ORIGINAL ARTICLE
Trans R Soc Trop Med Hyg 2014; 108: 692–698 doi:10.1093/trstmh/tru151
Bloodstream infections at a tertiary referral hospital in Yangon, Myanmar Tin Ohn Myata, Namrata Prasadb, Kyi Kyi Thinna, Kyu Kyu Wina, Wah Win Htikea, Khwar Nyo Zinc, David R. Murdochd and John A. Crumpb,* a
Department of Microbiology, University of Medicine 1, Yangon, Myanmar; bCentre for International Health, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand; cClinical Laboratory Department, Yangon General Hospital, Yangon, Myanmar; dDepartment of Pathology, University of Otago Christchurch, 2 Riccarton Avenue, P.O. Box 4345, Christchurch 8011, New Zealand *Corresponding author: Tel: +64 3 479 9460; Fax: +64 3 479 7298; E-mail: [email protected]
Background: Data regarding characteristics of bloodstream infections in Myanmar are limited. Methods: Blood culture results from all outpatients and inpatients were extracted from records of the Clinical Microbiology Laboratory, Yangon General Hospital, for the period 2005 through 2013. Results: Of 3865 blood cultures performed, 449 (11.6%) were positive for a pathogenic organism. Gram-negative bacteria was the most common organism group, accounting for 246 (55.5%) of 449 isolations. Staphylococcus aureus was the most common isolate, detected in 171 (38.1%) of 449 blood cultures. From 2005–2008 to 2009–2013 the proportion of all pathogenic isolates that were Gram-positive declined from 52.8% (167/316) to 20.3% (27/133) (p,0.001), whereas the proportion of Gram-negative bacteria rose from 45.6% (144/316) to 78.9% (105/133) (p,0.001), with non-fermentative bacilli accounting for much of this increase. Antimicrobial susceptibility testing demonstrated a high prevalence resistance of S. aureus to first-line antimicrobials such as erythromycin, penicillin and oxacillin. More than half of tested Escherichia coli and Citrobacter species showed resistance to amoxicillin-clavulanic acid, ceftriaxone or gentamicin. Conclusions: Bloodstream infections are common among patients receiving blood culture at a tertiary hospital in Yangon, Myanmar. Our findings suggest that antimicrobial resistance among invasive bacteria is common, similar to patterns described elsewhere in the region, and highlight the need for locally adapted antimicrobial guidelines for sepsis management. Keywords: Antimicrobial drug resistance, Bacteremia, Fever, Myanmar
Introduction Bacterial sepsis is a serious and life-threatening condition.1,2 A robust understanding of locally important causes of bloodstream infection and patterns of antimicrobial resistance among bacteria isolated from the bloodstream is essential to form empiric treatment strategies for patients with suspected sepsis.1 However, data on causes of bloodstream infection in south and southeast Asia, including Myanmar, are limited.3 The few published data from Myanmar suggest that bloodstream infections are common, with 35–50% of inpatients tested having bacteremia in some series.4,5 Myanmar has a central role in the history of communityacquired invasive bacterial infections in the tropics. Notably, Pseudomonas pseudomallei, now known as Burkholderia pseudomallei, the cause of meliodosis was first described by Whitmore and Krishnaswami in Yangon in 1911.6 Furthermore, healthcareassociated infections, including bloodstream infections, are known to be more common in low-resource areas than in
wealthier countries.7 In addition, Asia has emerged as an epicentre of antimicrobial drug resistance.8 Penicillin and erythromycinresistant Streptococcus pneumoniae;9 ampicillin-resistant Haemophilus influenzae; 10,11 multidrug-resistant (MDR) and carbapenem-resistant Acinetobacter baumannii; 12,13 extendedspectrum b-lactamase producing Enterobacteriaceae;14 New Delhi metallo-b-lactamase producing Enterobacteriaceae;15 MDR Salmonella enterica serovars Choleraesuis and Typhi;16,17 and azole-resistant Candida glabrata 18 have all been described in the region. However, few studies have reported the current situation in Myanmar with respect to community-acquired and healthcare associated bloodstream infections. Furthermore, the extent to which invasive pathogens in Myanmar reflect regional patterns of antimicrobial resistance is unknown. This study sought to describe data about invasive bacterial infections in Yangon, Myanmar. To provide a contemporary picture of causes of bloodstream infection and patterns of antimicrobial resistance at a tertiary hospital, blood culture records of the Yangon General Hospital were reviewed.
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
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Received 10 July 2014; revised 27 August 2014; accepted 1 September 2014
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Methods Study site
Patient enrolment and blood culture collection At Yangon General Hospital, blood cultures are collected from any outpatient or inpatient with suspected bloodstream infection or prolonged fever. Approximately five millilitres of blood was collected aseptically from each patient and inoculated into trypticase soy broth (HiMedia, Mumbai, India). Microbiological methods Blood cultures were incubated aerobically at 37.08C for up to 7 days. When growth was observed, broth was inoculated onto human blood agar and McConkey agar (HiMedia). Terminal subculture was performed for bottles with no signs of growth by day 7.
Data collection The clinical microbiology laboratory of Yangon General Hospital maintained records of isolated organisms and their antimicrobial susceptibility patterns. Data were extracted from records including date of blood culture collection, age, sex and ward of patient, organism identification and the results of antimicrobial susceptibility testing.
Statistical analysis All data were double entered into an electronic database (Excel, Microsoft, Redmond, WA, USA). The x2 test of statistical
Figure 1. Map of southeast Asia showing Yangon, Myanmar where this study was performed.
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The country of Myanmar (formerly known as Burma; Figure 1) has an estimated population of 59.1 million. Yangon, the former capital, has a population .6 million people and is the country’s largest city and most important commercial centre.19 Yangon has a tropical monsoon climate with a rainy season from May through October, and a dry season from November through April.20 The national HIV seroprevalence among adults in Myanmar was estimated at 0.6% in 2012, with 46% of those in need of antiretroviral therapy receiving it.21 The Yangon General Hospital is the biggest tertiary referral hospital in Yangon, Myanmar, serving adolescent and adult patients, aged ≥12 years. It is estimated that in 2013 approximately 51 877 patients were admitted to the hospital.
