A portable label-free electrochemical DNA biosensor for rapid detection of Salmonella Typhi.

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi (S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 µM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.

Modelling Salmonella Typhi in high-density urban Blantyre neighbourhood, Malawi, using point pattern methods.

Salmonella Typhi is a human-restricted pathogen that is transmitted by the faecal-oral route and causative organism of typhoid fever. Using health facility data from 2016 to 2020, this study focuses on modelling the spatial variation in typhoid risk in Ndirande township in Blantyre. To pursue this objective, we developed a marked inhomogeneous Poisson process model that allows us to incorporate both individual-level and environmental risk factors. The results from our analysis indicate that typhoid cases are spatially clustered, with the incidence decreasing by 54% for a unit increase in the water, sanitation, and hygiene (WASH) score. Typhoid intensity was also higher in children aged below 18 years than in adults. However, our results did not show evidence of a strong temporal variation in typhoid incidence. We also discuss the inferential benefits of using point pattern models to characterise the spatial variation in typhoid risk and outline possible extensions of the proposed modelling framework.

Extensively Drug-Resistant Salmonella Typhi: A National Disaster.

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Molecular Detection of Salmonella typhi Isolated from Patients Undergoing Gallbladder Cholecystectomy in Baghdad.

BACKGROUND: Salmonella typhi is a specific strain of the Salmonella bacterium, responsible for triggering typhoid fever; a significant public health concern in developing nations. OBJECTIVE: The current study aimed to identify the bacteria from the gallbladder, taken during cholecystectomies of patients, by isolating Salmonella typhi and by using microscopic characteristics, biochemical and polymerase chain reaction (PCR) tests. METHODS: A total of 120 specimens were collected from the Baghdad Teaching Hospital, Iraq. A cross-sectional descriptive study was carried out from October, 2021, to July, 2022. During that study, 26 (54.2%) male patient tested positive for Salmonella typhias well as 22 (45.8%) female patients. The age of the patients varied from < 30 to > 60 years. p-value > 0.05 was considered significant to confirm a relationship between age and Salmonella typhi effect for patients. RESULTS: Out of the 120 blood samples taken for this study, 48 (40%) tested positive by use of PCR test, 40 (33.3%) tested positive by use of the Widal test, 35 (29.1%) were positive for biopsy culture, and 35 (29.1%) were positive for blood culture. All Salmonella typhi isolates were found to be sensitive to the imipenem, cefepime, and ceftriaxone, but were resistant to gentamycin, ciprofloxacin, amikacin, erythromycin, and tetracycline (72%, 29%, 43%, 100%, 100%, respectively). CONCLUSIONS: The real time polymerase chain reaction (RT-PCR) tests and the Vitek 2 compact system showed a high level of accuracy in the detection of Salmonella typhi. Multidrug resistance was observed, which should be a signal to reduce antibiotic consumption.

Salmonella Typhi genotypic diversity, cluster identification and antimicrobial resistance determinants in Mukuru settlement, Nairobi Kenya.

BACKGROUND: Understanding the source of typhoid infections and the genetic relatedness of Salmonella Typhi (S. Typhi) by cluster identification in endemic settings is critical for establishing coordinated public health responses for typhoid fever management. This study investigated the genotypic diversity, antibiotic resistance mechanisms, and clustering of 35 S.Typhi strains isolated from cases and carriers in the Mukuru Informal Settlement. METHODS: We studied 35 S.Typhi isolates, including 32 from cases and 3 from carriers, from study participants in the informal settlement of Mukuru, Nairobi, Kenya. Genomic DNA was extracted, and whole-genome sequencing (WGS) was performed to determine the phylogenetic relatedness of strains and detect antimicrobial resistance determinants (AMR). WGS data were analyzed using bioinformatics tools available at the Center for Genomic Epidemiology and Pathogenwatch platforms. RESULTS: Genotype 4.3.1.2 EA3 was found to be dominant at 46% (16/35), followed by 4.3.1.2 EA2 at 28% (10/35), and 4.3.1.1 EA1 at 27% (9/35). A comparison of the isolates with global strains from Pathogenwatch identified close clustering with strains from Uganda, Tanzania, Rwanda, and India. Three isolates (9%) distributed in each cluster were isolated from carriers. All genotype 4.3.1.2 EA3 isolates were genotypically multidrug-resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Single mutations in the quinolone resistance-determining region were identified in the gyrA (S83Y) and gyrB (S464F) genes. All isolates associated with multidrug resistance showed the presence of the IncQ1 plasmid with the following genes: blaTEM-1B, catA1, sul1, sul2, and dfrA7. CONCLUSION: The close phylogenetic relatedness between antimicrobial-resistant case isolates and carriage isolates indicates that typhoid carriage is a possible source of infection in the community. Comparative analysis with global isolates revealed that the Kenyan isolates share common lineages with strains from neighboring East African countries and India, suggesting regional dissemination of specific MDR clones. AMR was a major feature of the isolates. Surveillance and testing for antimicrobial susceptibility should inform options for the management of cases.

