Background
Exposure to enteric pathogens is one of the major causes of diarrheal infections in both traveler and military populations [
1]. Previous studies have reported that military personnel acquired infectious diarrhea during military exercises [
2]. The risk of diarrheal infection is regionally dependent, particularly for civilian travelers and military personnel in transition from industrialized countries into developing countries [
3,
4]. Reported incident rates for bacterial diarrheal disease in military and travelers caused by enterotoxigenic
Escherichia coli (ETEC),
Campylobacter, and
Shigella were between 38 and 45% in previous reports from various countries [
5]. Typical treatment for traveler’s diarrhea includes the use antibiotics to include ciprofloxacin, azithromycin and rifaximin [
6]. However, enteric pathogens and their associated antibiotic resistance patterns evolve over time and vary by region [
7,
8]; therefore, access to up-to-date data on the global epidemiology of present diarrheal agents and their respective resistances are vital for diminishing the risk of diarrheal infection [
6].
There are five Pacific region countries with which the US has a functional security alliance, including Thailand. The Armed Forces Research Institute for Medical Sciences (AFRIMS), based in Thailand, has coordinated studies of deployed US military to Thailand (i.e., the annual US–Thai “Cobra Gold” joint military forces exercise) for several years. Documented studies from previous exercises in Thailand demonstrated that US soldiers suffer consistent diarrhea attack rates during their first few weeks in country [
9‐
11]. Despite modern preventive methods, diarrhea remains a primary concern for force health protection and therefore mission success for deployed military personnel in Thailand. Thus, the main objective of this study was to report the prevalence and clinical symptoms of diarrheal etiologic agents and bacterial pathogen antimicrobial susceptibility (AST) patterns affecting deployed US military personnel in Thailand for Cobra Gold exercises conducted in 2013 to 2017. This information will be useful in formulating more effective prevention and treatment strategies for these acute illnesses in deployed US forces.
Methods
Study design
A prospective acute diarrhea study was conducted in February of each calendar year, 2013–2017, at the following field sites: Lopburi, Phitsanulok, Chonburi (Samaesarn/Utapao), and Chanthaburi (Baan Chan Khem). Diarrheal cases were defined as three or more loose stool in the previous 24 h, starting no more than 72 h before presentation, with concurrent clinical symptoms such as nausea, vomiting, and abdominal or bowel pain. After obtaining informed consent, US military service members who presented with these criteria and symptoms self-reported on an administered questionnaire the following: stool frequency and description, poultry exposure, local food consumption, and any additional clinical symptoms. Stool grading (formed, soft, loose, or watery) was assessed by US military medical staff. The stool grading “loose” is described the stools that appear softer than normal whereas “watery” is specified to the stools appearance that no solid pieces and all liquid. The study was approved yearly by the Walter Reed Army Institute of Research institutional review board, Silver Spring, Maryland, USA.
Laboratory methods
Microbiological methods
Approximately 4–10 grams of stool was collected from each subject. Stool samples were tested for the presence of enteric bacteria pathogens by inoculating onto the following media: MacConkey agar (MC), Hektoen (HE), thiosulfate citrate bile salt sucrose (TCBS), modified semi-solid Rappaport–Vassiliadis (MSRV), modified charcoal cefoperazone deoxycholate agar (mCCDA), buffered peptone water, alkali peptone water and Preston selective enrichment broth. Cellulose acetate membrane (Sartorius, Germany) was used to filter inoculated stool samples on Brucella agar plate (BAP) with sheep blood. Subsequently, identification of
Shigella, Salmonella, Vibrio,
Aeromonas, Plesiomonas, Yersinia, Campylobacter and
Escherichia coli was performed as previously described [
12].
Antimicrobial susceptibility testing
Isolated enteric pathogens, except
Campylobacter and
Arcobacter, were evaluated by AST following standard Kirby-Bauer method to the following antibiotics, ampicillin (AMP), azithromycin (AZM), ceftriaxone (CRO), ciprofloxacin (CIP), nalidixic acid (NA) and co-trimoxazole (SXT), using commercially prepared discs according to the manufacturer’s instructions (Becton, Dickinson and Company, USA). Susceptibility results were interpreted following the Clinical and Laboratory Standards Institute (CLSI) guidelines [
13].
Campylobacter and
Arcobacter isolates were evaluated using E-tests (Biomérieux, NC, USA). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of an antimicrobial agent that completely inhibited visible growth and was read at the point where the elliptical zone of inhibition intersected the MIC scale on the strip. Due to the limitation of CLSI guidelines for
Campylobacter, the National Antimicrobial Resistance Monitoring System (NARM) 2013 criteria for AZM, CIP, erythromycin (ERY) and NA were followed for these isolates [
14].
