. Three sets of primers were designed and their specificity was confirmed using BLAST. Positive LAMP products were successfully digested with HinfI enzyme that was specific only to the amplified STBHUCCB_38510 gene of the S. Typhi, thus allowing its differentiation from closely related bacteria species (e.g., S. Typhimurium and S. Paratyphi A). After in-vitro confirmation of binding characteristics of both LAMP primer sets with culture isolates (n = 112), sensitivity and specificity were 100%. However, low sensitivity and specificity were recorded in an assay of 170 EDTA blood samples using one of the primer sets and some cross-reactivity was also observed.

Kaur et al.,2017 in a  research to develop an in-house optical detection system to detect signals from LAMP assay, targeted the Gene STY2879 that encodes for reverse transcriptase protein confirmed to be present in all S. typhi isolates. Highly sensitive detection of Salmonella typhi (S. typhi) in blood of 50 CFU/mL was achieved using their protocol involving a magnetic nanoparticle-based preconcentration. The specificity and cross-reactivity were tested against other bacterial isolates of E. coli, S. aureus, P. aeruginosa, A. baumanni, E. faecalis, S. paratyphi A and K. pneumonia. The proposed method was also evaluated against clinical samples and no cross-reactivity was observed at a high concentration of 106 CFU/mL of the pathogenic bacterial species as compared to a positive control (S. typhi DNA). The experiments were conducted in triplicates.

Fan et al., 2015, in a research to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection, performed regular PCR using 123 strains to test the specificity of their selected target genes primers. The results show that the STY1607 gene was amplified from 48 Salmonella Typhi strains. Seventy-five strains that comprised 34 other common Salmonella serotypes, primary enteric pathogens, and some critical febrile microorganisms yielded negative amplification results. The sequencing results of the PCR amplicons (531 bp) randomly selected from 15 Salmonella Typhi strains also showed 100% sequence identity in these strains. Their finding indicated that STY1607 is conserved and can be used as a marker gene for detecting Salmonella Typhi. Also using Human-simulated clinical blood/stool samples, and mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity. The STY1607-RT-LAMP detection limit was 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi–simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method.

In a well-established and evaluated Salmonella enterica ssp. enterica ser. Typhi-specific qPCR targeting the STY0201 region in studies carried out by Nga et al., (2010); Tennant et al.,(2015), reported positive LAMP signals with some LAMP primer sets for 35 out of 35 incubated blood culture materials from which Salmonella enterica ssp. enterica ser. Typhi had been isolated.  However, some false-positive results were recorded for other primer sets reported by Frickmann et al., 2019. Even though their focus was on specificity, Both LAMP primer sets showed excellent sensitivity with incubated blood culture materials using the Genie II Mk2 system.

5. InvA- Secretory protein S. Typhi

Francois et al., 2011 in a research to evaluate the robustness of loop-mediated isothermal amplification (LAMP) of DNA for Salmonella enterica serovar Typhi, designed Primers and probes specific for the invA gene of S. Typhi. The primers exhibited excellent performance (accurate, sensitive, and specific detection) in LAMPs typhoid diagnostic analysis as positive signals were observed at a DNA concentration of 0.5pg and also after manipulation sample and reaction parameters.  Although their cross-reactivity wasn’t extensively tested as only E. coli strains were used.

6. SSPA2539-Hypothetical Protein S. Paratyphi

Kaur et al., 2019 in their work on a Simultaneous and highly sensitive detection Salmonella paratyphi A in human clinical blood samples using an affordable portable device (SMOL), targeted the SPA2539 gene.  Primers specific for the SPA2539 gene in S. paratyphi A were used for LAMP and Within 6 h SMOL was able to detect positive and negative samples from 55 human clinical blood culture samples with very good sensitivity and specificity. It was also able to detect the viability of the cells. The results were concordant with culture and biochemical tests as well as by qPCR. To check for cross-reactivity, Common pathogenic bacterial species observed in human blood samples of patients were used with S. paratyphi A primers. The performance of the primers was tested against Escherichia coli, Salmonella havana, Pseudomonas aeruginosa, Salmonella typhimurium, Acetinobacter baumanni, Enterococcus faecalis, Klebsiella pneumonia, Staphylococcus aureus, and Salmonella paratyphi B. No cross-reactivity was observed at high concentrations (i.e. 106 CFU/mL) of the above bacterial pathogens. All the experiments were carried out in triplicates.

