Plugging the leaks: antibiotic resistance at human-animal interfaces in low-resource settings.

Antibiotic resistance is one of the greatest public health challenges of our time. International efforts to curb resistance have largely focused on drug development and limiting unnecessary antibiotic use. However, in areas where water, sanitation, and hygiene infrastructure is lacking, we propose that bacterial flow between humans and animals can exacerbate the emergence and spread of resistant pathogens. Here, we describe the consequences of poor environmental controls by comparing mobile resistance elements among Escherichia coli recovered from humans and meat in Cambodia, a middle-income country with substantial human-animal connectivity and unregulated antibiotic use. We identified identical mobile resistance elements and a conserved transposon region that were widely dispersed in both humans and animals, a phenomenon rarely observed in high-income settings. Our findings indicate that plugging leaks at human-animal interfaces should be a critical part of addressing antibiotic resistance in low- and especially middle-income countries.

Etiological spectrum of persistent fever in the tropics and predictors of ubiquitous infections: a prospective four-country study with pooled analysis.

BACKGROUND: Persistent fever, defined as fever lasting for 7 days or more at first medical evaluation, has been hardly investigated as a separate clinical entity in the tropics. This study aimed at exploring the frequencies and diagnostic predictors of the ubiquitous priority (i.e., severe and treatable) infections causing persistent fever in the tropics. METHODS: In six different health settings across four countries in Africa and Asia (Sudan, Democratic Republic of Congo [DRC], Nepal, and Cambodia), consecutive patients aged 5 years or older with persistent fever were prospectively recruited from January 2013 to October 2014. Participants underwent a reference diagnostic workup targeting a pre-established list of 12 epidemiologically relevant priority infections (i.e., malaria, tuberculosis, HIV, enteric fever, leptospirosis, rickettsiosis, brucellosis, melioidosis, relapsing fever, visceral leishmaniasis, human African trypanosomiasis, amebic liver abscess). The likelihood ratios (LRs) of clinical and basic laboratory features were determined by pooling all cases of each identified ubiquitous infection (i.e., found in all countries). In addition, we assessed the diagnostic accuracy of five antibody-based rapid diagnostic tests (RDTs): Typhidot Rapid IgM, Test-itTM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-itTM Leptospira IgM Lateral Flow Assay and SD Bioline Leptospira IgG/IgM for leptospirosis. RESULTS: A total of 1922 patients (median age: 35 years; female: 51%) were enrolled (Sudan, n = 667; DRC, n = 300; Nepal, n = 577; Cambodia, n = 378). Ubiquitous priority infections were diagnosed in 452 (23.5%) participants and included malaria 8.0% (n = 154), tuberculosis 6.7% (n = 129), leptospirosis 4.0% (n = 77), rickettsiosis 2.3% (n = 44), enteric fever 1.8% (n = 34), and new HIV diagnosis 0.7% (n = 14). The other priority infections were limited to one or two countries. The only features with a positive LR ≥ 3 were diarrhea for enteric fever and elevated alanine aminotransferase level for enteric fever and rickettsiosis. Sensitivities ranged from 29 to 67% for the three RDTs targeting S. Typhi and were 9% and 16% for the two RDTs targeting leptospirosis. Specificities ranged from 86 to 99% for S. Typhi detecting RDTs and were 96% and 97% for leptospirosis RDTs. CONCLUSIONS: Leptospirosis, rickettsiosis, and enteric fever accounted each for a substantial proportion of the persistent fever caseload across all tropical areas, in addition to malaria, tuberculosis, and HIV. Very few discriminative features were however identified, and RDTs for leptospirosis and Salmonella Typhi infection performed poorly. Improved field diagnostics are urgently needed for these challenging infections. TRIAL REGISTRATION: NCT01766830 at ClinicalTrials.gov.

Meat and Fish as Sources of Extended-Spectrum ß-Lactamase-Producing Escherichia coli, Cambodia.

We compared extended-spectrum ß-lactamase-producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum ß-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route.

Diagnostic accuracy of the InBiOS AMD rapid diagnostic test for the detection of Burkholderia pseudomallei antigen in grown blood culture broth.