Pathogen diversity and antimicrobial resistance transmission of Salmonella enterica serovars Typhi and Paratyphi A in Bangladesh, Nepal, and Malawi: a genomic epidemiological study.

BACKGROUND: Enteric fever is a serious public health concern. The causative agents, Salmonella enterica serovars Typhi and Paratyphi A, frequently have antimicrobial resistance (AMR), leading to limited treatment options and poorer clinical outcomes. We investigated the genomic epidemiology, resistance mechanisms, and transmission dynamics of these pathogens at three urban sites in Africa and Asia. METHODS: S Typhi and S Paratyphi A bacteria isolated from blood cultures of febrile children and adults at study sites in Dhaka (Bangladesh), Kathmandu (Nepal), and Blantyre (Malawi) during STRATAA surveillance were sequenced. Isolates were charactered in terms of their serotypes, genotypes (according to GenoTyphi and Paratype), molecular determinants of AMR, and population structure. We used phylogenomic analyses incorporating globally representative genomic data from previously published surveillance studies and ancestral state reconstruction to differentiate locally circulating from imported pathogen AMR variants. Clusters of sequences without any single-nucleotide variants in their core genome were identified and used to explore spatiotemporal patterns and transmission dynamics. FINDINGS: We sequenced 731 genomes from isolates obtained during surveillance across the three sites between Oct 1, 2016, and Aug 31, 2019 (24 months in Dhaka and Kathmandu and 34 months in Blantyre). S Paratyphi A was present in Dhaka and Kathmandu but not Blantyre. S Typhi genotype 4.3.1 (H58) was common in all sites, but with different dominant variants (4.3.1.1.EA1 in Blantyre, 4.3.1.1 in Dhaka, and 4.3.1.2 in Kathmandu). Multidrug resistance (ie, resistance to chloramphenicol, co-trimoxazole, and ampicillin) was common in Blantyre (138 [98%] of 141 cases) and Dhaka (143 [32%] of 452), but absent from Kathmandu. Quinolone-resistance mutations were common in Dhaka (451 [>99%] of 452) and Kathmandu (123 [89%] of 138), but not in Blantyre (three [2%] of 141). Azithromycin-resistance mutations in acrB were rare, appearing only in Dhaka (five [1%] of 452). Phylogenetic analyses showed that most cases derived from pre-existing, locally established pathogen variants; 702 (98%) of 713 drug-resistant infections resulted from local circulation of AMR variants, not imported variants or recent de novo emergence; and pathogen variants circulated across age groups. 479 (66%) of 731 cases clustered with others that were indistinguishable by point mutations; individual clusters included multiple age groups and persisted for up to 2·3 years, and AMR determinants were invariant within clusters. INTERPRETATION: Enteric fever was associated with locally established pathogen variants that circulate across age groups. AMR infections resulted from local transmission of resistant strains. These results form a baseline against which to monitor the impacts of control measures. FUNDING: Wellcome Trust, Bill & Melinda Gates Foundation, EU Horizon 2020, and UK National Institute for Health and Care Research.

Mobile genetic elements define the non-random structure of the Salmonella enterica serovar Typhi pangenome.

