The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: a dockyard of ß-lactamases periling nosocomial infections.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) constitutes a global public health threat challenging clinical treatment and infection control, especially in low- and middle-income countries such as India. We analyzed the antimicrobial susceptibility, major ß-lactamase genes, plasmid profiles, and genetic relatedness to understand the molecular epidemiology of CRECC clinical isolates (n = 44) in West Bengal, India, during 2021-2022. The majority (> 55%) of the isolates were resistant to fluoroquinolones, aminoglycosides, and co-trimoxazole, even > 20% for tigecycline and > 35% were extensively drug-resistant. Co-ß-lactamase production was categorized into twenty-seven types, importantly NDM (84%), OXA-48 (40%), TEM (61%), CTX-M (46%), OXA-1 (55%), and MIR (27%). The NDM-1 and OXA-181 were major variants with the first observations of NDM-24 and -29 variants in India. Wide-range of plasmids (2 to > 212 kb) were harbored by the ß-lactamase-producing isolates: small (91%), medium (27%), large (9%), and mega (71%). IncX3, ColE1, and HI2 were noted in about 30% of isolates, while IncF and R were carried by < 20% of isolates. The clonally diverse CRECC isolates were noted to cause cross-infections, especially at superficial site, bloodstream, and urinary-tract. This is the first molecular surveillance on CRECC in India. The study isolates serve as the dockyard of NDM, TEM, and CTX-M harboring a wide range of plasmids. The outcomes of the study may strengthen local and national policies for infection prevention and control practices, clarifying the genetic diversity among CRECC. Extensive genomic study may further intersect the relationships between these different plasmids, especially with their sizes, types, and antibiotic resistance markers.

Situation Report on mcr-Carrying Colistin-Resistant Clones of Enterobacterales: A Global Update Through Human-Animal-Environment Interfaces.

In the twenty-first century, antibiotic resistance (ABR) is one of the acute medical emergencies around the globe, overwhelming human-animal-environmental interfaces. Hit-or-mis use of antibiotics exacerbates the crisis of ABR, dispersing transferable resistance traits and challenging treatment regimens based on life-saving drugs such as colistin. Colistin is the highest priority critically important antimicrobials for human medicine, but its long use as a growth promoter in animal husbandry reduces clinical efficacy. Since 2015, the emergence and spread of mobile colistin resistance (mcr)-carrying colistin-resistant clones of Enterobacterales have been markedly sustained in both humans and animals, especially in developing countries. Hospital and community transmissions of mcr clones pose a high risk for infection prevention and outbreaks at the national and international levels. Several public health and limited one health studies have highlighted the genomic insights of mcr clones, clarifying the chromosomal sequence types (STs) and plasmid incompatibility (Inc) types. But this information is segregated into humans and animals, and rarely are environmental sectors complicating the understanding of possibly intercontinental and sectoral transmission of these clones. India is the hotspot for superbugs, including mcr-carrying colistin-resistant isolates that threaten cross-border transmission. The current review provided an up-to-date worldwide scenario of mcr-carrying STs and plasmid Inc types among the Gram-negative bacilli of Enterobacterales across human-animal-environmental interfaces and correlated with the available information from India.

Customized molecular diagnostics of bacterial bloodstream infections for carbapenem resistance: A convenient and affordable approach.

