• View in gallery
    Figure 1.

    Maximum-likelihood tree generated from single nucleotide polymorphisms in the core genome. (A) Phylogenetic tree of Salmonella Paratyphi A isolates from Tribhuvan University, Kathmandu, Nepal. (B) Phylogenetic sub-tree of major endemic S. Paratyphi A isolates.

  • 1.

    Crump JA, Luby SP, Mintz ED, 2004. The global burden of typhoid fever. Bull World Health Organ 82: 346353.

  • 2.

    Basnyat B, Maskey AP, Zimmerman MD, Murdoch DR, 2005. Enteric (typhoid) fever in travelers. Clin Infect Dis 41: 14671472.

  • 3.

    Karkey A, Aryjal A, Basnyat B, Baker S, 2008. Kathmandu, Nepal: still an enteric fever capital of the world. J Infect Dev Ctries 2: 461465.

  • 4.

    Li H, Durbin R, 2010. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26: 589595.

  • 5.

    Li H, 2011. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27: 29872993.

    • Search Google Scholar
    • Export Citation
  • 6.

    Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, Miller CA, Mardis ER, Ding L, Wilson RK, 2012. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22: 568576.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL, 2004. Versatile and open software for comparing large genomes. Genome Biol 5: R12.

    • Search Google Scholar
    • Export Citation
  • 8.

    Zhou Z, McCann A, Weill FX, Blin C, Nair S, Wain J, Dougan G, Achtman M, 2014. Transient Darwinian selection in Salmonella enterica serovar Paratyphi A during 450 years of global spread of enteric fever. Proc Natl Acad Sci USA 111: 1219912204.

    • Search Google Scholar
    • Export Citation
  • 9.

    Stamatakis A, 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 26882690.

    • Search Google Scholar
    • Export Citation
  • 10.

    Arjyal A et al. 2016. Gatifloxacin versus ceftriaxone for uncomplicated enteric fever in Nepal: an open-label, two-centre, randomised controlled trial. Lancet Infect Dis 16: 535545.

    • Search Google Scholar
    • Export Citation
  • 11.

    Shrestha KL, Pant ND, Bhandari R, Khatri S, Shrestha B, Lekhak B, 2016. Re-emergence of the susceptibility of the Salmonella spp. isolated from blood samples to conventional first line antibiotics. Antimicrob Resist Infect Control 5: 22.

    • Search Google Scholar
    • Export Citation
  • 12.

    Yan M et al. 2015. A large-scale community-based outbreak of paratyphoid fever caused by hospital-derived transmission in southern China. PLoS Negl Trop Dis 9: e0003859.

    • Search Google Scholar
    • Export Citation
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Molecular and Clinical Epidemiology of Salmonella Paratyphi A Isolated from Patients with Bacteremia in Nepal

Jatan Bahadur SherchanDepartment of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel, Nepal;

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Masatomo MoritaDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan;

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Takashi MatonoDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan;

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Hidemasa IzumiyaDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan;

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Makoto OhnishiDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan;

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Jeevan B. SherchandPublic Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal;

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Sarmila TandukarPublic Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal;

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Ujjwal LaghuPublic Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal;

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Maki NagamatsuDisease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan

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Yasuyuki KatoDisease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan

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Norio OhmagariDisease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan

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Kayoko HayakawaDisease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan

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Little is known about the epidemiology of typhoid and paratyphoid fever in Nepal. We aimed to elucidate the molecular and clinical epidemiology of Salmonella Paratyphi A in Nepal. Isolates were collected from 23 cases of bacteremia due to S. Paratyphi A between December 2014 and October 2015. Thirteen patients (57%) were male, and the median age was 21 years. None of the patients had an underlying chronic disease. All S. Paratyphi A isolates were sensitive to ampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, and chloramphenicol. All isolates were resistant to nalidixic acid and were categorized as intermediately susceptible to levofloxacin. Phylogenetic analysis revealed close relatedness among the isolates, including several clonal groups, suggesting local spread. Patients with bacteremia due to S. Paratyphi A in Kathmandu, Nepal, were relatively young and nondebilitated. Improving control of S. Paratyphi infections should focus on effective infection control measures and selection of empirical therapy based on current resistance patterns.

INTRODUCTION

Salmonella enterica serotype Typhi or S. enterica serotype Paratyphi cause an estimated 22 million new cases of enteric fever (typhoid or paratyphoid) annually, and 200,000 deaths.1 Nepal is located in South Asia where typhoid and paratyphoid fever are most prevalent.2 Especially, Kathmandu is known to have the significant burden of enteric fever caused by S. Typhi and S. Paratyphi A.3 The recent spread of multidrug resistant S. Typhi and S. Paratyphi A is a serious threat to public health; however, little is known about the epidemiology of typhoid and paratyphoid fever in Nepal. Previous reports have suggested an increase in the incidence of paratyphoid fever, against which currently available typhoid vaccines provide little to no protection.2,3 This study aimed to elucidate the molecular and clinical epidemiology including the prevalence of drug-resistant strains and phylogenetic analyses of S. Paratyphi A in Nepal.