Subcultures were incubated at 37.08C and visually inspected at 24 and 48 hours. Bacteria were identified using standard methods including, inspection of growth characteristics on solid media, direct microscopic examination of Gram stained smear of colonies and conventional biochemical testing.22 All media were manufactured in-house and were quality controlled. Antimicrobial susceptibility testing was done on Mueller-Hinton agar (HiMedia) by the modified Kirby-Bauer disk diffusion method and interpreted according to the standards of the Clinical and Laboratory Standards Institute (CLSI).23 Interpretive criteria for P. aeruginosa were applied to all Pseudomonas spp. and interpretive criteria for S. pneumoniae for alpha-hemolytic streptococci. Antimicrobial susceptibility testing was dependent on the availability of antimicrobial disks (HiMedia) at Yangon General Hospital. Quality control for susceptibility testing used was done according to CLSI guidelines using ATCC strains.
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Table 1. Antimicrobial discs tested and reported for each bacterial group at Yangon General Hospital, 2005–2013 Bacterial group
Antimicrobial discs used and reported
significance or Fisher’s exact test, when appropriate, was used to compare proportions and odd ratios were calculated to estimate effect sizes associated with bloodstream infections, using STATA software (StataCorp 2013, Stata Statistical Software: Release 13, College Station, TX, USA). A comparison between groups was done only when the sample sizes of both groups exceeded 10. For the purposes of analysis blood culture isolates were categorized into four groups: Gram-positive organisms, Enterobacteriaceae, non-fermenting Gram-negative bacilli and yeasts. Coagulasenegative Staphylococci, Corynebacterium species and Bacillus group isolates were considered contaminants. Trends in antimicrobial resistance were restricted predominantly to routinely reported, or Group A antimicrobials suggested by CLSI.24 The antimicrobial discs used and reported for each bacterial group are shown in Table 1. Time trends were assessed comparing the time period 2005 through 2008 with 2009 through 2013, reflecting the approximate mid-point of the dataset. The rainy season was defined as May through October and the dry season from November through April.
Results Patient characteristics Between 2005 and 2013, 3865 blood cultures were processed from 3858 patients attending Yangon General Hospital. Of the 3858 patients, 3604 (93.2%) were inpatients and the remainder from outpatients. The median (range) age of patients was 40 (12–100) years.
Bloodstream infections Of 3865 blood cultures performed, pathogenic bacteria were isolated from 449 (11.6%) samples, 229 (5.9%) grew probable contaminates and 3187 (82.5%) showed no growth. Table 2 shows the distribution of pathogenic bacterial and fungal species isolated from blood cultures at Yangon General Hospital by time period. Of 3865 blood culture collected during the study period, 1849 (47.8%) were collected from 2005 through 2008, and 2016 (52.2%) were collected from 2009 through 2013 (p,0.001).
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During the period 2005 through 2008, Gram-positive bacteria accounted for 52.8% (167/316) bloodstream infections as compared with 20.3% (27/133) during the period 2009 through 2013 (p,0.001). During 2005 through 2008, Staphylococcus aureus accounted for 46.7% (147/316) bloodstream infections compared with 18.0% (24/133) during 2009 through 2013 (p,0.001). From 2005 through 2008, Gram-negative bacteria accounted for 45.6% (144/316) bloodstream infections compared with 78.9% (105/133) from 2009 through 2013 (p,0.001). Enterobacteriaceae accounted for 26.9% (85/316) Gram-negative isolates from 2005 through 2008 compared with 36.8% (49/133) from 2009–2013 (p,0.001). Non-fermentative bacilli accounted for 18.7% (59/316) Gramnegative pathogens from 2005 through 2008 compared to 42.1% (56/133) from 2009 through 2013 (p,0.001). Streptococcus species were not identified to the species level. No H. influenzae isolates were identified in this study.
Patterns of antimicrobial resistance among bloodstream isolates Changes in availability of antimicrobial disks and antimicrobial agents in Myanmar influenced drugs selected for susceptibility testing during the study period. Of all tested S. aureus, 5 of 7 isolates were resistant to penicillin during period 2005 through 2008 period compared with 1 of 7 tested in the 2009 through 2013 time period. Over the entire study period, 10.4% (17/163) of the S. aureus tested were resistant to tetracycline and 36.7% (58/158) were resistant to oxacillin (i.e., methicillin-resistant S. aureus, MRSA). MRSA accounted for 38.7% (55/142) of S. aureus isolates during 2005 through 2008, compared with 18.8% (3/16) of isolates from 2009 through 2013 (p¼0.116). Erythromycin resistance was identified in 51.1% (68/133) of S. aureus isolates from 2005 through 2008, compared with 27.3% (6/22) of isolates from 2009 through 2013 (p,0.001). Over the entire study period, 30.8% (4/13) of Enterococcus species were found to be resistant to ampicillin and 68.8% (11/16) were resistant to erythromycin. Of tested Streptococcus species, 2 of 3 tested were resistant to penicillin. No vancomycin-resistant Gram-positive bacteria were found. Among Enterobactericeae isolated during the entire study period, 72% (13/18) E. coli were resistant to amoxicillin-clavulanic
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Gram-positive bacteria First line drugs: ampicillin, erythromycin, penicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, clindamycin, ciprofloxacin. Second line drugs: vancomycin, azithromycin, amoxicillin-clavulanic acid, levofloxacin. Enterobacteriaceae First line drugs: amikacin, gentamicin, amoxicillin-clavulanic acid, ceftriaxone, cefotaxime, ceftazidime, ciprofloxacin, gentamicin. Second line drugs: ampicillin-sulbactam, piperacillin-tazobactam, cefoperazone-sulbactam, levofloxacin, imipenem, cefipime. Gram-negative First line drugs: amikacin, gentamicin, cefoperazone-sulbactam, ceftazidme, ceftriaxone, cefotaxime, ciprofloxacin. non-fermentative Second line drugs: piperacillin- tazobactam, imipenem, cefipime, levofloxacin. bacilli
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Table 2. Distribution of pathogenic bacterial and fungal species isolated from blood cultures at Yangon General hospital, 2005 through 2008 compared with 2009 through 2013
Table 3. Seasonal variation of blood culture isolates at Yangon General Hospital, 2005 through 2013 Organism
Organism
Rainy season
Total
2005–2008
2009–2013
Total
n
(%)
n
n
n
n
n
(%)
167 (52.8) 147 (46.7)
(%) 27 (20.3) 24 (18.0)