The acute crisis of carbapenem resistance impedes the empirical use of carbapenems in medical emergencies, especially, bloodstream infections. Carbapenemase-producing carbapenem-resistant organisms (CP-CROs) attribute high case-fatality, necessitating rapid diagnostics to initiate early targeted antibiotics. Expensive diagnostics are the major driver of antibiotic misuse, neglecting evidence-based treatment in India. One in-house molecular diagnostics assay was customized for rapid detection of CP-CROs using positive blood-culture (BC) broths at a low-cost. The assay was validated using a known-set of isolates and evaluated on positive BC broths. DNA was extracted from positive BC broths using a modified alkali-wash/heat-lysis method. One end-point multiplex-PCR was customized targeting five carbapenemases (KPC, NDM, VIM, OXA-48-, and OXA-23-type) with 16S-rDNA as internal extraction control. Carbapenem resistance due to other carbapenemases, efflux-pump activity, and loss of porins was not under the scope of the assay. Promising analytical performances (sensitivity and specificity, >90%; kappa = 0.87), encouraged to assess diagnostic value, qualified the assay for the WHO minimal requirements (both≥95%) for a multiplex-PCR. Higher LR+ (>10) and lower LR- (<0.1) indicate a good diagnostic tool for ruling in or ruling out CRO bloodstream infections. Inclusion of OXA-23-type improved assay positivity. Multiple carbapenemases were detected in>30% of samples. Good concordance was found (kappa = 0.91) with twenty-six discrepant results. The results were available in 3 hours. The running cost of the assay was US$10 per sample. Fast and reliable detection of carbapenemase(s) allows clinicians and infection-control practitioners to execute early-directed therapy and containment measures. This convenient approach facilitates implementing the assay in resource-limited healthcare settings.

The crisis of carbapenemase-mediated carbapenem resistance across the human-animal-environmental interface in India.

Carbapenems are the decision-making antimicrobials used to combat severe Gram-negative bacterial infections in humans. Carbapenem resistance poses a potential public health emergency, especially in developing countries such as India, accounting for high morbidity, mortality, and healthcare cost. Emergence and transmission of plasmid-mediated "big five" carbapenemase genes including KPC, NDM, IMP, VIM and OXA-48-type among Gram-negative bacteria is spiralling the issue. Carbapenemase-producing carbapenem-resistant organisms (CP-CRO) cause multi- or pan-drug resistance by co-harboring several antibiotic resistance determinants. In addition of human origin, animals and even environmental sites are also the reservoir of CROs. Spillage in food-chains compromises food safety and security and increases the chance of cross-border transmission of these superbugs. Metallo-ß-lactamases, mainly NDM-1 producing CROs, are commonly shared between human, animal and environmental interfaces worldwide, including in India. Antimicrobial resistance (AMR) surveillance using the One Health approach has been implemented in Europe, the United-Kingdom and the United-States to mitigate the crisis. This concept is still not implemented in most developing countries, including India, where the burden of antibiotic-resistant bacteria is high. Lack of AMR surveillance in animal and environmental sectors underestimates the cumulative burden of carbapenem resistance resulting in the silent spread of these superbugs. In-depth indiscriminate AMR surveillance focusing on carbapenem resistance is urgently required to develop and deploy effective national policies for preserving the efficacy of carbapenems as last-resort antibiotics in India. Tracking and mapping of international high-risk clones are pivotal for containing the global spread of CP-CRO.

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata, India, 2014-2018.

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014-18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016-18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes blaTEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.

Colistin susceptibility testing of gram-negative bacilli: Better performance of vitek2 system than E-test compared to broth microdilution method as the gold standard test.

Introduction: Unavailability of optimal susceptibility testing (ST) challenges the clinical use of colistin. Broth microdilution (BMD), which is the reference for colistin ST, is inconvenient for diagnostics. Vitek2 and E-test although technically easier, are no longer recommended. Materials and Methods: For the evaluation of Vitek2 and E-test in reference with BMD, a total of 138 Gram-negative bacilli (GNB) especially carbapenem-resistant isolates from Tata Medical Center, Kolkata, India, were included during 2017-2018. The evaluation was performed only for Enterobacteriaceae (n = 102), but not for non-fermentative GNB (n = 36) due to lack of colistin-resistant (COLR) isolates. Results and Conclusion: Of 138 isolates, meropenem, colistin and dual resistance were detected in 110 (79.7%), 31 (22.5%) and 21 (15.2%) of isolates, respectively. Using the European Committee on Antimicrobial Susceptibility Testing guidelines (susceptible, ≤2 µg/ml), Vitek2 performed better than E-test (essential agreement, 92.2% vs. 63.7%; categorical agreement, 94.1% vs. 93.1%; very major error [VME], 10% vs. 23.3%). However, Vitek2 overcalled resistance than E-test (major error, 4.2% vs. 0%). Considering Chew et al. proposed breakpoints (susceptible, ≤1 µg/ml), VMEs declined for both test (6.7% vs. 10%), but still remained unacceptable. Of eight colistin-heteroresistant isolates, two VME were categorised by Vitek2, one VME was by E-test, and two were uninterpretable. Both Vitek2 and E-test are unreliable. Further studies correlating minimum inhibitory concentrations with clinical outcome are needed to determine the accurate breakpoints for better patient management.

Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

In silico identification and characterization of stress and virulence associated repeats in Salmonella.

So much genomic similarities yet causing different diseases, is like a paradox in Salmonella biology. Repeat is one of the probes that can explain such differences. Here, a comparative genomics approach is followed to identify and characterize repeats that might play role in adaptation and pathogenesis. Repeats are non-randomly distributed in the genomes except few typhoid causing strains. Perfect long repeats are rare compare to polymorphic ones and both are statistically consistent. Significant differences in repeat densities in stress related genes manifest its probable participation in survival and virulence. 573 and 1053 repeat loci have been identified which are exclusively associated with stress and virulent genes respectively. In Salmonella Typhi, an octameric VNTR locus is found in between acrD and yffB genes having more than 25 perfect copies across Salmonella Typhi but possesses only single copy in other serovars. This repeat can be used as a diagnostic probe for typhoid.

Molecular Subtyping of Salmonella enterica Serovar Typhi by Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis in India: Their Association with Antimicrobial Resistance Profiles.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NALR) and NALR-ciprofloxacin-resistant (CIPR) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NALR-CIPR, and most NALR isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NALR-CIPR isolates showed variation in single or double VNTR loci, whereas NALR isolates varied in more than 2 loci, reflecting higher genetic diversity among the NALR isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.

Antimicrobial resistance and molecular subtypes of Salmonella enterica serovar Typhi isolates from Kolkata, India over a 15 years period 1998-2012.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains an unresolved public health problem in India. Emergence of antimicrobial resistant strains poses a great concern for typhoid treatment and influences reshaping of current S. Typhi population. We included representative S. Typhi strains (n=164) from retrospective studies, both community and hospital based, conducted at National Institute of Cholera and Enteric Diseases, Kolkata during 15 years period (1998-2012) to analyze their antimicrobial resistance (AMR) profiles, mechanism of AMR and molecular subtypes of the strains. More than 60% of the S. Typhi isolates were obtained from community based studies. During the study period, steady decline (46.4%-15.6%) in isolation of multidrug-resistant (MDR, resistant to ampicillin, chloramphenicol and co-trimoxazole) S. Typhi was noticed with parallel increase of nalidixic acid-resistant (NALR) strains (60.7%-93.8%) and ciprofloxacin resistant (CIPR) strains (0%-25%). Of 53 MDR strains, 46 (86.8%) were NALR showing decreased ciprofloxacin susceptible (DCS) (MIC for ciprofloxacin 0.12-0.5µg/ml) phenotype. Conjugative IncHI1 (230kb) and non-conjugative non-IncHI1 (180kb) plasmids were found in 23 (43.4%) and 14 (26.4%) MDR strains respectively, plasmid was absent in 16 (30.2%) MDR strains. MDR strains with or without plasmid shared the same set of resistance genes (blaTEM-1, catA1, sul1, sul2, strA and strB) and class 1 integron possessing dfrA7 gene cassette. Two S. Typhi strains harbored 50kb transferrable plasmids carrying dfrA15 and aadA1 gene cassettes in class 1 integron. The majority of the strains (135/164, 82.3%) belonged to H58 haplotype. Among the MDR isolates, fluoroquinolone resistant or combined resistant isolates (n=147), 127 (86.4%) were H58 and 20 (13.6%) belonged to non-H58. NALRS. Typhi strains with decreased susceptibility or resistance to ciprofloxacin had point mutation(s) in quinolone resistance-determining region of gyrA and parC genes. Pulsed-field gel electrophoresis showed more diversity among NALRS. Typhi than MDR strains. Results of this study generated information useful for better understanding of the disease epidemiology and its control in endemic settings.

