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.

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.

Diagnostic accuracy of antigen-based immunochromatographic rapid diagnostic tests for the detection of Salmonella in blood culture broth.

BACKGROUND: In low resource settings, Salmonella serovars frequently cause bloodstream infections. This study investigated the diagnostic performance of immunochromatographic rapid diagnostic tests (RDTs), which detect Salmonella antigens, when applied to stored grown blood culture broth. MATERIAL/METHODS: The SD Bioline One Step Salmonella Typhi Ag Rapid Detection Kit (Standard Diagnostics, Republic of Korea), marketed for the detection of Salmonella enterica serovar Typhi (Salmonella Typhi) in stool and the Salmonella Ag Rapid Test (Creative Diagnostics, USA), marketed for the detection of all Salmonella serotypes in stool, were selected for evaluation based on a pre-test evaluation of six RDT products. The limits of detection (LOD) for culture suspensions were established and the selected RDT products were assessed on 19 freshly grown spiked blood culture broth samples and 413 stored clinical blood culture broth samples, collected in Cambodia and the Democratic Republic of the Congo. RESULTS: The LOD of both products was established as 107-108 CFU/ml. When applied to clinical blood culture broth samples, the diagnostic sensitivity and specificity of the SD Bioline RDT were respectively 100% and 79.7% for the detection of Salmonella Typhi; 94.4% (65/69) of false-positive results were caused by Salmonella Enteritidis. When considering the combined detection of Salmonella Typhi and Enteritidis (both group D Salmonella), sensitivity and specificity were 97.9% and 98.5% respectively. For Creative Diagnostics, diagnostic sensitivity was 78.3% and specificity 91.0% for all Salmonella serotypes combined; 88.3% (53/60) of false negative results were caused by Salmonella Paratyphi A. CONCLUSIONS: When applied to grown blood culture broths, the SD Bioline RDT had a good sensitivity and specificity for the detection of Salmonella Typhi and Salmonella Enteritidis. The Creative Diagnostics product had a moderate sensitivity and acceptable specificity for the detection of all Salmonella serovars combined and needs further optimization. A RDT that reliably detects Salmonella Paratyphi A is needed.

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.

Whole-genome sequencing confirms that Burkholderia pseudomallei multilocus sequence types common to both Cambodia and Australia are due to homoplasy.

Burkholderia pseudomallei isolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial diversity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy.

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.

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.

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.

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.