Multiplex polymerase chain reaction (multiplex-PCR)
Isolated 5–10 lactose fermenting colonies were inoculated onto MacConkey agar (MC) and Hektoen (HE) media, cultured for 18–24 h at 37 ℃, and sub-cultured onto trypticase soy agar (TSA) with 5% sheep blood. Lactose-fermenting colonies were picked for nucleic acid by boiling extraction method. Identification of diarrheagenic
Escherichia coli (Enteropathogenic
E.coli (EPEC) [
15], Enteroinvasive
E.coli (EIEC) [
16], Enteroaggregative
E.coli (EAEC) [
17,
18], Enterotoxigenic
E. coli (ETEC) [
19‐
21], and Enterohemorrhagic
E. coli (EHEC) [
15,
22] was performed using multiplex PCR.
Enzyme-linked immunosorbent assay (ELISA)
Qualitative Enzyme-linked immunosorbent assay (ELISA) kits (TechLab, Inc., USA) were utilized to detect Giardia lamblia, Cryptosporidium, and Entamoeba histolytica in fecal specimens. ELISA kits (Ridascreen® and R-Biopharm; Germany) were used for the detection of rotavirus, astrovirus, adenovirus, and Campylobacter according to the manufacturer’s instructions.
TaqMan® array card
Total nucleic acid was extracted from frozen stool using the QiaAmp stool DNA kit (Qiagen, Valencia, California) and used in the Enteric Pathogen TaqMan
® Array Card (TAC) as previously described [
23]. Briefly, 40 μL of extracted nucleic acid from each stool sample was mixed with 60 μL of Ag-Path-ID One-Step RT-PCR kit (Applied Biosystems, Foster City, CA) and this mixture was loaded onto the eight ports of the TAC, sealed, and loaded into the ViiATM7 instrument (Applied Biosystems). TAC can detect the following pathogens: bacteria:
Aeromonas,
Bacteroides fragilis,
Campylobacter (
C. jejuni and
C. coli),
Clostridium difficile, EAEC, EPEC, ETEC,
Helicobacter pylori,
Salmonella,
Shigella/EIEC, STEC, and
Vibrio cholera, fungi:
Encephalitozoon intestinalis and
Enterocytozoon bieneusi, nematodes:
Ancylostoma duodenale,
Ascaris lumbricoides,
Necator americanus,
Strongyloides stercoralis, and
Trichuris trichiura, protozoan parasites:
Cryptosporidium,
Cyclospora,
Entameoba histolytica,
Giardia A/B, and
Isospora and viruses: adenovirus, astrovirus, norovirus GI/GII, rotavirus, and sapovirus [
23]. Analysis of raw data files were processed using ViiATM7 software version 1.2.2 (Applied Biosystems) as previously described [
23]. A threshold cycle (Ct) greater than 35 was used as the analytical cutoff (lower limit of detection).
Statistical methods
Statistical analysis was conducted using IBM SPSS Statistics version 24.0. Chi squared tests were sued to determine if the association between patient questionnaire data and subsequent pathogen identification was significant.
Results
A total of 48 acute diarrhea cases were enrolled from 2013 to 2017 (47 completed questionnaire), with 9 cases in 2013, followed by 8 (2014), 18 (2015), 9 (2016) and 4 (2017) cases respectively. 38% (18/47) of the stool samples were described as “loose”, 21% (10/47) as “soft”, 23% (11/47) as “watery”, 6% (3/47) as “no record’ and the remaining as “formed”. 89.4% (42/47) of the subjects consumed locally prepared food. Primary self-reported complaints included: 72% (34/47) abdominal pain, 64% (30/47) nausea and 34% (16/47) vomiting. Bowel movement frequencies varied between 2 and 20 times in 48 h. Seven patients suffered from bloody diarrhea and three of the seven presenting with bloody diarrhea accompanied by nausea, vomiting, abdominal pain, and bowel movement pain. The clinical presenting data has been described on Table
1.