(%)
(37.7) (36.3) (24.0)
139 (45.9) 81 (26.7) 80 (26.4)
194 (43.2) 171 (38.1)
15 5 144 85 31 22 6 7
(4.7) (1.6) (45.6) (26.9) (9.8) (7.0) (1.9) (2.2)
2 1 105 49 17 23 3 0
(1.5) (0.8) (78.9) (36.8) (12.8) (17.3) (2.3) (0)
17 6 249 134 48 45 9 7
(3.8) (1.3) (55.5) (29.8) (10.7) (10.0) (2.0) (1.6)
5 3 11 59
(1.6) (0.9) (3.5) (18.7)
1 0 5 56
(0.8) (0) (3.8) (42.1)
6 3 16 115
(1.3) (0.7) (3.6) (25.6)
27 23 1 8 5 4
(8.5) (7.3) (0.3) (2.5) (1.6) (1.3)
36 8 2 10 1 1
(27.1) (6.0) (1.5) (7.5) (0.8) (0.8)
63 31 3 18 6 5
(14.0) (6.9) (0.7) (4.0) (1.3) (1.1)
1 (0.3) 0 (0) 1 (0.2) 316 (100.0) 133 (100.0) 449 (100.0)
a
Salmonella enterica consisted of 5 Salmonella enterica serovar Typhi and 2 Salmonella enterica that were not serotyped. b Others: organisms that could not be speciated, included 18 non-fermentative Gram-negative bacilli and 17 Enterobacteriaceae.
acid, 82% (37/45) were resistant to ceftriaxone and 57% (25/44) were resistant to gentamicin. Among Citrobacter species, 83% (15/18) were resistant to amoxicillin-clavulanic acid, 65% (30/46) were resistant to ceftriaxone and 66% (29/44) were resistant to gentamicin. Among Gram-negative non-fermentative bacilli, 4 of 8 Acinetobacter species were resistant to ceftazidime, 35% (9/26) to gentamicin and 26% (6/23) were resistant to amikacin. Among Pseudomonas species, 51% (30/59) were resistant ceftazidime, 40% (24/60) resistant to gentamicin and 24% (11/46) were resistant to amikacin. Of Pseudomonas species 5% (1/21) were resistant to levoflaxcin during the period 2005 through 2008 compared with 19% (5/27) from the time period 2009 through 2013 (p,0.001). Of 20 Enterobacteriaceae and 15 non-fermentative
Gram-positive bacteria 55 Enterobacteriaceae 53 Gram-negative 35 non-fermentative bacilli Yeasts 3 Total 146
(%)
(%)
194 (43.2) 134 (29.8) 115 (25.6)
(2.1) 3 (1.0) 6 (1.3) (100.0) 303 (100.0) 449 (100.0)
Gram-negative bacilli, none were found to be resistant to meropenem or imipenem.
Seasonal variation Of the 449 pathogenic blood culture isolates, 146 (32.5%) were found in the dry season compared to 303 (67.5%) in the rainy or monsoon season (OR¼1.9; 95% CI 1.5–2.6; p,0.001). Gram-positive bacteria (OR¼2.2; 95% CI 0.9–2.1; p¼0.110) and Gram-negative non-fermentative bacilli (OR¼4.6; 95% CI 2.8–7.5; p,0.001) were more likely to be isolated from patients during the wet season compared to the dry season. Enterobacteriaceae were more likely to be isolated from patients during the dry season compared to the wet season (OR¼0.6; 95% CI 0.4–0.99; p¼0.047) No significant seasonal association was found for yeasts (Table 3).
Discussion A high prevalence of bloodstream infections among patients receiving blood cultures at a tertiary hospital in Yangon, Myanmar, was found in this study. Although S. aureus was the most frequently isolated pathogen, Gram-negative bacteria were the most common group of organisms accounting for 55.5% of isolated pathogens. While community-acquired pathogens such as Salmonella typhi and B. pseudomallei were uncommon in this study, pathogens that are often healthcare-associated were frequently isolated and demonstrated patterns of antimicrobial resistance similar to those common elsewhere in the south and southeast Asian region. There were significant differences in the prevalence of groups of bloodstream pathogens between the earlier and later time period, with a decrease in Gram-positive isolates and an increase in Gram-negative isolates over time. A growing proportion of non-fermentative bacilli drove the increase in Gram-negative organisms in the later period. Although the increase in the Gram-negative bloodstream infections is likely to be real, it is possible that changes in the use of antimicrobial agents over time could have played a role. It is possible that healthcare-associated infections might also have contributed to this trend.25 Differences in bloodstream infections between the wet and dry seasons were also observed, with patients found to be
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Gram-positive Staphylococcus aureus Enterococcus spp Streptococcus spp Gram-negative Enterobacteriaceae Citrobacter spp Escherichia coli Klebsiella spp Salmonella enterica a Serratia spp Enterobacter spp Otherb Non-fermentative bacilli Pseudomonas spp Acinetobacter spp Burkholderia spp Otherb Yeasts Non-albicans Candida Candida Total
Dry season
Time period
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However, the recent emergence of carbapenem-resistant bacterial strains in neighboring China43 and India15 warrant vigilance and judicious use of this class of antimicrobials. The study had a number of limitations. It was retrospective, so patient selection for blood culture and clinical and laboratory methods were not standardized. This, in turn, contributed to relative small numbers of isolates of some species. Limited access to relevant clinical information meant that we were unable to distinguish community-acquired infections from healthcare-associated infections. Key determinants of blood culture sensitivity such as blood culture volume adequacy and antimicrobial exposure prior to blood culture were not known. Furthermore, only a single blood culture done for each patient and blood culture contamination at specimen collection could not be controlled.44,45 The microbiologic methods of this study were not optimized in a number of respects. For example, the use of human blood agar and lack of use of chocolate agar, and CO2 incubation is likely to have diminished the yield of a number of relevant pathogens including fastidious organisms such as S. pneumoniae and H. influenzae.46 Limited capacity to serogroup or serotype S. enterica and to identify non-fermentative Gram-negative bacteria to the species level affected our ability to detect regionally relevant pathogens, including Salmonella typhi and B. pseudomallei. Furthermore, changes in drugs and methods selected for antimicrobial susceptibility testing constrained our ability to measure time trends in antimicrobial resistance. In conclusion, this study describes the epidemiology of bloodstream infections among patients seen at a tertiary hospital in Yangon, Myanmar. MRSA and drug-resistant Gram-negative organisms were found to have emerged in the study area. Our findings suggest that patterns of antimicrobial resistance among invasive organism are similar to those described elsewhere in the region,8,47 and provide useful information for the development of locallyadapted antimicrobial guidelines for the management of sepsis. However, our study had a number of limitations that could be addressed in future studies with prospective and standardized investigation of bloodstream infections using optimized laboratory methods.