Bacterial relatedness measured using select chromosomal loci forms the basis of public health genomic surveillance. While approximating vertical evolution through this approach has proven exceptionally valuable for understanding pathogen dynamics, it excludes a fundamental dimension of bacterial evolution-horizontal gene transfer. Incorporating the accessory genome is the logical remediation and has recently shown promise in expanding epidemiological resolution for enteric pathogens. Employing k-mer-based Jaccard index analysis, and a novel genome length distance metric, we computed pangenome (i.e., core and accessory) relatedness for the globally important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both vertical (homology-by-descent) and horizontal (homology-by-admixture) evolutionary relationships in a reticulate network of over 2,200 U.S. Typhi genomes. This analysis revealed non-random structure in the Typhi pangenome that is driven predominantly by the gain and loss of mobile genetic elements, confirming and expanding upon known epidemiological patterns, revealing novel plasmid dynamics, and identifying avenues for further genomic epidemiological exploration. With an eye to public health application, this work adds important biological context to the rapidly improving ways of analyzing bacterial genetic data and demonstrates the value of the accessory genome to infer pathogen epidemiology and evolution.IMPORTANCEGiven bacterial evolution occurs in both vertical and horizontal dimensions, inclusion of both core and accessory genetic material (i.e., the pangenome) is a logical step toward a more thorough understanding of pathogen dynamics. With an eye to public, and indeed, global health relevance, we couple contemporary tools for genomic analysis with decades of research on mobile genetic elements to demonstrate the value of the pangenome, known and unknown, annotated, and hypothetical, for stratification of Salmonella enterica serovar Typhi (Typhi) populations. We confirm and expand upon what is known about Typhi epidemiology, plasmids, and antimicrobial resistance dynamics, and offer new avenues of exploration to further deduce Typhi ecology and evolution, and ultimately to reduce the incidence of human disease.

Tracking the shift in enteric fever trends and evolving antibiotic sensitivity patterns.

Objective: This study aims to examine the frequency of Salmonella Paratyphi found in blood cultures and evaluate the antibiotic susceptibility pattern of Salmonella isolates to different antibiotics. Additionally, the study aims to assess the paradigm shift in the trend of enteric fever caused by Salmonella Typhi (S. Typhi) to Salmonella Paratyphi(S. Paratyphi) . Study Design: Retrospective study. Participant: The study enrolled patients aged 12 years and above diagnosed with enteric fever (positive blood culture) and admitted to Peelamedu Samanaidu Govindasamy Naidu (PSG) Hospital. Interventions: The study analyzed demographic and antibiotic susceptibility profiles of Salmonella isolates collected from 106 enteric fever patients in the hospital between 2010 and 2022. The susceptibility profiles of Salmonella isolates to multiple antibiotics were assessed. Results: There were 106 participants, and 95 (89.62%) of them had enteric fever linked to Salmonella Typhi, while only 11 (10.38%) had enteric fever linked to Salmonella Paratyphi A. From 2010 to 2022, the study discovered a general decline in the prevalence of enteric fever caused by Salmonella species. But between 2014 and 2022, the incidence of enteric fever linked to S. Typhi rapidly increased. Azithromycin (100% , n = 106) and ceftriaxone (99% , n = 105) were highly effective against the Salmonella isolates, whereas nalidixic acid was resisted by 3 isolates (4.72%, n = 3). Conclusion: The study observed a higher incidence of Salmonella Typhi in comparison to Paratyphi A and a greater susceptibility of males to enteric fever. Funding: None declared.

Linking epidemiological and genomic data in cases of enteric fever in England to inform clinical management and public health action.

OBJECTIVES: To explore the feasibility of linking data from enhanced surveillance patient questionnaires from each enteric fever case in England with genome sequencing data, including antimicrobial resistance (AMR) profiles, from the corresponding isolate of typhoidal salmonellae. METHODS: After linking data we interrogated the merged dataset and assessed the utility of passive surveillance data to match and monitor antimicrobial treatment regimens in enteric fever patients with the AMR profiles of the infectious agent. RESULTS: A high proportion of cases were given antibiotics (n = 1230/1415; 86.9%); half of the cases stated the class of antibiotic they were given (n = 630/1239) and half were prescribed cephalosporins (n = 316/630). Reported treatment with a combination of antibiotics increased with symptom severity. Nearly half of isolates (n = 644/1415; 45.5%) had mutations conferring resistance to ciprofloxacin. Based on genome-derived AMR profiles, typhoidal salmonellae isolates inferred to be susceptible to the recommended first-line antimicrobials were twice as likely to be isolated from individuals residing in the least deprived areas compared with the most deprived (n = 26/169; 15.4% versus n = 32/442; 7.2%). CONCLUSIONS: Due to the high proportion of missing data obtained from patient interviews, we recommend a more transparent and systematic approach to recording the antibiotic prescription details by healthcare professionals in primary and secondary care. A more robust approach to data capture at this point in the care pathway would enable us to audit inconsistencies in the prescribing algorithms across England and ensure equitable treatment across all sections of society. Integrating prescribing data with the genome-derived AMR profiles of the causative agent at the individual patient level provides an opportunity to monitor the impact of treatment on clinical outcomes, and to promote best practice in real time.