Evaluation of fliC-d based direct blood PCR assays for typhoid diagnosis.

BACKGROUND: Typhoid cases need to be diagnosed accurately for early antibiotic therapy and reducing mortality. Identification of Salmonella Typhi (S. Typhi) in blood culture is conclusive, but has poor sensitivity. Detection of S. Typhi by PCR from blood sample has shown promise. Real-time quantitative PCR (Q-PCR) has been widely used in diagnostics for its rapidity and reliability. In the present study, the performance of molecular methods like conventional PCR (C-PCR), nested PCR (N-PCR) and Q-PCR were investigated and compared by targeting S. Typhi specific flagellar fliC-d gene directly in blood samples for typhoid diagnosis. RESULTS: Analytical sensitivities and specificities of the PCR assays were determined under laboratory condition followed by diagnostic performances were demonstrated in 110 clinically diagnosed typhoid fever (CDTF) cases included as study subjects. The DNA detection limit of C-PCR was observed 3 × 10(4) copies/reaction; those of N-PCR and Q-PCR (cutoff Ct value, ≤37) were 3 copies/reaction. The C-PCR was not further evaluated since it showed negative results with all clinical samples due to low sensitivity. Low isolation rate (21.8 %, 24/110) of S. Typhi by blood culture did not reflect the true burden of typhoid fever among the study subjects. Hence diagnostic performances of N-PCR and Q-PCR were determined considering CDTF cases positive by any of the diagnostic assay methods (n = 81) as true positives. Laboratory confirmed non-typhoidal cases (n = 29) were included as true negatives. On comparison, although both the assays were 100 % specific; sensitivity (91.4 % vs. 81.5 %) and efficiency (93.6 % vs. 86.4 %) of Q-PCR were better, but statistically not significant (p > 0.1) than N-PCR. The positive and negative likelihood ratios of Q-PCR were ∞ and 0.09 which indicated the potential clinical utility of Q-PCR for typhoid diagnosis. Q-PCR was more rapid than N-PCR (2 h vs. 6 h) in obtaining test results. CONCLUSIONS: This study demonstrates for the first time that TaqMan-based Q-PCR assay performs more favorably than N-PCR for direct detection of S. Typhi DNA in blood samples. Direct and quantitative blood Q-PCR is a rapid and reliable method for diagnosis of typhoid fever.

Revisit of fluoroquinolone and azithromycin susceptibility breakpoints for Salmonella enterica serovar Typhi.

In recent years, increase in occurrence of fluoroquinolone (FQ)-resistant S almonella Typhi isolates has caused considerable inconvenience in selecting appropriate antimicrobials for treatment of typhoid. The World Health Organization (WHO) recommends azithromycin for the empirical treatment option of uncomplicated typhoid. The CLSI updated the breakpoints of disc diffusion (DD) and MIC results of FQs and azithromycin for Salmonella Typhi in 2015, but DD breakpoints of ofloxacin and levofloxacin were not included. In this study, the inhibition zone diameters and MICs of nalidixic acid, ciprofloxacin, ofloxacin, levofloxacin and azithromycin were determined in Salmonella Typhi Kolkata isolates (n =146) over a 16-year period (1998 to 2013) and the data were compared with the available CLSI breakpoints. Very major error and major error (ME) of FQs were not observed in the study isolates, but the minor error of ciprofloxacin (15.8 %) and ME of azithromycin (3.5 %) exceeded the acceptable limit. A positive correlation between MICs of FQ and mutations in the quinolone-resistance-determining region (QRDR) showed the reliability of MIC results to determine FQ susceptibility of Salmonella Typhi (n =74). Isolates showing decreased ciprofloxacin susceptibility (MIC 0.125-0.5 µg ml-1) were likely to have at least one mutation in the QRDR region. The results on DD breakpoints of ofloxacin (resistant, ≤15 mm; intermediate, 16-24 mm, and susceptible, ≥25 mm) and levofloxacin (resistant, ≤18 mm; intermediate, 19-27 mm, and susceptible, ≥28 mm) corroborated those of earlier studies. In view of the emerging FQ- and azithromycin-resistant Salmonella Typhi isolates, DD and MIC breakpoints of those antimicrobials should be revisited routinely.