METHODS

From December 2014 to October 2015, S. Paratyphi A isolates were collected from patients with bacteremia at Tribhuvan University, Kathmandu, Nepal. For patients from whom more than one S. Paratyphi A strain was isolated during the study period, only the first episode was analyzed (i.e., unique patient episodes). Institutional review boards at the Tribhuvan University approved the study before its initiation. Parameters retrieved from the patient records included demographics, background conditions and clinical symptoms, duration of hospital stay (for patients who were hospitalized), empiric antimicrobial treatment; information on occupation and exposure (e.g., animal contact, contact to similar cases), and clinical outcome.

Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method using the Dry Plate Eiken (Eiken Chemical, Tokyo, Japan), in accordance with the Clinical and Laboratory Standard Institutions (CLSI) criteria (M100-S26) at the National Center for Global Health and Medicine. Whole-genome sequencing and phylogenetic analysis were conducted at the National Institute of Infectious Diseases. Genomic DNA was prepared using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Genomic DNA libraries were prepared using the Nextera XT DNA sample prep kit (Illumina, San Diego, CA) and paired-end (300 × 2 bp) short reads for each library were sequenced on a MiSeq instrument (Illumina). Sufficient DNA sequence reads were generated to cover the genome at least 60 folds. Sequence reads were assembled with the de novo genome assembly programCLC Genomics Workbench v.8.5.1 (CLC Bio, Aarhus, Denmark) to generate a multicontig draft genome for each sample. All contigs were compared with the reference genome for S. Paratyphi A strain ATCC 9150 (CP000026.1) to detect mutations on gyrA, gyrB, parC, and parE genes. To compare short-read mapping data for all strains with the reference chromosomal sequence of S. Paratyphi A strain ATCC 9150 (CP000026.1), bwasw4 and samtools5 software were used with default parameters. Single nucleotide polymorphisms (SNPs) were extracted with VarScan v.2.3.46 using default parameters. The SNPs in repetitive and recombination regions were excluded for further analyses. Exact and inexact repeat regions were detected using the MUMmer v.3.23.7 RecHMM was used to identify recombination regions.8 The remaining 254 SNPs were concatenated to generate a pseudosequence for phylogenetic analysis; maximum likelihood phylogenetic analysis was performed using RAxML v.8.2.09 with 1,000 bootstrap iterations.

RESULTS

During the study period, 23 cases of bacteremia due to S. Paratyphi A, and 86 cases of bacteremia due to S. Typhi were identified. Among the 23 cases of bacteremia due to S. Paratyphi A, 13 patients (56.5%) were male, the median age was 21 years (interquartile range [IQR]: 17–23 years, range: 5–68 years). Among the 86 cases of bacteremia due to S. Typhi, 51 patients (59.3%) were male, the median age was 21 years (IQR: 17–26 years, range: 4–70 years). Clinical characteristics are summarized in Table 1. Twelve (52.2%) patients were hospitalized, the other were outpatients. None of the patients had an underlying chronic disease or immunosuppressive status. Twelve patients (52.2%) were students, four (17.4%) were housewives, three were farmers or gardeners, two (8.7%) were drivers, one (4.3%) was a nurse, and one (4.3%) worked at a pastry shop. The most common clinical symptom was fever (N = 23, 100%), followed by abdominal pain (N = 10, 43.5%), diarrhea (N = 3, 13%), and vomiting (N = 2, 8.7%). Most commonly prescribed empiric antimicrobial treatment was fluoroquinolone (N = 12, 52.2%), followed by cephalosporins (cefixime [N = 4, 17.4%], ceftriaxone [N = 2, 8.7%]), and azithromycin (N = 1, 4.3%). The median duration of hospitalization was 5 (IQR: 3–6) days, and no patient had complications or died.

Table 1

Clinical characteristics of patients with bacteremia due to Salmonella Paratyphi A (N = 23)

Number of patients (%)
Demographics
 Age, median (IQR)21 (17–23)
 Male patients13 (56.5)
 Inpatients/out patients12 (52.2)/11 (47.8)
Occupation
 Student12 (52.2)
 House wife4 (17.4)
 Farmer/gardener3 (13)
 Driver2 (8.7)
 Nurse1 (4.3)
 Pastry shop worker1 (4.3)
Clinical symptom
 Fever23 (100)
 Abdominal pain10 (43.5)
 Diarrhea3 (13)
 Vomiting2 (8.7)
Empiric antimicrobial treatment
 Fluoroquinolone*12 (52.2)
 Cefixime4 (17.4)
 Ceftriaxone2 (8.7)
 Azithromycin1 (4.3)
Duration of hospitalization, median (IQR), days5 (3–6)

IQR = interquartile range. One patient received cefixime, azithromycin, ceftriaxone, and another patient received ceftriaxone, azithromycin, and levofloxacin.