The origins of haplotype 58 (H58) Salmonella enterica serovar Typhi.

Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi (S. Typhi) is commonly associated with the H58 lineage, a lineage that arose comparatively recently before becoming globally disseminated. To better understand when and how H58 emerged and became dominant, we performed detailed phylogenetic analyses on contemporary genome sequences from S. Typhi isolated in the period spanning the emergence. Our dataset, which contains the earliest described H58 S. Typhi organism, indicates that ancestral H58 organisms were already multi-drug resistant (MDR). These organisms emerged spontaneously in India in 1987 and became radially distributed throughout South Asia and then globally in the ensuing years. These early organisms were associated with a single long branch, possessing mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent use of fluoroquinolones led to several independent mutations in gyrA. The ability of H58 to acquire and maintain AMR genes continues to pose a threat, as extensively drug-resistant (XDR; MDR plus resistance to ciprofloxacin and third generation cephalosporins) variants, have emerged recently in this lineage. Understanding where and how H58 S. Typhi originated and became successful is key to understand how AMR drives successful lineages of bacterial pathogens. Additionally, these data can inform optimal targeting of typhoid conjugate vaccines (TCVs) for reducing the potential for emergence and the impact of new drug-resistant variants. Emphasis should also be placed upon the prospective identification and treatment of chronic carriers to prevent the emergence of new drug resistant variants with the ability to spread efficiently.

The frequency and associated factors of typhoid carriage in patients undergoing cholecystectomy for gallbladder disease in Pakistan: A cross-sectional study.

BACKGROUND: Enteric fever is caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A, B, and C. It continues to be a significant cause of morbidity and mortality worldwide. In highly endemic areas, children are disproportionately affected, and antimicrobial resistance reduces therapeutic options. It is estimated that 2-5% of enteric fever patients develop chronic asymptomatic infection. These carriers may act as reservoirs of infection; therefore, the prospective identification and treatment of carriers are critical for long-term disease control. We aimed to find the frequency of Salmonella Typhi carriers in patients undergoing cholecystectomy. We also compared the detection limit of culturing versus qPCR in detecting S. Typhi, performed a geospatial analysis of the carriers identified using this study, and evaluated the accuracy of anti-Vi and anti-YncE in identifying chronic typhoid carriage. METHODS: We performed a cross-sectional study in two centers in Pakistan. Gallbladder specimens were subjected to quantitative PCR (qPCR) and serum samples were analyzed for IgG against YncE and Vi by ELISA. We also mapped the residential location of those with a positive qPCR result. FINDINGS: Out of 988 participants, 3.4% had qPCR-positive gallbladder samples (23 S. Typhi and 11 S. Paratyphi). Gallstones were more likely to be qPCR positive than bile and gallbladder tissue. Anti-Vi and YncE were significantly correlated (r = 0.78 p<0.0001) and elevated among carriers as compared to qPCR negative controls, except for anti-Vi response in Paratyphi A. But the discriminatory values of these antigens in identifying carriers from qPCR negative controls were low. CONCLUSION: The high prevalence of typhoid carriers observed in this study suggests that further studies are required to gain information that will help in controlling future typhoid outbreaks in a superior manner than they are currently being managed.

Case Report: Salmonella typhi Iliopsoas Abscess with Concomitant Sacroiliitis in a Young Immunocompetent Male: A Rare Case.

Iliopsoas abscess is an infrequent condition characterized by the collection of pus in the iliopsoas compartment. The prevalence of the disease has been increasing in recent years with the emergence of various comorbidities and risk factors. The availability of newer imaging modalities has also improved the detection of new cases. Salmonellosis is an uncommon etiology in iliopsoas abscess and sacroiliitis. Most cases reported in the literature are associated with Staphylococcus aureus, Streptococci species, and Escherichia coli. Diabetes, hematological malignancies, HIV, and other immunocompromised states are important comorbidities/risk factors for iliopsoas abscess. We report a case of an 18-year-old male who presented with a history of fever and right hip pain for 10 days. Radioimaging revealed right sacroiliitis and iliopsoas abscess. Blood culture revealed pan-sensitive Salmonella typhi. After the prolonged course of antibiotics (intravenous ceftriaxone followed by oral levofloxacin), the patient improved with no further relapse in symptoms. Salmonella typhi should be an important differential of iliopsoas abscess in endemic regions after ruling out the common etiology such as S. aureus and Mycobacterium tuberculosis.