To assess the diagnostic and operational performance of the InBiOS AMD rapid diagnostic test (RDT) (Seattle, USA) for the detection of B. pseudomallei in grown blood culture broth. The InBiOS RDT is a lateral flow immunoassay in a strip format detecting B. pseudomallei capsular polysaccharide in culture fluids, marketed for research only. Broth of blood culture bottles (BacT/Alert, bioMérieux, Marcy L'Etoile, France) sampled in adult patients at the Sihanouk Hospital Center of HOPE, Phnom Penh, Cambodia, during 2010-2017 and stored at - 80 °C was tested. They included samples grown with B. pseudomallei (n = 114), samples with no growth (n = 12), and samples with growth of other pathogens (n = 139, among which Burkholderia cepacia (n = 5)). Diagnostic sensitivity and specificity were 96.5% [95% confidence interval (CI): 91.3-98.6%] and 100% [CI: 97.5-100%] respectively. Background clearance and line intensities were good and very good. The RDT's test strip, not housed in a cassette, caused difficulties in manipulation and biosafety. The centrifugation step prescribed by the procedure challenged biosafety, but processing of 19 B. pseudomallei samples without centrifugation showed similar results for line intensity and background clearance, compared to centrifugation. The InBiOS RDT showed excellent accuracy for detection of B. pseudomallei in grown blood culture broth. Provided operational adaptations such as cassette housing, it has the potential to reduce time to diagnosis of melioidosis.

Does HIV status affect the aetiology, bacterial resistance patterns and recommended empiric antibiotic treatment in adult patients with bloodstream infection in Cambodia?

OBJECTIVE: The microbiologic causes of bloodstream infections (BSI) may differ between HIV-positive and HIV-negative patients and direct initial empiric antibiotic treatment (i.e. treatment before culture results are available). We retrospectively assessed community-acquired BSI episodes in adults in Cambodia according to HIV status for spectrum of bacterial pathogens, antibiotic resistance patterns and appropriateness of empiric antibiotics. METHODS: Blood cultures were systematically performed in patients suspected of BSI in a referral hospital in Phnom Penh, Cambodia. Data were collected between 1 January 2009 and 31 December 2011. RESULTS: A total of 452 culture-confirmed episodes of BSI were recorded in 435 patients, of whom 17.9% and 82.1% were HIV-positive and HIV-negative, respectively. Escherichia coli accounted for one-third (n = 155, 32.9%) of 471 organisms, with similar rates in both patient groups. Staphylococcus aureus and Salmonella cholereasuis were more frequent in HIV-positive vs. HIV-negative patients (17/88 vs. 38/383 (P = 0.02) and 10/88 vs. 5/383 (P < 0.001)). Burkholderia pseudomallei was more common in HIV-negative than in HIV-positive patients (39/383 vs. 2/88, P < 0.001). High resistance rates among commonly used antibiotics were observed, including 46.6% ceftriaxone resistance among E. coli isolates. Empiric antibiotic treatments were similarly appropriate in both patient groups but did not cover antibiotic-resistant E. coli (both patient groups), S. aureus (both groups) and B. pseudomallei (HIV-negative patients). CONCLUSION: The present data do not warrant different empiric antibiotic regimens for HIV-positive vs. HIV-negative patients in Cambodia. The overall resistance rates compromise the appropriateness of the current treatment guidelines.

Melioidosis, phnom penh, Cambodia.

We describe 58 adult patients with melioidosis in Cambodia (2007-2010). Diabetes was the main risk factor (59%); 67% of infections occurred during the rainy season. Bloodstream infection was present in 67% of patients, which represents 12% of all bloodstream infections. The case-fatality rate was 52% and associated with inappropriate empiric treatment.

Nonautomated Blood Cultures in a Low-Resource Setting: Optimizing the Timing of Blind Subculture.