Authors’ contributions: TOM and NP contributed equally to this work; JAC, DRM and KKT conceived the study; TOM, NP, KKT, DRM and JAC designed the study protocol; TOM, NP, KKW, WWH and KNZ collected the data; KNZ oversaw laboratory assessments; NP and TOM carried out data analysis; NP and JAC drafted the manuscript; all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript. NP, TOM and JAC are guarantors of the paper. Acknowledgements: We are grateful to the clinical and laboratory staff and patients of Yangon General Hospital for their contributions to this study. Funding: This research was supported in part by a grant from the University of Otago Development Office, New Zealand. Competing interests: None declared. Ethical approval: Ethical approval was obtained from the Research and Ethical Committee, University of Medicine, Yangon, Myanmar and the Human Ethics Committee, University of Otago, Dunedin, New Zealand.
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approximately twice as likely to have a bacterial bloodstream infection in the rainy season as compared to the dry season. More specifically, Gram-positive bacteria and non-fermenting Gram-negative bacilli were found to account for a larger proportion of bloodstream pathogens during the rainy season compared to the dry season at Yangon General Hospital. A variety of reasons have been suggested for seasonal patterns of bloodstream infections and include behavior, environmental factors such as weather, and occurrence of underlying diseases, variation in virulence factors of bacterial pathogens, host susceptibility and patient demographics.26,27 Increased humidity is also found to be associated with increased incidence of P. aeruginosa bloodstream infection.28 Humidity is high in Yangon during the wet season and may contribute to patterns observed in our study. Our findings differed from those of bloodstream infection studies elsewhere in Myanmar and in the south and southeast Asia region. The proportion of blood cultures positive for a pathogen was 11.6% in our study, but ranged from 15–50% in studies done elsewhere.5,29–31 Salmonella typhi and Paratyphi A were isolated less often in our study than in studies elsewhere in Myanmar4,5 and Asia.3,30–32 Given the history of meliodosis in Yangon, it was surprising that there were no confirmed B. pseudomallei isolates. However, the disease is described among travellers returning from Myanmar33 and elsewhere in the country.34 Laboratory methods used during the study period were suboptimal for the isolation and identification of B. pseudomallei 22 and it is likely that cases of melioidosis were missed. It is also possible that melioidosis is less common in the catchment population of Yangon General Hospital than it once was. Antimicrobial resistance was common among a range of important pathogens and to a number of key antimicrobial agents relevant to clinical management. Resistance of S. aureus to erythromycin, penicillin, oxacillin and trimethoprim-sulfamethoxazole was common. Notably, almost one third of S. aureus isolates were methicillin-resistant, a proportion similar to other studies in the region.35 However, unlike studies from neighbouring countries36–38 no Gram-positive organisms demonstrated decreased susceptibility or resistance to vancomycin, which may have been due to the limited availability of the drug in the country during the study period. Among Gram-negative bacteria, more than half of tested E. coli and Citrobacter species showed resistance to amoxicillin-clavulanic acid, ceftriaxone or gentamicin. The small number of Enterobacter and S. enterica isolates and limited testing for antimicrobial resistance precluded analysis of resistance patterns among these organisms. Of Gram-negative non-fermentative bacilli, 20–60% of all tested Acinetobacter and Pseudomonas species were found to be resistant to the commonly recommended antimicrobials ceftazidime, gentamicin and amikacin. Resistance to imipenem was not detected among the 20 Enterobacteriaceae and 15 nonfermentative bacilli tested. Although unconfirmed, it is likely that resistance to ceftriaxone among Enterobacteriaceae is mediated by a range of extended-spectrum b-lactamases as high numbers of such isolates have been found in other studies in Asia.14 The presence of resistance to extended-spectrum cephalosporins is of particular concern because of their role in the empiric therapy of serious community-onset infections in low-resource areas.39,40 Furthermore, this pattern of antimicrobial resistance may fuel the use of carbapenems as the drug class of first choice for the treatment of serious infections.41,42 Notably, carbapenem-resistant bloodstream isolates were not found during the study period.
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44 Hall KK, Lyman JA. Updated review of blood culture contamination. Clin Microbiol Rev 2006;19:788–802. 45 Weinstein MP. Current blood culture methods and systems: clinical concepts, technology, and interpretation of results. Clin Infect Dis 1996;23:40–6. 46 Castillo D, Harcourt B, Hatcher C et al. Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Geneva: World Health Organization; 2011. 47 Lai CC, Lee K, Xiao Y et al. High burden of antimicrobial drug resistance in Asia. J Glob Antimicrob Resist 2014;2:141–7.
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41 Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159–66. 42 Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 2005;18:657–86.
43 Xiao YH, Giske CG, Wei ZQ et al. Epidemiology and characteristics of antimicrobial resistance in China. Drug Resist Updat 2011;14:236–50.
Trans R Soc Trop Med Hyg 2014; 108: 692–698 doi:10.1093/trstmh/tru151
Bloodstream infections at a tertiary referral hospital in Yangon, Myanmar Tin Ohn Myata, Namrata Prasadb, Kyi Kyi Thinna, Kyu Kyu Wina, Wah Win Htikea, Khwar Nyo Zinc, David R. Murdochd and John A. Crumpb,* a
Department of Microbiology, University of Medicine 1, Yangon, Myanmar; bCentre for International Health, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand; cClinical Laboratory Department, Yangon General Hospital, Yangon, Myanmar; dDepartment of Pathology, University of Otago Christchurch, 2 Riccarton Avenue, P.O. Box 4345, Christchurch 8011, New Zealand *Corresponding author: Tel: +64 3 479 9460; Fax: +64 3 479 7298; E-mail: [email protected]
Background: Data regarding characteristics of bloodstream infections in Myanmar are limited. Methods: Blood culture results from all outpatients and inpatients were extracted from records of the Clinical Microbiology Laboratory, Yangon General Hospital, for the period 2005 through 2013. Results: Of 3865 blood cultures performed, 449 (11.6%) were positive for a pathogenic organism. Gram-negative bacteria was the most common organism group, accounting for 246 (55.5%) of 449 isolations. Staphylococcus aureus was the most common isolate, detected in 171 (38.1%) of 449 blood cultures. From 2005–2008 to 2009–2013 the proportion of all pathogenic isolates that were Gram-positive declined from 52.8% (167/316) to 20.3% (27/133) (p,0.001), whereas the proportion of Gram-negative bacteria rose from 45.6% (144/316) to 78.9% (105/133) (p,0.001), with non-fermentative bacilli accounting for much of this increase. Antimicrobial susceptibility testing demonstrated a high prevalence resistance of S. aureus to first-line antimicrobials such as erythromycin, penicillin and oxacillin. More than half of tested Escherichia coli and Citrobacter species showed resistance to amoxicillin-clavulanic acid, ceftriaxone or gentamicin. Conclusions: Bloodstream infections are common among patients receiving blood culture at a tertiary hospital in Yangon, Myanmar. Our findings suggest that antimicrobial resistance among invasive bacteria is common, similar to patterns described elsewhere in the region, and highlight the need for locally adapted antimicrobial guidelines for sepsis management. Keywords: Antimicrobial drug resistance, Bacteremia, Fever, Myanmar
Introduction Bacterial sepsis is a serious and life-threatening condition.1,2 A robust understanding of locally important causes of bloodstream infection and patterns of antimicrobial resistance among bacteria isolated from the bloodstream is essential to form empiric treatment strategies for patients with suspected sepsis.1 However, data on causes of bloodstream infection in south and southeast Asia, including Myanmar, are limited.3 The few published data from Myanmar suggest that bloodstream infections are common, with 35–50% of inpatients tested having bacteremia in some series.4,5 Myanmar has a central role in the history of communityacquired invasive bacterial infections in the tropics. Notably, Pseudomonas pseudomallei, now known as Burkholderia pseudomallei, the cause of meliodosis was first described by Whitmore and Krishnaswami in Yangon in 1911.6 Furthermore, healthcareassociated infections, including bloodstream infections, are known to be more common in low-resource areas than in
wealthier countries.7 In addition, Asia has emerged as an epicentre of antimicrobial drug resistance.8 Penicillin and erythromycinresistant Streptococcus pneumoniae;9 ampicillin-resistant Haemophilus influenzae; 10,11 multidrug-resistant (MDR) and carbapenem-resistant Acinetobacter baumannii; 12,13 extendedspectrum b-lactamase producing Enterobacteriaceae;14 New Delhi metallo-b-lactamase producing Enterobacteriaceae;15 MDR Salmonella enterica serovars Choleraesuis and Typhi;16,17 and azole-resistant Candida glabrata 18 have all been described in the region. However, few studies have reported the current situation in Myanmar with respect to community-acquired and healthcare associated bloodstream infections. Furthermore, the extent to which invasive pathogens in Myanmar reflect regional patterns of antimicrobial resistance is unknown. This study sought to describe data about invasive bacterial infections in Yangon, Myanmar. To provide a contemporary picture of causes of bloodstream infection and patterns of antimicrobial resistance at a tertiary hospital, blood culture records of the Yangon General Hospital were reviewed.
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
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Received 10 July 2014; revised 27 August 2014; accepted 1 September 2014
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Methods Study site
Patient enrolment and blood culture collection At Yangon General Hospital, blood cultures are collected from any outpatient or inpatient with suspected bloodstream infection or prolonged fever. Approximately five millilitres of blood was collected aseptically from each patient and inoculated into trypticase soy broth (HiMedia, Mumbai, India). Microbiological methods Blood cultures were incubated aerobically at 37.08C for up to 7 days. When growth was observed, broth was inoculated onto human blood agar and McConkey agar (HiMedia). Terminal subculture was performed for bottles with no signs of growth by day 7.
Data collection The clinical microbiology laboratory of Yangon General Hospital maintained records of isolated organisms and their antimicrobial susceptibility patterns. Data were extracted from records including date of blood culture collection, age, sex and ward of patient, organism identification and the results of antimicrobial susceptibility testing.
Statistical analysis All data were double entered into an electronic database (Excel, Microsoft, Redmond, WA, USA). The x2 test of statistical
Figure 1. Map of southeast Asia showing Yangon, Myanmar where this study was performed.
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The country of Myanmar (formerly known as Burma; Figure 1) has an estimated population of 59.1 million. Yangon, the former capital, has a population .6 million people and is the country’s largest city and most important commercial centre.19 Yangon has a tropical monsoon climate with a rainy season from May through October, and a dry season from November through April.20 The national HIV seroprevalence among adults in Myanmar was estimated at 0.6% in 2012, with 46% of those in need of antiretroviral therapy receiving it.21 The Yangon General Hospital is the biggest tertiary referral hospital in Yangon, Myanmar, serving adolescent and adult patients, aged ≥12 years. It is estimated that in 2013 approximately 51 877 patients were admitted to the hospital.
Subcultures were incubated at 37.08C and visually inspected at 24 and 48 hours. Bacteria were identified using standard methods including, inspection of growth characteristics on solid media, direct microscopic examination of Gram stained smear of colonies and conventional biochemical testing.22 All media were manufactured in-house and were quality controlled. Antimicrobial susceptibility testing was done on Mueller-Hinton agar (HiMedia) by the modified Kirby-Bauer disk diffusion method and interpreted according to the standards of the Clinical and Laboratory Standards Institute (CLSI).23 Interpretive criteria for P. aeruginosa were applied to all Pseudomonas spp. and interpretive criteria for S. pneumoniae for alpha-hemolytic streptococci. Antimicrobial susceptibility testing was dependent on the availability of antimicrobial disks (HiMedia) at Yangon General Hospital. Quality control for susceptibility testing used was done according to CLSI guidelines using ATCC strains.
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Table 1. Antimicrobial discs tested and reported for each bacterial group at Yangon General Hospital, 2005–2013 Bacterial group
Antimicrobial discs used and reported
significance or Fisher’s exact test, when appropriate, was used to compare proportions and odd ratios were calculated to estimate effect sizes associated with bloodstream infections, using STATA software (StataCorp 2013, Stata Statistical Software: Release 13, College Station, TX, USA). A comparison between groups was done only when the sample sizes of both groups exceeded 10. For the purposes of analysis blood culture isolates were categorized into four groups: Gram-positive organisms, Enterobacteriaceae, non-fermenting Gram-negative bacilli and yeasts. Coagulasenegative Staphylococci, Corynebacterium species and Bacillus group isolates were considered contaminants. Trends in antimicrobial resistance were restricted predominantly to routinely reported, or Group A antimicrobials suggested by CLSI.24 The antimicrobial discs used and reported for each bacterial group are shown in Table 1. Time trends were assessed comparing the time period 2005 through 2008 with 2009 through 2013, reflecting the approximate mid-point of the dataset. The rainy season was defined as May through October and the dry season from November through April.
Results Patient characteristics Between 2005 and 2013, 3865 blood cultures were processed from 3858 patients attending Yangon General Hospital. Of the 3858 patients, 3604 (93.2%) were inpatients and the remainder from outpatients. The median (range) age of patients was 40 (12–100) years.
Bloodstream infections Of 3865 blood cultures performed, pathogenic bacteria were isolated from 449 (11.6%) samples, 229 (5.9%) grew probable contaminates and 3187 (82.5%) showed no growth. Table 2 shows the distribution of pathogenic bacterial and fungal species isolated from blood cultures at Yangon General Hospital by time period. Of 3865 blood culture collected during the study period, 1849 (47.8%) were collected from 2005 through 2008, and 2016 (52.2%) were collected from 2009 through 2013 (p,0.001).
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During the period 2005 through 2008, Gram-positive bacteria accounted for 52.8% (167/316) bloodstream infections as compared with 20.3% (27/133) during the period 2009 through 2013 (p,0.001). During 2005 through 2008, Staphylococcus aureus accounted for 46.7% (147/316) bloodstream infections compared with 18.0% (24/133) during 2009 through 2013 (p,0.001). From 2005 through 2008, Gram-negative bacteria accounted for 45.6% (144/316) bloodstream infections compared with 78.9% (105/133) from 2009 through 2013 (p,0.001). Enterobacteriaceae accounted for 26.9% (85/316) Gram-negative isolates from 2005 through 2008 compared with 36.8% (49/133) from 2009–2013 (p,0.001). Non-fermentative bacilli accounted for 18.7% (59/316) Gramnegative pathogens from 2005 through 2008 compared to 42.1% (56/133) from 2009 through 2013 (p,0.001). Streptococcus species were not identified to the species level. No H. influenzae isolates were identified in this study.
Patterns of antimicrobial resistance among bloodstream isolates Changes in availability of antimicrobial disks and antimicrobial agents in Myanmar influenced drugs selected for susceptibility testing during the study period. Of all tested S. aureus, 5 of 7 isolates were resistant to penicillin during period 2005 through 2008 period compared with 1 of 7 tested in the 2009 through 2013 time period. Over the entire study period, 10.4% (17/163) of the S. aureus tested were resistant to tetracycline and 36.7% (58/158) were resistant to oxacillin (i.e., methicillin-resistant S. aureus, MRSA). MRSA accounted for 38.7% (55/142) of S. aureus isolates during 2005 through 2008, compared with 18.8% (3/16) of isolates from 2009 through 2013 (p¼0.116). Erythromycin resistance was identified in 51.1% (68/133) of S. aureus isolates from 2005 through 2008, compared with 27.3% (6/22) of isolates from 2009 through 2013 (p,0.001). Over the entire study period, 30.8% (4/13) of Enterococcus species were found to be resistant to ampicillin and 68.8% (11/16) were resistant to erythromycin. Of tested Streptococcus species, 2 of 3 tested were resistant to penicillin. No vancomycin-resistant Gram-positive bacteria were found. Among Enterobactericeae isolated during the entire study period, 72% (13/18) E. coli were resistant to amoxicillin-clavulanic
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Gram-positive bacteria First line drugs: ampicillin, erythromycin, penicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, clindamycin, ciprofloxacin. Second line drugs: vancomycin, azithromycin, amoxicillin-clavulanic acid, levofloxacin. Enterobacteriaceae First line drugs: amikacin, gentamicin, amoxicillin-clavulanic acid, ceftriaxone, cefotaxime, ceftazidime, ciprofloxacin, gentamicin. Second line drugs: ampicillin-sulbactam, piperacillin-tazobactam, cefoperazone-sulbactam, levofloxacin, imipenem, cefipime. Gram-negative First line drugs: amikacin, gentamicin, cefoperazone-sulbactam, ceftazidme, ceftriaxone, cefotaxime, ciprofloxacin. non-fermentative Second line drugs: piperacillin- tazobactam, imipenem, cefipime, levofloxacin. bacilli
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Table 2. Distribution of pathogenic bacterial and fungal species isolated from blood cultures at Yangon General hospital, 2005 through 2008 compared with 2009 through 2013
Table 3. Seasonal variation of blood culture isolates at Yangon General Hospital, 2005 through 2013 Organism
Organism
Rainy season
Total
2005–2008
2009–2013
Total
n
(%)
n
n
n
n
n
(%)
167 (52.8) 147 (46.7)
(%) 27 (20.3) 24 (18.0)
(%)
(37.7) (36.3) (24.0)
139 (45.9) 81 (26.7) 80 (26.4)
194 (43.2) 171 (38.1)
15 5 144 85 31 22 6 7
(4.7) (1.6) (45.6) (26.9) (9.8) (7.0) (1.9) (2.2)
2 1 105 49 17 23 3 0
(1.5) (0.8) (78.9) (36.8) (12.8) (17.3) (2.3) (0)
17 6 249 134 48 45 9 7
(3.8) (1.3) (55.5) (29.8) (10.7) (10.0) (2.0) (1.6)
5 3 11 59
(1.6) (0.9) (3.5) (18.7)
1 0 5 56
(0.8) (0) (3.8) (42.1)
6 3 16 115
(1.3) (0.7) (3.6) (25.6)
27 23 1 8 5 4
(8.5) (7.3) (0.3) (2.5) (1.6) (1.3)
36 8 2 10 1 1
(27.1) (6.0) (1.5) (7.5) (0.8) (0.8)
63 31 3 18 6 5
(14.0) (6.9) (0.7) (4.0) (1.3) (1.1)
1 (0.3) 0 (0) 1 (0.2) 316 (100.0) 133 (100.0) 449 (100.0)
a
Salmonella enterica consisted of 5 Salmonella enterica serovar Typhi and 2 Salmonella enterica that were not serotyped. b Others: organisms that could not be speciated, included 18 non-fermentative Gram-negative bacilli and 17 Enterobacteriaceae.
acid, 82% (37/45) were resistant to ceftriaxone and 57% (25/44) were resistant to gentamicin. Among Citrobacter species, 83% (15/18) were resistant to amoxicillin-clavulanic acid, 65% (30/46) were resistant to ceftriaxone and 66% (29/44) were resistant to gentamicin. Among Gram-negative non-fermentative bacilli, 4 of 8 Acinetobacter species were resistant to ceftazidime, 35% (9/26) to gentamicin and 26% (6/23) were resistant to amikacin. Among Pseudomonas species, 51% (30/59) were resistant ceftazidime, 40% (24/60) resistant to gentamicin and 24% (11/46) were resistant to amikacin. Of Pseudomonas species 5% (1/21) were resistant to levoflaxcin during the period 2005 through 2008 compared with 19% (5/27) from the time period 2009 through 2013 (p,0.001). Of 20 Enterobacteriaceae and 15 non-fermentative
Gram-positive bacteria 55 Enterobacteriaceae 53 Gram-negative 35 non-fermentative bacilli Yeasts 3 Total 146
(%)
(%)
194 (43.2) 134 (29.8) 115 (25.6)
(2.1) 3 (1.0) 6 (1.3) (100.0) 303 (100.0) 449 (100.0)
Gram-negative bacilli, none were found to be resistant to meropenem or imipenem.
Seasonal variation Of the 449 pathogenic blood culture isolates, 146 (32.5%) were found in the dry season compared to 303 (67.5%) in the rainy or monsoon season (OR¼1.9; 95% CI 1.5–2.6; p,0.001). Gram-positive bacteria (OR¼2.2; 95% CI 0.9–2.1; p¼0.110) and Gram-negative non-fermentative bacilli (OR¼4.6; 95% CI 2.8–7.5; p,0.001) were more likely to be isolated from patients during the wet season compared to the dry season. Enterobacteriaceae were more likely to be isolated from patients during the dry season compared to the wet season (OR¼0.6; 95% CI 0.4–0.99; p¼0.047) No significant seasonal association was found for yeasts (Table 3).