Table 1
Clinical symptom summary of US military personnel deployed in Thailand from 2013–2017 with diarrhea
2013 | 9 | 0 | 7 | 6 | 6 | 2–10 |
2014 | 8 | 2 | 7 | 4 | 7 | 4–20 |
2015 | 18 | 2 | 10 | 3 | 12 | 3–20 |
2016 | 9 | 2 | 5 | 1 | 6 | 4–20 |
2017 | 4 | 1 | 1 | 2 | 3 | 7–20 |
Pathogen detection for stool samples collected from 2013 and 2017 was performed using ELISAs and TAC. Microbiological culture and multiplex PCR were added to the diagnostic panel for samples collected in 2015–2017. Of the 48 acute diarrhea stool samples, enteric pathogens were identified in 79.2% (38/48) of the samples while an etiologic agent was not detected in 20.8% (10/48) of the stool samples. The pathogenic profile of the 48 study samples from 2013 to 2017 is summarized in Table
2. Briefly, the most common detected pathogen was
Campylobacter spp. (43.8%; 21/48), followed by the diarrheagenic
E. coli (42%; 20/48) and
Salmonella spp. (23%; 11/48). The most common
Campylobacter species was
C. jejuni at 25% (12/48), whereas
Campylobacter spp. was identified in 18.8% (9/48) of the samples. Of the twenty diarrheagenic
E. coli cases, 65% (13/20) were EPEC, followed by 15% (3/20) ETEC, 15% (3/20) EAEC, and 5% (1/20 EIEC).
Salmonella spp. and norovirus were detected in 23% (11/48) and 15% (7/48) of the stool samples respectively (Table
2). Additionally,
Vibrio cholera and
V. parahaemolyticus were detected in US military personnel stationed at the Chonburi province, a coastal city, in 2016.
Table 2
Summary of identified pathogens present in stool samples collected during 2013 to 2017 from US military personnel presenting with diarrheal disease while deployed in Thailand
Campylobacter | Campylobacter jejuni | 12/48 (25%) |
Campylobacter species | 9/48 (18.8%) |
Total | 21/48 (43.8%) |
Diarrheagenic E. coli* | EPEC | 13/48 (27%) |
ETEC | 3/48 (6%) |
EAEC | 3/48 (6%) |
EIEC | 1/48 (2%) |
Total | 20/48 (42%) |
Salmonella spp. | Salmonella group B | 5/48 (10%) |
Salmonella group c | 4/48 (8%) |
Salmonella species | 2/48 (4%) |
Total | 11/48 (23%) |
Norovirus | Norovirus GII | 5/48 (10%) |
Norovirus GI | 2/48 (4%) |
Total | 7/48 (15%) |
Plesiomonas shigelloides | | 6/48 (13%) |
Aeromonas species | | 6/48 (13%) |
Vibrio species | | 3/48 (6%) |
Rotavirus | | 2/48 (4%) |
Helicobacter pylori | | 2/48 (4%) |
Shigella species | | 1/48 (2%) |
Arcobacter butzleri | | 1/48 (2%) |
No pathogen detected | | 10/48 (21%) |
Co-infections (defined as more than one etiologic agent) were detected in 46% (22/48) of the study samples with one sample from 2016 containing seven enteric pathogens: Aeromonas veronii bv sorbria, Arcobacter butzleri, C. jejuni, EPEC, Plesiomonas shigelloides, V. cholera, and V. parahaemolyticus. Stool samples containing the diarrheagenic E. coli and P. shigelloides were found to be most commonly associated with those US service members who were exposed to poultry (p = 0.02). One surprising observation was the absence of any typical etiologic agents for 2/10 samples that were classified as bloody diarrhea.
All pathogenic bacterial isolates obtained from 2015 to 2017 were further sub-cultured to perform AST, with resulting antibiotic resistance profiles contained in Table
3. 100% of the
Salmonella isolates were resistant to AMP and 44.4% resistant to SXT. 52.9% (9/17) of the diarrheagenic
E. coli isolates were resistant to AMP and 100% (4/4) of the EPEC isolates resistant to SXT. One
C. jejuni and one
A. butzleri isolate were resistant to AZM. The AZM-resistant
C. jejuni was also resistant to ERY. 100% of the
C. jejuni isolates were resistant to CIP and NA. All of the
Plesiomonas and
Aeromonas isolates were susceptible to all tested antibiotics.