Laboratory procedures for blood cultures in a hospital in Phnom Penh were adapted to optimize detection of Burkholderia pseudomallei, an important pathogen in this setting. The effects of these changes are analyzed in this study. Blood cultures consisted of two BacT/ALERT bottles (bioMérieux, Marcy-l'Etoile, France). Growth was detected visually by daily inspection of the bottles. In 2016, the aerobic-anaerobic pair (FA/FN FAN) was substituted by an aerobic pair of BacT/ALERT FA Plus bottles. Blind subculture (BS) (subculture in the absence of visual growth) was advanced from day 3 to day 2 of incubation in July 2016. In July 2018, it was further advanced to day 1 of incubation. From July 2016 to October 2019, 9,760 blood cultures were sampled. The proportion of cultures showing pathogen growth decreased from 9.6% to 6.8% after the implementation of the laboratory changes (P < 0.001). Advancing the BS from day 3 to day 2 led to an increased proportion of pathogens detected by day 3 (92.8% versus 82.3%; P < 0.001); for B. pseudomallei, this increase was even more remarkable (92.0% versus 18.2%). Blind subculture on day 1 similarly increased the proportion of pathogens detected by day 2 (82.9% versus 69.0% overall, 66.7% versus 10.0% for B. pseudomallei; both P < 0.001). However, after implementation of day 1 subculture, a decrease in recovery of B. pseudomallei was observed (12.4% of all pathogens versus 4.3%; P < 0.001). In conclusion, earlier subculture significantly shortens time to detection and time to actionable results. Some organisms may be missed by performing an early subculture, especially those that grow more slowly.

Diagnostic Bacteriology in District Hospitals in Sub-Saharan Africa: At the Forefront of the Containment of Antimicrobial Resistance.

This review provides an update on the factors fuelling antimicrobial resistance and shows the impact of these factors in low-resource settings. We detail the challenges and barriers to integrating clinical bacteriology in hospitals in low-resource settings, as well as the opportunities provided by the recent capacity building efforts of national laboratory networks focused on vertical single-disease programmes. The programmes for HIV, tuberculosis and malaria have considerably improved laboratory medicine in Sub-Saharan Africa, paving the way for clinical bacteriology. Furthermore, special attention is paid to topics that are less familiar to the general medical community, such as the crucial role of regulatory frameworks for diagnostics and the educational profile required for a productive laboratory workforce in low-resource settings. Traditionally, clinical bacteriology laboratories have been a part of higher levels of care, and, as a result, they were poorly linked to clinical practices and thus underused. By establishing and consolidating clinical bacteriology laboratories at the hospital referral level in low-resource settings, routine patient care data can be collected for surveillance, antibiotic stewardship and infection prevention and control. Together, these activities form a synergistic tripartite effort at the frontline of the emergence and spread of multi-drug resistant bacteria. If challenges related to staff, funding, scale, and the specific nature of clinical bacteriology are prioritized, a major leap forward in the containment of antimicrobial resistance can be achieved. The mobilization of resources coordinated by national laboratory plans and interventions tailored by a good understanding of the hospital microcosm will be crucial to success, and further contributions will be made by market interventions and business models for diagnostic laboratories. The future clinical bacteriology laboratory in a low-resource setting will not be an "entry-level version" of its counterparts in high-resource settings, but a purpose-built, well-conceived, cost-effective and efficient diagnostic facility at the forefront of antimicrobial resistance containment.

Slow growth of Burkholderia pseudomallei compared to other pathogens in an adapted blood culture system in Phnom Penh, Cambodia.

PURPOSE: Burkholderia pseudomallei is a key pathogen causing bloodstream infections at Sihanouk Hospital Center of Hope, Phnom Penh, Cambodia. Here, visual instead of automated detection of growth of commercial blood culture bottles is done. The present study assessed the performance of this system. METHODOLOGY: Blood culture sets, consisting of paired adult aerobic and anaerobic bottles (bioMérieux, FA FAN 259791 and FN FAN 252793) were incubated in a standard incubator for 7 days after reception. Each day, the bottle growth indicator was visually inspected for colour change indicating growth. Blind subculture was performed from the aerobic bottle at day 3. RESULTS: From 2010 to 2015, 11  671 sets representing 10  389 suspected bloodstream infection episodes were documented. In 1058 (10.2  %) episodes, pathogens grew; they comprised Escherichia coli (31.7 %), Salmonella Paratyphi A (13.9 %), B. pseudomallei (8.5 %), Staphylococcus aureus (7.8 %) and Klebsiella pneumoniae (7.0 %). Blind subculture yielded 72 (4.1  %) pathogens, mostly (55/72, 76.4 %) B. pseudomallei. Cumulative proportions of growth at day 2 were as follows: E. coli: 85.0 %, Salmonella Paratyphi A: 85.0 %, K. pneumoniae: 76.3  % and S. aureus: 52.2  %; for B. pseudomallei, this was only 4.0  %, which increased to 70.1  % (70/99) at day 4 mainly by detection on blind subculture (55/99). Compared to the anaerobic bottles, aerobic bottles had a higher yield and a shorter time-to-detection, particularly for B. pseudomallei. CONCLUSIONS: Visual inspection for growth of commercial blood culture bottles in a low-resource setting provided satisfactory yield and time-to-detection. However, B. pseudomallei grew slowly and was mainly detected by blind subculture. The aerobic bottle outperformed the anaerobic bottle.