Transcriptional profiling links unique human macrophage phenotypes to the growth of intracellular Salmonella enterica serovar Typhi.

Macrophages provide a crucial environment for Salmonella enterica serovar Typhi (S. Typhi) to multiply during typhoid fever, yet our understanding of how human macrophages and S. Typhi interact remains limited. In this study, we delve into the dynamics of S. Typhi replication within human macrophages and the resulting heterogeneous transcriptomic responses of macrophages during infection. Our study reveals key factors that influence macrophage diversity, uncovering distinct immune and metabolic pathways associated with different stages of S. Typhi intracellular replication in macrophages. Of note, we found that macrophages harboring replicating S. Typhi are skewed towards an M1 pro-inflammatory state, whereas macrophages containing non-replicating S. Typhi exhibit neither a distinct M1 pro-inflammatory nor M2 anti-inflammatory state. Additionally, macrophages with replicating S. Typhi were characterized by the increased expression of genes associated with STAT3 phosphorylation and the activation of the STAT3 transcription factor. Our results shed light on transcriptomic pathways involved in the susceptibility of human macrophages to intracellular S. Typhi replication, thereby providing crucial insight into host phenotypes that restrict and support S. Typhi infection.

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi.

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

Enteric Fever in Children: An Epidemiological and Clinical Review of Cases in Southeast Texas.

Extensively drug-resistant (XDR) strains of Salmonella enterica serotype Typhi have emerged in Pakistan and Iraq. We report 13 children with enteric fever in Southeast Texas seen over 3.5 years, of whom 23.1% had XDR isolates.

The Salmonella typhi Cell Division Activator Protein StCAP Impacts Pathogenesis by Influencing Critical Molecular Events.

Conserved molecular signatures in multidrug-resistant Salmonella typhi can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the S. typhi cell division activator protein (StCAP) as a conserved target across S. typhi variants. From in silico and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg34 of StCAP with Ala34 showed a dramatic (15-fold) increase in Kd value compared to the wild type (Kd 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone S. typhi culture with an IC50 of 38 µM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with S. typhi where colony formation was severely hindered with IC50 reduced further to 10 µM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to S. typhi during infection, thereby making it a potent alternative therapeutic target.

Uncovering the growing burden of enteric fever: A molecular analysis of Salmonella Typhi antimicrobial resistance.

Enteric fever, a persistent public health challenge in developing regions, is exacerbated by suboptimal socioeconomic conditions, contaminated water and food sources, and insufficient sanitation. This study delves into the antimicrobial susceptibility of Salmonella Typhi, uncovering the genetic underpinnings of its resistance. Analyzing 897 suspected cases, we identified a significant prevalence of typhoid fever, predominantly in males (58.3 %) and younger demographics. Alarmingly, our data reveals an escalation in resistance to both primary and secondary antibiotics, with cases of multi-drug resistant (MDR) and extensively drug-resistant (XDR) S. Typhi reaching 14.7 % and 43.4 %, respectively, in 2021. The Multiple Antibiotic Resistance (MAR) index exceeded 0.2 in over half of the isolates, signaling widespread antibiotic misuse. The study discerned 47 unique antibiotic resistance patterns and pinpointed carbapenem and macrolide antibiotics as the remaining effective treatments against XDR strains, underlining the critical need to preserve these drugs for severe cases. Molecular examinations identified blaTEM, blaSHV, and blaCTX-M genes in ceftriaxone-resistant strains, while qnrS was specific to ciprofloxacin-resistant variants. Notably, all examined strains exhibited a singular mutation in the gyrA gene, maintaining wild-type gyrB and parC genes. The erm(B) gene emerged as the primary determinant of azithromycin resistance. Furthermore, a distressing increase in resistance genes was observed over three years, with erm(B), blaTEM and qnrS showing significant upward trends. These findings are a clarion call for robust antimicrobial stewardship programs to curtail inappropriate antibiotic use and forestall the burgeoning threat of antibiotic resistance in S. Typhi.

Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water.

Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor.

The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3'-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence.