenes shared between serovars. By determining whether each shared and serovar-specific pseudogene had been recombined between Paratyphi A and Typhi, we found evidence that most pseudogenes have accumulated after the recombination between serovars. We also divided pseudogenes into relative-time groups: ancestral pseudogenes inherited from a common ancestor, pseudogenes recombined between serovars which likely arose between initial divergence and later recombination, serovar-specific pseudogenes arising after recombination but prior to the last evolutionary bottlenecks in each population, and more recent strain-specific pseudogenes. CONCLUSION:Recombination and pseudogene-formation have been important mechanisms of genetic convergence between Paratyphi A and Typhi, with most pseudogenes arising independently after extensive recombination between the serovars. The recombination events, along with divergence of and within each serovar, provide a relative time scale for pseudogene-forming mutations, affording rare insights into the progression of functional gene loss associated with host adaptation in Salmonella.

Project description:BackgroundEnteric fever, a systemic infection caused by Salmonella enterica serovars Typhi and Paratyphi A, remains a major cause of morbidity and mortality in low-income and middle-income countries. Enteric fever is preventable through the provision of clean water and adequate sanitation and can be successfully treated with antibiotics. However, high levels of antimicrobial resistance (AMR) compromise the effectiveness of treatment. We provide estimates of the prevalence of AMR S Typhi and S Paratyphi A in 75 endemic countries, including 30 locations without data.MethodsWe used a Bayesian spatiotemporal modelling framework to estimate the percentage of multidrug resistance (MDR), fluoroquinolone non-susceptibility (FQNS), and third-generation cephalosporin resistance in S Typhi and S Paratyphi A infections for 1403 administrative level one districts in 75 endemic countries from 1990 to 2019. We incorporated data from a comprehensive systematic review, public health surveillance networks, and large multicountry studies on enteric fever. Estimates of the prevalence of AMR and the number of AMR infections (based on enteric fever incidence estimates by the Global Burden of Diseases study) were produced at the country, super-region, and total endemic area level for each year of the study.FindingsWe collated data from 601 sources, comprising 184 225 isolates of S Typhi and S Paratyphi A, covering 45 countries over 30 years. We identified a decline of MDR S Typhi in south Asia and southeast Asia, whereas in sub-Saharan Africa, the overall prevalence increased from 6·0% (95% uncertainty interval 4·3-8·0) in 1990 to 72·7% (67·7-77·3) in 2019. Starting from low levels in 1990, the prevalence of FQNS S Typhi increased rapidly, reaching 95·2% (91·4-97·7) in south Asia in 2019. This corresponded to 2·5 million (1·5-3·8) MDR S Typhi infections and 7·4 million (4·7-11·3) FQNS S Typhi infections in endemic countries in 2019. The prevalence of third-generation cephalosporin-resistant S Typhi remained low across the whole endemic area over the study period, except for Pakistan where prevalence of third-generation cephalosporin resistance in S Typhi reached 61·0% (58·0-63·8) in 2019. For S Paratyphi A, we estimated low prevalence of MDR and third-generation cephalosporin resistance in all endemic countries, but a drastic increase of FQNS, which reached 95·0% (93·7-96·1; 3·5 million [2·2-5·6] infections) in 2019.InterpretationThis study provides a comprehensive and detailed analysis of the prevalence of MDR, FQNS, and third-generation cephalosporin resistance in S Typhi and S Paratyphi A infections in endemic countries, spanning the last 30 years. Our analysis highlights the increasing levels of AMR in this preventable infection and serves as a resource to guide urgently needed public health interventions, such as improvements in water, sanitation, and hygiene and typhoid fever vaccination campaigns.FundingFleming Fund, UK Department of Health and Social Care; Wellcome Trust; and Bill and Melinda Gates Foundation.

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Clinical Evaluation of a Multiplex PCR for the Detection of Salmonella enterica Serovars Typhi and Paratyphi A from Blood Specimens in a High-Endemic Setting.

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