Escherichia coli ST410 among humans and the environment in Southeast Asia.

Escherichia coli ST410 (Ec-ST410) is an emerging, multidrug-resistant clone. Recent investigations of its global epidemiology and evolution have been based almost exclusively on isolates from Europe and North America. It is unclear whether Southeast Asian-origin Ec-ST410 (SEA-Ec-ST410) belong to these same clones or represent regionally disseminated variants. Antimicrobial resistance mechanisms among SEA-Ec-ST410 were characterised, and whether they belonged to regional variants was investigated by contextualising them within a global collection. Seven Ec-ST410 were identified among a recent collection of expanded-spectrum cephalosporin-resistant E. coli recovered from 91 healthy women (stool) and 26 infected patients (blood and urine) living in Phnom Penh, Cambodia. Nine additional Ec-ST410 genomes were identified from Thailand (n = 7) and Vietnam (n = 2) through EnteroBase and PubMed searches. The assembled genomes were characterised and a SNP-based phylogenetic tree was created comparing these 16 SEA-Ec-ST410 with a previously published Ec-ST410 collection, primarily sourced from Europe (97/128) and North America (24/128). SEA-Ec-ST410 belonged to several distinct branches within previously described clonal clades. SEA-Ec-ST410 within the B3/H24Rx sublineage encoded blaCTX-M-55 (8/12) and F18:A-:B1 plasmid replicons (6/12), neither of which were detected among other Ec-ST410 belonging to this clade. Three of four SEA-Ec-ST410 within the B4/H24RxC sublineage lacked both blaOXA-181 and an IncX3 plasmid replicon that were harboured by 97% and 100% of all other Ec-ST410 in this sublineage (n = 64), respectively. In conclusion, Ec-ST410 are present in Southeast Asia following multiple introductions. The unique pattern of antimicrobial resistance elements harboured by SEA-Ec-ST410 suggests independent circulation in the region.

Antimicrobial resistance in Cambodia: a review.

OBJECTIVES: Following the launch of the Global Antimicrobial Resistance Surveillance System (GLASS), antimicrobial resistance (AMR) rates in many countries remain poorly described. This review provides an overview of published AMR data from Cambodia in the context of recently initiated national human and food-animal surveillance. METHODS: PubMed and the Cochrane Database of Systematic Reviews were searched for articles published from 2000 to 2018, which reported antimicrobial susceptibility testing (AST) data for GLASS specific organisms isolated from Cambodia. Articles were screened using strict inclusion/exclusion criteria. AST data was extracted, with medians and ranges of resistance rates calculated for specific bug-drug combinations. RESULTS: Twenty-four papers were included for final analysis, with 20 describing isolates from human populations. Escherichia coli was the most commonly described organism, with median resistance rates from human isolates of 92.8% (n=6 articles), 46.4% (n=4), 55.4% (n=8), and 46.4% (n=5) to ampicillin, 3rd generation cephalosporins, fluoroquinolones, and gentamicin respectively. CONCLUSIONS: Whilst resistance rates are high for several GLASS organisms, there were insufficient data to draw robust conclusions about the AMR situation in Cambodia. The recently implemented national AMR surveillance systems will begin to address this data gap.

Serotype Distribution of Clinical Streptococcus pneumoniae Isolates before the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Cambodia.

Childhood vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in Cambodia in January 2015. Baseline data regarding circulating serotypes are scarce. All microbiology laboratories in Cambodia were contacted for identification of stored isolates of Streptococcus pneumoniae from clinical specimens taken before the introduction of PCV13. Available isolates were serotyped using a multiplex polymerase chain reaction method. Among 166 identified isolates available for serotyping from patients with pneumococcal disease, 4% were isolated from upper respiratory samples and 80% were from lower respiratory samples, and 16% were invasive isolates. PCV13 serotypes accounted for 60% (95% confidence interval [CI] 52-67) of all isolates; 56% (95% CI 48-64) of noninvasive and 77% (95% CI 57-89) of invasive isolates. Antibiotic resistance was more common among PCV13 serotypes. This study of clinical S. pneumoniae isolates supports the potential for high reduction in pneumococcal disease burden and may serve as baseline data for future monitoring of S. pneumoniae serotypes circulation after implementation of PCV13 childhood vaccination in Cambodia.

The clinical and microbiological characteristics of enteric fever in Cambodia, 2008-2015.

BACKGROUND: Enteric fever remains a major public health problem in low resource settings and antibiotic resistance is increasing. In Asia, an increasing proportion of infections is caused by Salmonella enterica serovar Paratyphi A, which for a long time was assumed to cause a milder clinical syndrome compared to Salmonella enterica serovar Typhi. METHODOLOGY: A retrospective chart review study was conducted of 254 unique cases of blood culture confirmed enteric fever who presented at a referral adult hospital in Phnom Penh, Cambodia between 2008 and 2015. Demographic, clinical and laboratory data were collected from clinical charts and antibiotic susceptibility testing was performed. Whole genome sequence analysis was performed on a subset of 121 isolates. RESULTS: One-hundred-and-ninety unique patients were diagnosed with Salmonella Paratyphi A and 64 with Salmonella Typhi. In the period 2008-2012, Salmonella Paratyphi A comprised 25.5% of 47 enteric fever cases compared to 86.0% of 207 cases during 2013-2015. Presenting symptoms were identical for both serovars but higher median leukocyte counts (6.8 x 109/L vs. 6.3 x 109/L; p = 0.035) and C-reactive protein (CRP) values (47.0 mg/L vs. 36 mg/L; p = 0.034) were observed for Salmonella Typhi infections. All but one of the Salmonella Typhi isolates belonged to haplotype H58 associated with multidrug resistance (MDR) (i.e. resistance to ampicillin, chloramphenicol and co-trimoxazole).;42.9% actually displayed MDR compared to none of the Salmonella Paratyphi A isolates. Decreased ciprofloxacin susceptibility (DCS) was observed in 96.9% (62/64) of Salmonella Typhi isolates versus 11.5% (21/183) of Salmonella Paratyphi A isolates (all but one from 2015). All isolates were susceptible to azithromycin and ceftriaxone. CONCLUSIONS: In Phnom Penh, Cambodia, Salmonella Paratyphi A now causes the majority of enteric fever cases and decreased susceptibility against ciprofloxacin is increasing. Overall, Salmonella Typhi was significantly more associated with MDR and DCS compared to Salmonella Paratyphi A.

Genomic analysis of Salmonella enterica serotype Paratyphi A during an outbreak in Cambodia, 2013-2015.

In 2013, an unusual increase in the number of Salmonella enterica serotype Paratyphi A (Salmonella Paratyphi A) infections was reported in patients in Phnom Penh, Cambodia, and in European, American and Japanese travellers returning from Cambodia. Epidemiological investigations did not identify a common source of exposure. To analyse the population structure and genetic diversity of these Salmonella Paratyphi A isolates, we used whole-genome sequencing on 65 isolates collected from 1999 to 2014: 55 from infections acquired in Cambodia and 10 from infections acquired in other countries in Asia, Africa and Europe. Short-read sequences from 80 published genomes from around the world and from 13 published genomes associated with an outbreak in China were also included. Pulsed-field gel electrophoresis (PFGE) was performed on a subset of isolates. Genomic analyses were found to provide much more accurate information for tracking the individual strains than PFGE. All but 2 of the 36 isolates acquired in Cambodia during 2013-2014 belonged to the same clade, C5, of lineage C. This clade has been isolated in Cambodia since at least 1999. The Chinese outbreak isolates belonged to a different clade (C4) and were resistant to nalidixic acid, whereas the Cambodian outbreak isolates displayed pan-susceptibility to antibiotics. Since 2014, the total number of cases has decreased, but there has been an increase in the frequency with which nalidixic acid-resistant C5 isolates are isolated. The frequency of these isolates should be monitored over time, because they display decreased susceptibility to ciprofloxacin, the first-choice antibiotic for treating paratyphoid fever.

Diagnosis of Persistent Fever in the Tropics: Set of Standard Operating Procedures Used in the NIDIAG Febrile Syndrome Study.