Discussion A high prevalence of bloodstream infections among patients receiving blood cultures at a tertiary hospital in Yangon, Myanmar, was found in this study. Although S. aureus was the most frequently isolated pathogen, Gram-negative bacteria were the most common group of organisms accounting for 55.5% of isolated pathogens. While community-acquired pathogens such as Salmonella typhi and B. pseudomallei were uncommon in this study, pathogens that are often healthcare-associated were frequently isolated and demonstrated patterns of antimicrobial resistance similar to those common elsewhere in the south and southeast Asian region. There were significant differences in the prevalence of groups of bloodstream pathogens between the earlier and later time period, with a decrease in Gram-positive isolates and an increase in Gram-negative isolates over time. A growing proportion of non-fermentative bacilli drove the increase in Gram-negative organisms in the later period. Although the increase in the Gram-negative bloodstream infections is likely to be real, it is possible that changes in the use of antimicrobial agents over time could have played a role. It is possible that healthcare-associated infections might also have contributed to this trend.25 Differences in bloodstream infections between the wet and dry seasons were also observed, with patients found to be
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Gram-positive Staphylococcus aureus Enterococcus spp Streptococcus spp Gram-negative Enterobacteriaceae Citrobacter spp Escherichia coli Klebsiella spp Salmonella enterica a Serratia spp Enterobacter spp Otherb Non-fermentative bacilli Pseudomonas spp Acinetobacter spp Burkholderia spp Otherb Yeasts Non-albicans Candida Candida Total
Dry season
Time period
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However, the recent emergence of carbapenem-resistant bacterial strains in neighboring China43 and India15 warrant vigilance and judicious use of this class of antimicrobials. The study had a number of limitations. It was retrospective, so patient selection for blood culture and clinical and laboratory methods were not standardized. This, in turn, contributed to relative small numbers of isolates of some species. Limited access to relevant clinical information meant that we were unable to distinguish community-acquired infections from healthcare-associated infections. Key determinants of blood culture sensitivity such as blood culture volume adequacy and antimicrobial exposure prior to blood culture were not known. Furthermore, only a single blood culture done for each patient and blood culture contamination at specimen collection could not be controlled.44,45 The microbiologic methods of this study were not optimized in a number of respects. For example, the use of human blood agar and lack of use of chocolate agar, and CO2 incubation is likely to have diminished the yield of a number of relevant pathogens including fastidious organisms such as S. pneumoniae and H. influenzae.46 Limited capacity to serogroup or serotype S. enterica and to identify non-fermentative Gram-negative bacteria to the species level affected our ability to detect regionally relevant pathogens, including Salmonella typhi and B. pseudomallei. Furthermore, changes in drugs and methods selected for antimicrobial susceptibility testing constrained our ability to measure time trends in antimicrobial resistance. In conclusion, this study describes the epidemiology of bloodstream infections among patients seen at a tertiary hospital in Yangon, Myanmar. MRSA and drug-resistant Gram-negative organisms were found to have emerged in the study area. Our findings suggest that patterns of antimicrobial resistance among invasive organism are similar to those described elsewhere in the region,8,47 and provide useful information for the development of locallyadapted antimicrobial guidelines for the management of sepsis. However, our study had a number of limitations that could be addressed in future studies with prospective and standardized investigation of bloodstream infections using optimized laboratory methods.
Authors’ contributions: TOM and NP contributed equally to this work; JAC, DRM and KKT conceived the study; TOM, NP, KKT, DRM and JAC designed the study protocol; TOM, NP, KKW, WWH and KNZ collected the data; KNZ oversaw laboratory assessments; NP and TOM carried out data analysis; NP and JAC drafted the manuscript; all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript. NP, TOM and JAC are guarantors of the paper. Acknowledgements: We are grateful to the clinical and laboratory staff and patients of Yangon General Hospital for their contributions to this study. Funding: This research was supported in part by a grant from the University of Otago Development Office, New Zealand. Competing interests: None declared. Ethical approval: Ethical approval was obtained from the Research and Ethical Committee, University of Medicine, Yangon, Myanmar and the Human Ethics Committee, University of Otago, Dunedin, New Zealand.
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approximately twice as likely to have a bacterial bloodstream infection in the rainy season as compared to the dry season. More specifically, Gram-positive bacteria and non-fermenting Gram-negative bacilli were found to account for a larger proportion of bloodstream pathogens during the rainy season compared to the dry season at Yangon General Hospital. A variety of reasons have been suggested for seasonal patterns of bloodstream infections and include behavior, environmental factors such as weather, and occurrence of underlying diseases, variation in virulence factors of bacterial pathogens, host susceptibility and patient demographics.26,27 Increased humidity is also found to be associated with increased incidence of P. aeruginosa bloodstream infection.28 Humidity is high in Yangon during the wet season and may contribute to patterns observed in our study. Our findings differed from those of bloodstream infection studies elsewhere in Myanmar and in the south and southeast Asia region. The proportion of blood cultures positive for a pathogen was 11.6% in our study, but ranged from 15–50% in studies done elsewhere.5,29–31 Salmonella typhi and Paratyphi A were isolated less often in our study than in studies elsewhere in Myanmar4,5 and Asia.3,30–32 Given the history of meliodosis in Yangon, it was surprising that there were no confirmed B. pseudomallei isolates. However, the disease is described among travellers returning from Myanmar33 and elsewhere in the country.34 Laboratory methods used during the study period were suboptimal for the isolation and identification of B. pseudomallei 22 and it is likely that cases of melioidosis were missed. It is also possible that melioidosis is less common in the catchment population of Yangon General Hospital than it once was. Antimicrobial resistance was common among a range of important pathogens and to a number of key antimicrobial agents relevant to clinical management. Resistance of S. aureus to erythromycin, penicillin, oxacillin and trimethoprim-sulfamethoxazole was common. Notably, almost one third of S. aureus isolates were methicillin-resistant, a proportion similar to other studies in the region.35 However, unlike studies from neighbouring countries36–38 no Gram-positive organisms demonstrated decreased susceptibility or resistance to vancomycin, which may have been due to the limited availability of the drug in the country during the study period. Among Gram-negative bacteria, more than half of tested E. coli and Citrobacter species showed resistance to amoxicillin-clavulanic acid, ceftriaxone or gentamicin. The small number of Enterobacter and S. enterica isolates and limited testing for antimicrobial resistance precluded analysis of resistance patterns among these organisms. Of Gram-negative non-fermentative bacilli, 20–60% of all tested Acinetobacter and Pseudomonas species were found to be resistant to the commonly recommended antimicrobials ceftazidime, gentamicin and amikacin. Resistance to imipenem was not detected among the 20 Enterobacteriaceae and 15 nonfermentative bacilli tested. Although unconfirmed, it is likely that resistance to ceftriaxone among Enterobacteriaceae is mediated by a range of extended-spectrum b-lactamases as high numbers of such isolates have been found in other studies in Asia.14 The presence of resistance to extended-spectrum cephalosporins is of particular concern because of their role in the empiric therapy of serious community-onset infections in low-resource areas.39,40 Furthermore, this pattern of antimicrobial resistance may fuel the use of carbapenems as the drug class of first choice for the treatment of serious infections.41,42 Notably, carbapenem-resistant bloodstream isolates were not found during the study period.
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