Work-exposure to PM10 and aromatic volatile organic compounds, excretion of urinary biomarkers and effect on the pulmonary function and heme-metabolism: A study of petrol pump workers and traffic police personnel in Kolkata City, India.

This study focused work-exposure to particulate matter ≤ 10 µm (PM10), volatile organic compounds (VOCs) and biological monitoring of major VOCs (BTEX) to observe the significant effects of traffic related pollutants on respiratory and hematological systems of workers engaged in two occupational settings, petrol pumps and traffic areas of Kolkata metropolitan city, India. PM10 was assessed by personal sampling and particle size distribution by 8-stage Cascade Impactor. VOCs were analysed by gas chromatography-flame ionization detector (GC-FID) and five urinary metabolites, trans trans- mercapturic acid (tt-MA), S-phenyl mercapturic acid (SPMA), hippuric acid (HA), mandelic acid (MA) and methyl hippuric acid (MHA) of VOCs, benzene, toluene, ethyl benzene and xylenes (BTEX) by reverse phase high performance liquid chromatography (HPLC). Pulmonary functions test (PFT) was measured Spirometrically. ∂-aminoleavulinic acid (ALA) and porphobilinogen (PBG) in lymphocytes were measured spectrophometrically following column chromatographic separation. High exposure to PM10, having 50% of particles, ≤ 5.0 µm in both the occupational settings. Exposure to toluene was highest in petrol pumps whereas benzene was highest (104.6 ± 99.0 µg m-3) for traffic police personnel. Workplace Benzene is found many fold higher than the National ambient standard. Air-benzene is correlated significantly with pre- and post-shift tt-MA (p < 0.001) and SPMA (p < 0.001) of exposed workers. Blood cell counts indicated benzene induced hematotoxicity. ALA and PBG accumulation in lymphocytes indicated alteration in heme-metabolism, especially among traffic police. Significant reduction of force exploratory volume in one second (FEV1) and forced vital capacity (FVC) of fuel fillers are observed with increased tt-MA and SPMA. Study revealed PFT impairments 11.11% (6.66% restrictive and 2.22% obstructive and combined restrictive and obstructive type, each) among petrol pumps and 8.3% obstructive type among traffic police.

Antimicrobial resistance, virulence profiles and molecular subtypes of Salmonella enterica serovars Typhi and Paratyphi A blood isolates from Kolkata, India during 2009-2013.

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (blaTEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.

Validation of a new serology-based dipstick test for rapid diagnosis of typhoid fever.

Currently, no reliable diagnostic test is available for typhoid fever. One serology-based dipstick test, developed indigenously, was validated in this study. Preserved sera from 336 fever patients with known culture results for Salmonella Typhi were blindly tested by the Widal test and the new assay. Analytical sensitivities, specificities, and efficiencies for the new assay versus the Widal test were 68.8% versus 62.5%, 71.1% versus 37.1%, and 70.5% versus 43.2%, respectively (p < 0.001), considering S. Typhi-positive samples as gold standards. Thereafter, fresh sera from 102 hospital-attending children with clinical typhoid fever (including 20 confirmed nontyphoidal cases as control) were tested by both methods and analyzed statistically. The diagnostic sensitivity, specificity, and efficiency were 51.2%, 85%, and 57.8% for the new assay, and 43.9%, 65%, and 48% for the Widal test, respectively. Overall performance ability of the new assay was not better than the Widal test (p > 0.5). Further improvement of the new point-of-care typhoid assay is recommended before implementation in the field setup.