In resource-limited settings, the scarcity of skilled personnel and adequate laboratory facilities makes the differential diagnosis of fevers complex [1-5]. Febrile illnesses are diagnosed clinically in most rural centers, and both Rapid Diagnostic Tests (RDTs) and clinical algorithms can be valuable aids to health workers and facilitate therapeutic decisions [6,7]. The persistent fever syndrome targeted by NIDIAG is defined as presence of fever for at least one week. The NIDIAG clinical research consortium focused on potentially severe and treatable infections and therefore targeted the following conditions as differential diagnosis of persistent fever: visceral leishmaniasis (VL), human African trypanosomiasis (HAT), enteric (typhoid and paratyphoid) fever, brucellosis, melioidosis, leptospirosis, malaria, tuberculosis, amoebic liver abscess, relapsing fever, HIV/AIDS, rickettsiosis, and other infectious diseases (e.g., pneumonia). From January 2013 to October 2014, a prospective clinical phase III diagnostic accuracy study was conducted in one site in Cambodia, two sites in Nepal, two sites in Democratic Republic of the Congo (DRC), and one site in Sudan (clinicaltrials.gov no. NCT01766830). The study objectives were to (1) determine the prevalence of the target diseases in patients presenting with persistent fever, (2) assess the predictive value of clinical and first-line laboratory features, and (3) assess the diagnostic accuracy of several RDTs for the diagnosis of the different target conditions.

Risk factors of treatment-limiting anemia after substitution of zidovudine for stavudine in HIV-infected adult patients on antiretroviral treatment.

BACKGROUND: Anemia is the main concern among patients using a zidovudine (AZT)-based antiretroviral treatment (ART). Some studies suggested weight-adjusted AZT dosing as a way to reduce toxicity. We analyzed the risk factors associated with AZT-induced anemia in a cohort using AZT as substitution for stavudine (D4T). METHODS: We retrospectively studied HIV-infected patients in a referral hospital in Phnom Penh, Cambodia between 2003 and 2011. Factors associated with AZT-related anemia requiring AZT-discontinuation within the first year after AZT initiation were analyzed using Cox regression. RESULTS: Overall, 1180 patients, 60.5% female, were included. At AZT initiation, the median hemoglobin was 12.7 g/dL (IQR 11.7-13.9), the median weight: 51 kg (IQR 45-58) and the median time on ART prior to AZT substitution: 1.4 years (IQR 1.0-2.0). Within one year follow-up, 139 patients (11.8%) developed anemia requiring AZT discontinuation. Overall, there was no independent association of body weight with AZT discontinuation. AZT discontinuation was associated with lower hemoglobin level when starting AZT; older age and taking D4T-based ART less than one year prior to AZT. In exploratory analysis, a linear increase in risk of grade 2-4 anemia with lower body weight was seen if starting AZT substitution within less than one year of D4T-based ART. CONCLUSION: Our findings argue against the need of weight-based dosing of AZT to reduce anemia among patients using AZT as substitution for D4T. Whether this also applies to ART-naïve individuals remains to be assessed. Future studies with AZT dose reduction should assess efficacy and overall tolerance of AZT.

Bloodstream infection among adults in Phnom Penh, Cambodia: key pathogens and resistance patterns.

BACKGROUND: Bloodstream infections (BSI) cause important morbidity and mortality worldwide. In Cambodia, no surveillance data on BSI are available so far. METHODS: From all adults presenting with SIRS at Sihanouk Hospital Centre of HOPE (July 2007-December 2010), 20 ml blood was cultured. Isolates were identified using standard microbiological techniques; antibiotic susceptibilities were assessed using disk diffusion and MicroScan®, with additional E-test, D-test and double disk test where applicable, according to CLSI guidelines. RESULTS: A total of 5714 samples from 4833 adult patients yielded 501 clinically significant organisms (8.8%) of which 445 available for further analysis. The patients' median age was 45 years (range 15-99 y), 52.7% were women. HIV-infection and diabetes were present in 15.6% and 8.8% of patients respectively. The overall mortality was 22.5%. Key pathogens included Escherichia coli (n = 132; 29.7%), Salmonella spp. (n = 64; 14.4%), Burkholderia pseudomallei (n = 56; 12.6%) and Staphylococcus aureus (n = 53; 11.9%). Methicillin resistance was seen in 10/46 (21.7%) S. aureus; 4 of them were co-resistant to erythromycin, clindamycin, moxifloxacin and sulphamethoxazole-trimethoprim (SMX-TMP). We noted combined resistance to amoxicillin, SMX-TMP and ciprofloxacin in 81 E. coli isolates (62.3%); 62 isolates (47.7%) were confirmed as producers of extended spectrum beta-lactamase. Salmonella isolates displayed high rates of multidrug resistance (71.2%) with high rates of decreased ciprofloxacin susceptibility (90.0%) in Salmonella Typhi while carbapenem resistance was observed in 5.0% of 20 Acinetobacter sp. isolates. CONCLUSIONS: BSI in Cambodian adults is mainly caused by difficult-to-treat pathogens. These data urge for microbiological capacity building, nationwide surveillance and solid interventions to contain antibiotic resistance.