Fluoroquinolones include ciprofloxacin (N = 10), ofloxacin (N = 1), and levofloxacin (N = 1).

Antibiotic susceptibility and MICs of the S. Paratyphi A isolates are listed in Table 2. All isolates were sensitive to ampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, and chloramphenicol, and had an azithromycin MIC of less than 16 μg/mL. All isolates were resistant to nalidixic acid. The susceptibilities to fluoroquinolones differed, i.e., all isolates were resistant to ofloxacin based on CLSI criteria (M100-S26), four (17.4%) were resistant to ciprofloxacin, and all isolates were categorized as intermediately susceptible to levofloxacin.

Table 2

Antibiotic susceptibility and minimal inhibitory concentration (MIC) of Salmonella Paratyphi A (N = 23)

AntibioticsAntibiotic susceptibility, number of resistant* isolates (%)MIC (μg/mL)
SIRMIC50MIC90
Nalidixic acid23 (100)> 128> 128
Ciprofloxacin19 (82.6)4 (17.4)0.51
Levofloxacin23 (100)11
Ofloxacin23 (100)22
Norfloxacin44
Gatifloxacin0.51
Prulifloxacin0.50.5
Tosufloxacin0.50.5
Gentamicin23 (100)0.120.25
Kanamycin23 (100)0.51
Trimethoprim- sulfamethoxazole23 (100)0.12/2.380.25/4.75
Tetracycline23 (100)24
Minocycline23 (100)44
Azithromycin23 (100)88
Cefotaxime23 (100)0.120.25
Ceftriaxone23 (100)0.120.25
Ampicillin23 (100)44
Aztreonam0.120.12
Imipenem23 (100)0.250.25
Panipenem0.120.12
Biapenem0.250.25
Chloramphenicol23 (100)88

Based on Clinical and Laboratory Standard Institutions (CLSI) criteria 2016 (M100-S26) unless otherwise noted.

CLSI criteria 2016 (M100-S26) did not specify Salmonella spp. as they did for other fluoroquinolones, and thus, antibiotic susceptibility was left blank.

No breakpoint is available for S. Paratyphi based on CLSI criteria 2016 (M100-S26).

Phylogenetic analyses showed that except for one strain (151006PA), all isolates clustered together, with SNP distances of 0–9 SNPs (Figure 1). Clonal isolates (genetically closest isolates with 0 SNPs) were detected in five groups, whereas definitive outbreak from food handlers (the housewives and pastry shop worker) and family infection were not found. Among the five groups of clonal isolates, each isolate was predominantly detected 1 month or more apart, and only three paired isolates had a near sampling dates: 151080PA (on July 1, 2015) and 151086PA (on July 16, 2015); 151029PA (on March 5, 2015) and 151032PA (on March 12, 2015); and 151033PA (March 15, 2015) and 151031PA (March 8, 2015). All S. Paratyphi A isolates had a same single mutation C248T in gyrA encoding Ser83Phe; however, no mutation was found in gyrB, parC, and parE.

Figure 1.
Figure 1.

Maximum-likelihood tree generated from single nucleotide polymorphisms in the core genome. (A) Phylogenetic tree of Salmonella Paratyphi A isolates from Tribhuvan University, Kathmandu, Nepal. (B) Phylogenetic sub-tree of major endemic S. Paratyphi A isolates.

Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0227

DISCUSSION

In this study, we identified the molecular and clinical epidemiology of S. Paratyphi A in Kathmandu, Nepal. As we included sequential patients with bacteremia due to S. Paratyphi A, our findings would reflect the epidemiologic characteristics of this endemic region. It was revealed that S. Paratyphi A mainly affects relatively young subjects. The high prevalence of fever is consistent with recent reports on S. Typhi and S. Paratyphi bacteremia.10 In our cohort, the prevalence of abdominal pain was slightly higher, whereas diarrhea and vomiting were less prevalent than in previous reports.10 This might be due to differences in baseline characteristics, including age, of the tested population.