7&8. SSPA2308-Hypothetical Protein and SSPA 1724-ATP Dependent Protease Binding Unit of S. paratyphi 

Rojak et al., 2019 in a research to design and use an In-house loop-mediated isothermal amplification (LAMP) procedure for the detection of paratyphoid fever-associated bacteria. They designed LAMP primers targeting two genes from S. Paratyphi A, SSPA 2308 hypothetical protein and the SSPA 1724-ATP Dependent Protease Binding Unit.  The primers were tested with DNA from culture isolates from strain collections and spiked blood cultures against published PCR protocols targeting the same micro-organisms. LAMP proved to be more sensitive and specific (80.0 to 100.0% and 96.1 to 100.0%) using these targets than PCR (65 to 100% and 98.7 to 100%). For the spiked blood LAMP also showed similar results although slightly lower with some primer pairs than PCR. Some cross-reactivity was also observed with one of the LAMP primers for S. Paratyphi A with S. havana.

Fluoroquinolones and cephalosporins have been reported to be dominantly used antibiotics for the treatment of typhoid fever in Cameroon (Stakeholders engagement). We  selected AMR target genes affiliated with resistance to fluoroquinolones and cephalosporins. Studies of AMR in Cameroon are limited, so our choice of AMR genes was largely based on studies done in countries neighboring Cameroon and other African countries. our choice of AMR genes was larIn this study, we test for three AMR target regions in S.typi : blaCTX-M gene for class A extended-spectrum beta-lactamase CTX-M-1, plasmid mediated qnrS gene and point mutation in gyrA leading to fluoroquinolone resistance (summarized in Table \ref{386515}).  

1. Nonsynonymous mutations in the DNA gyrase genes (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) is likely to confer Salmonella reduced susceptibility to ciprofloxacin (quinolone).  The mutation results in a significant reduction of the drug-enzyme binding, and as such, the ability for fluoroquinolones to inhibit DNA ligation is totally restricted \citep{Aldred2012,Barnard_2001}\citep{Aldred2012,Barnard_2001}.  AMR to quinolones due to mutations in the DNA gyrase genes (gyrA and gyrB), and DNA topoisomerase IV (parC and parC) have been reported in several African regions including Ghana    \citet{Acheampong2019}\citet{Acheampong2019}, Congo \citet{Phoba2017}\citet{Phoba2017}, Kenya \citet{Al-Emran2016} and South Africa \citet{Smith2010}. A point mutation in the 83Ser in gyrA being the most commonly reported. In this test, we design our primers to address two challenges that would be widely applied to detect point mutations:

 2. Plasmid-mediated quinolone resistance genes (PMQR) are also responsible for conferring resistance to fluoroquinolones. This is done by encoding quinolone resistance proteins (encoded by qnr genes (qnrA, qnrB, qnrC, qnrD, qnrS) that shield DNA gyrase from the effect of fluoroquinolones. A case report by \citet{Nkemngu_2005} demonstrates a case of ciprofloxacin treatment failure in Cameroon.  This may indicate the presence of nalidixic acid-resistant S. enterica serovar Typhi (NARST) with reduced susceptibility to ciprofloxacin (quinolone) in Cameroon. Plasmids encoding the qnrB2, qnrB4 and qnrS1 determinants have been reported in Africa \citet{Britto2018}. Primers to detect the presence of qnrS gene have been designed (see table 1) 

3. Extended-spectrum β lactamase (ESBL) producing S. Typhi isolates, confer resistance to third-generation cephalosporins; the drug of choice for fluoroquinolone-resistant Salmonella isolates and isolates resistant to first-line antimicrobials. It has been reported in the Democratic Republic of Congo encoding ESBL (blaCTX-M15) on an Inc Y type plasmid  \citet{Phoba2017}  and   \citet{Akinyemi_2015} reveal in their study the emergence of beta-lactamase-producing gene blaCTX-M-1 S. Typhi strain in Lagos, Nigeria as an agent of persistent pyrexia. Primers to detect the blaCTX-M gene for class A extended-spectrum beta-lactamase CTX-M-1    have been designed (see table 1)