Azithromycin and ciprofloxacin resistance in Salmonella bloodstream infections in Cambodian adults.

BACKGROUND: Salmonella enterica is a frequent cause of bloodstream infection (BSI) in Asia but few data are available from Cambodia. We describe Salmonella BSI isolates recovered from patients presenting at Sihanouk Hospital Centre of Hope, Phnom Penh, Cambodia (July 2007-December 2010). METHODOLOGY: Blood was cultured as part of a microbiological prospective surveillance study. Identification of Salmonella isolates was performed by conventional methods and serotyping. Antibiotic susceptibilities were assessed using disk diffusion, MicroScan and E-test macromethod. Clonal relationships were assessed by Pulsed Field Gel Electrophoresis; PCR and sequencing for detection of mutations in Gyrase and Topoisomerase IV and presence of qnr genes. PRINCIPAL FINDINGS: Seventy-two Salmonella isolates grew from 58 patients (mean age 34.2 years, range 8-71). Twenty isolates were identified as Salmonella Typhi, 2 as Salmonella Paratyphi A, 37 as Salmonella Choleraesuis and 13 as other non-typhoid Salmonella spp. Infection with human immunodeficiency virus (HIV) was present in 21 of 24 (87.5%) patients with S. Choleraesuis BSI. Five patients (8.7%) had at least one recurrent infection, all with S. Choleraesuis; five patients died. Overall, multi drug resistance (i.e., co-resistance to ampicillin, sulphamethoxazole-trimethoprim and chloramphenicol) was high (42/59 isolates, 71.2%). S. Typhi displayed high rates of decreased ciprofloxacin susceptibility (18/20 isolates, 90.0%), while azithromycin resistance was very common in S. Choleraesuis (17/24 isolates, 70.8%). Two S. Choleraesuis isolates were extended spectrum beta-lactamase producer. CONCLUSIONS AND SIGNIFICANCE: Resistance rates in Salmonella spp. in Cambodia are alarming, in particular for azithromycin and ciprofloxacin. This warrants nationwide surveillance and revision of treatment guidelines.

Evaluation of a systematic substitution of zidovudine for stavudine-based HAART in a program setting in rural Cambodia.

OBJECTIVE: To evaluate a treatment strategy of substituting zidovudine (ZDV) for stavudine (d4T)-based highly active antiretroviral therapy (HAART), aimed at preventing d4T-associated toxicity, in a programmatic setting in rural Cambodia. METHODS: Survival probability, CD4 gain, anemia incidence, and factors associated with severe anemia were analyzed in a cohort of adult patients switched from d4T- to ZDV-containing regimens from March 2006 to March 2007. RESULTS: Among 527 patients systematically switched to ZDV after d4T-based HAART for a median of 18 months, 4 (0.8%) patients died, 2 (0.4%) were lost to follow-up, 18 (3.4%) were transferred out, and 503 (95.4%) remained on HAART. Median CD4 gain was +263.5 cells/microL (interquartile range: 89.25-369.5) at 24 months. Within 1 year after the switch, 21.9% and 7.1% of patients developed anemia (grades 1-4) and severe anemia (grades 3-4), respectively. Low body mass index (< or =18) and low CD4 count (<200 cells/microL) at the time of switch were factors associated with severe anemia. Additional follow-up visits for laboratory monitoring and adherence counseling, increased absenteeism from work, and transportation costs for the patients were noted. CONCLUSIONS: The switch strategy of substituting ZDV for d4T-based HAART led to satisfactory overall clinical outcomes. However, it resulted in a relatively high incidence of mild to severe anemia and increased burden for the program and the patients.