Table 3
Antibiotic susceptibility profile of enteric bacteria detected in stool samples from US military personnel presenting with diarrheal disease while deployed to Thailand from 2015–2017
Campylobacter jejuni | 11 | 11 | – | 1 | 1 | 11 | 11 | – | – |
Arcobacter butzleri | 1 | 1 | – | 1 | 0 | 0 | 0 | – | – |
Salmonella species | 9 | 9 | 9 | – | – | 0 | – | 4 | 0 |
ETEC | 3 | 2 | 2 | – | – | 0 | – | 0 | 0 |
EAEC | 3 | 3 | 3 | – | – | 0 | – | 2 | 0 |
EPEC | 11 | 4 | 4 | – | – | 1 | – | 4 | 0 |
Shigella sonnei | 1 | 1 | 0 | – | – | 0 | – | 1 | 0 |
Vibrio species | 3 | 2 | 1 | – | – | 0 | – | 0 | – |
Discussion
Diarrhea remains a leading cause of acute morbidity and chronic health effects, negatively impacting the health and functionality of both traveler and military populations. US military service members often deploy into developing regions in which enteric pathogens associated with diarrheal disease are prevalent.
Campylobacter was the most frequent pathogen identified in this study, which correlates to the high prevalence in travelers with acute diarrhea in previous studies in Thailand in travelers and US military service members participating in previous Cobra Gold exercises [
5,
24,
25].
Campylobacter isolates from this study were also entirely resistant to quinolones (NA) and fluoroquinolone (CIP) antibiotics, which is of additional concern based upon recent evidence indicating that quinolone- and fluoroquinolone-resistant
Campylobacter infections are associated with the development of post-infectious long term sequelae to include Guillen Barre Syndrome [
26,
27].
A. butzleri, a member of the
Campylobacteraceae family, was isolated from one stool samples.
Arcobacter species are not typically associated with diarrheal disease, however, previous studies showed an 8% prevalence of traveler’s diarrhea associated with
A. butzleri in Mexico, Guatemala, and India [
28]. Study of tourist restaurants in Thailand suggested that
Arcobacter was a food-borne pathogen and its isolates were frequently resistant to AZM which is the common therapeutic recommendation for the treatment of diarrhea in Asia [
29]. The AZM-resistant
C. jejuni was also resistant to ERY, the recommended antimicrobial treatment in invasive cases or to eliminate carrier states. Erythromycin resistance has been reported in Thailand previously [
30].
The second most common etiologic diarrhea agent identified in this study was
E. coli. ETEC is the leading cause of childhood diarrhea and the most frequent cause of diarrhea in travelers to developing countries [
31]. ETEC contribution to diarrheal disease is dependent upon the region of interest and seasonality [
32‐
34]. In this study, EPEC was detected more commonly in cases than ETEC. A previous study noted that EPEC was dependent upon co-infection with other pathogenic bacteria to include
Aeromonas and
Salmonella in travelers who developed travelers’ diarrhea [
35]. However in our study, EPEC was detected in only one co-infected sample. Non-typhoidal
Salmonella (NTS) was the third most common pathogen detected and previous epidemiological studies demonstrated that infection with drug-resistant NTS enterica serotypes was associated with excess morbidity [
36]. Based on the antibiotic profiles in this study highlighted that AZM should remain first-line treatment for travelers’ diarrhea to Thailand [
37]. Norovirus genogroups II and I were detected in several of the cases, but are usually associated with outbreaks of diarrhea. Nevertheless, previous studies have shown that norovirus is becoming commonly detected in both children and adults returning from tropical settings though most laboratories do not commonly test for norovirus in a hospital, clinical setting [
38].
Plesiomonas and
Aeromonas are not normally associated with travelers’ diarrhea though this study indicated that these pathogens were detected in samples with other enteric pathogens. These co-infection results, associated with clinical diarrhea in military patients, support evidence from previous studies that
Aeromonas contribute towards the development of diarrhea [
39]. Meng et al. reported that synergy or antagonism among pathogens likely affected the degree of diarrheal disease severity more than a single infection in children [
40], and that the presence of multiple infections dramatically challenged the ability to properly identify the actual etiological agents of diarrhea disease.
There were several limitations to the study. A relatively small number of diarrheal stool samples were collected with no matched control sample which makes stating that the identified pathogen (s) were truly the cause of the diarrhea. Another limitation is the lack of antibiotic profiles for the bacterial pathogens detected in samples from 2013 to 2014 as the main diagnostic methodology used in these years were ELISAs and TAC. Inclusion of conventional microbiological methods allowed for the determination of antibiotic susceptibility profiles. Due to diagnostic limitations, some pathogens remain undetectable by these methods because they require challenging or unknown unfavorable growth conditions. A previous study indicates that
C. consisus and
C. ureolyticus are emergent-bacterial diarrheal pathogens [
41]. However, these organisms are obligate anaerobes that require a H
2-enriched atmosphere for optimum growth [
42]. Methods to identify these pathogens were not used in this study.
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