In this study, the antibiotic susceptibility and MICs revealed patterns of re-emergence of susceptibility to conventional antibiotics (e.g., chloramphenicol, trimethoprim/sulfamethoxazole, and ampicillin) similar to those described previously.11 On the other hand, MICs for quinolones were elevated. Importantly, S. Paratyphi A isolates of 10 patients who received ciprofloxacin as empiric therapy showed ciprofloxacin MICs ≥ 0.5 μg/mL, and one patient who received ofloxacin carried S. Paratyphi A with an ofloxacin MIC of 2 μg/mL. Along with recent trial results on the use of gatifloxacin,10 reconsideration is warranted in terms of empiric antimicrobial treatment against S. Paratyphi A bacteremia in Nepal.

Based on phylogenetic analyses, there seem to be two distinct endemic strains of S. Paratyphi A in the study region. The major endemic strain was genetically diverged as compared with outbreak-associated isolates in China.12 The relatively high prevalence of patients who handle food (e.g., food-handling business, housewives) and patients who have close contact with other people (e.g., students) in this study suggests a potential point of intervention to reduce the transmission of S. Paratyphi, for which no vaccine is readily available.

High relatedness, coupled with high prevalence of reduced susceptibility to fluoroquinolones, was observed in S. Paratyphi isolated from patients with bacteremia in Nepal, Kathmandu. Further studies are warranted in terms of appropriate effective therapy and effective infection control approaches in this region.

REFERENCES

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    Crump JA, Luby SP, Mintz ED, 2004. The global burden of typhoid fever. Bull World Health Organ 82: 346353.

  • 2.

    Basnyat B, Maskey AP, Zimmerman MD, Murdoch DR, 2005. Enteric (typhoid) fever in travelers. Clin Infect Dis 41: 14671472.

  • 3.

    Karkey A, Aryjal A, Basnyat B, Baker S, 2008. Kathmandu, Nepal: still an enteric fever capital of the world. J Infect Dev Ctries 2: 461465.

  • 4.

    Li H, Durbin R, 2010. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26: 589595.

  • 5.

    Li H, 2011. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27: 29872993.

    • Search Google Scholar
    • Export Citation
  • 6.

    Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, Miller CA, Mardis ER, Ding L, Wilson RK, 2012. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22: 568576.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL, 2004. Versatile and open software for comparing large genomes. Genome Biol 5: R12.

    • Search Google Scholar
    • Export Citation
  • 8.

    Zhou Z, McCann A, Weill FX, Blin C, Nair S, Wain J, Dougan G, Achtman M, 2014. Transient Darwinian selection in Salmonella enterica serovar Paratyphi A during 450 years of global spread of enteric fever. Proc Natl Acad Sci USA 111: 1219912204.

    • Search Google Scholar
    • Export Citation
  • 9.

    Stamatakis A, 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 26882690.

    • Search Google Scholar
    • Export Citation
  • 10.

    Arjyal A et al. 2016. Gatifloxacin versus ceftriaxone for uncomplicated enteric fever in Nepal: an open-label, two-centre, randomised controlled trial. Lancet Infect Dis 16: 535545.

    • Search Google Scholar
    • Export Citation
  • 11.

    Shrestha KL, Pant ND, Bhandari R, Khatri S, Shrestha B, Lekhak B, 2016. Re-emergence of the susceptibility of the Salmonella spp. isolated from blood samples to conventional first line antibiotics. Antimicrob Resist Infect Control 5: 22.

    • Search Google Scholar
    • Export Citation
  • 12.

    Yan M et al. 2015. A large-scale community-based outbreak of paratyphoid fever caused by hospital-derived transmission in southern China. PLoS Negl Trop Dis 9: e0003859.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Kayoko Hayakawa, Disease Control and Prevention Center, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: kayokohayakawa@gmail.com

Financial support: This work was supported by a grant in Clinical Epidemiology Research, St. Luke’s International University, Tokyo, Japan (2016).

Authors’ addresses: Jatan Bahadur Sherchan, Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel, Nepal, E-mail: jatansherchan@gmail.com. Masatomo Morita, Takashi Matono, Hidemasa Izumiya, and Makoto Ohnishi, Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan, E-mails: mmorita@niid.go.jp, tmatono@niid.go.jp, izumiya@nih.go.jp, and ohnishi7@nih.go.jp. Jeevan B. Sherchand, Sarmila Tandukar, and Ujjwal Laghu, Department of Microbiology and Parasitology, Tribhuvan University Teaching Hospital, Kathmandu, Nepal, E-mails: jeevansherchand@hotmail.com, sar1234tan@gmail.com, and ujjwal111@iom.edu.np. Maki Nagamatsu, Yasuyuki Kato, Norio Ohmagari, and Kayoko Hayakawa, Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan, E-mails: dfrmm217@yahoo.co.jp, ykato@hosp.ncgm.go.jp, nohmagari@hosp.ncgm.go.jp, and kayokohayakawa@gmail.com.

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