Exploring the pediatric nasopharyngeal bacterial microbiota with culture-based MALDI-TOF mass spectrometry and targeted metagenomic sequencing.

The nasopharynx is an important reservoir of disease-associated and antimicrobial-resistant bacterial species. This proof-of-concept study assessed the utility of a combined culture, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and targeted metagenomic sequencing workflow for the study of the pediatric nasopharyngeal bacterial microbiota. Nasopharyngeal swabs and clinical metadata were collected from Cambodian children during a hospital outpatient visit and then biweekly for 12 weeks. Swabs were cultured on chocolate and blood-gentamicin agar, and all colony morphotypes were identified by MALDI-TOF MS. Metagenomic sequencing was done on a scrape of all colonies from a chocolate agar culture and processed using the mSWEEP pipeline. One hundred one children were enrolled, yielding 620 swabs. MALDI-TOF MS identified 106 bacterial species/40 genera: 20 species accounted for 88.5% (2,190/2,474) of isolates. Colonization by Moraxella catarrhalis (92.1% of children on ≥1 swab), Haemophilus influenzae (87.1%), and Streptococcus pneumoniae (83.2%) was particularly common. In S. pneumoniae-colonized children, a median of two serotypes [inter-quartile range (IQR) 1-2, range 1-4] was detected. For the 21 bacterial species included in the mSWEEP database and identifiable by MALDI-TOF, detection by culture + MALDI-TOF MS and culture + mSWEEP was highly concordant with a median species-level agreement of 96.9% (IQR 86.8%-98.8%). mSWEEP revealed highly dynamic lineage-level colonization patterns for S. pneumoniae which were quite different to those for S. aureus. A combined culture, MALDI-TOF MS, targeted metagenomic sequencing approach for the exploration of the young child nasopharyngeal microbiome was technically feasible, and each component yielded complementary data. IMPORTANCE: The human upper respiratory tract is an important source of disease-causing and antibiotic-resistant bacteria. However, understanding the interactions and stability of these bacterial populations is technically challenging. We used a combination of approaches to determine colonization patterns over a 3-month period in 101 Cambodian children. The combined approach was feasible to implement, and each component gave complementary data to enable a better understanding of the complex patterns of bacterial colonization.

Detection of maternal transmission of resistant Gram-negative bacteria in a Cambodian hospital setting.

Infection with Extended-spectrum beta-lactamase -producing Enterobacterales (ESBL-E) is common in infants and leads to increased intensive care unit admission and mortality, but the role of maternal transmission in colonization of infants is unclear. Using paired isolates from 50 pairs of mothers and neonates admitted to a Cambodian hospital, we investigated antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae using whole genome sequencing. We detected a wide variety of ESBL-E genes present in this population along with high levels of multidrug resistance. From 21 pairs where the same organism was present in both mother and neonate, we identified eight pairs with identical or near-identical isolates from both individuals suggestive of transmission at or around birth, including a pair with transmission of multiple strains. We found no evidence for transmission of plasmids only from mother to infant. This suggests vertical transmission outside hospitals as a common cause of ESBL-E colonization in neonates.

Transmission dynamics and control of multidrug-resistant Klebsiella pneumoniae in neonates in a developing country.

Multidrug-resistant Klebsiella pneumoniae is an increasing cause of infant mortality in developing countries. We aimed to develop a quantitative understanding of the drivers of this epidemic by estimating the effects of antibiotics on nosocomial transmission risk, comparing competing hypotheses about mechanisms of spread, and quantifying the impact of potential interventions. Using a sequence of dynamic models, we analysed data from a one-year prospective carriage study in a Cambodian neonatal intensive care unit with hyperendemic third-generation cephalosporin-resistant K. pneumoniae. All widely-used antibiotics except imipenem were associated with an increased daily acquisition risk, with an odds ratio for the most common combination (ampicillin + gentamicin) of 1.96 (95% CrI 1.18, 3.36). Models incorporating genomic data found that colonisation pressure was associated with a higher transmission risk, indicated sequence type heterogeneity in transmissibility, and showed that within-ward transmission was insufficient to maintain endemicity. Simulations indicated that increasing the nurse-patient ratio could be an effective intervention.

Healthcare workers' perceptions of an organizational quality assurance program implemented in a resource-limited setting: a qualitative study.

Background:There is increasing awareness of the need to implement quality assurance programs in developing countries. Healthcare staff are the primary drivers of improving the quality of care,but little is known about how they perceive quality assurance programs in resource-limited settings. This study aims to evaluate healthcare workers' perceptions of the organizational quality assurance program (OQA) at Angkor Hospital for Children (AHC), Cambodia. The OQA involves regular data collection and monitoring of quality indicators, to assess whether agreed quality standards are being met. Methods: This qualitative study consisted of four focus group discussions (FGDs) with 29 hospital staff (convenience sampling) from medical, nursing and non-medical departments. Staff members' understanding of quality assurance and perceptions of the strengths and weaknesses of the OQA were explored. Thematic content analysis was used to identify key themes. Results: Participants emphasized that quality indicators must include physical and psychological well-being. Strengths of the OQA included shared understanding amongst all groups of participants of its goals, committed leadership, that it was locally-relevant and that target indicators were developed from a "ground-up" approach. On-going challenges included that there was a gap in understanding of the OQA processes and overall running of the OQA across the organization between managers and staff. Conclusion: The introduction of the OQA at AHC has been well-received by staff members.Overall, the program is perceived to be valuable. Healthcare provision in resource-limited settings increasingly needs to demonstrate quality assurance. The model of OQA developed at AHC is one way to achieve this.

Transmission Dynamics of Hyper-Endemic Multi-Drug Resistant Klebsiella pneumoniae in a Southeast Asian Neonatal Unit: A Longitudinal Study With Whole Genome Sequencing.

Background:Klebsiella pneumoniae is an important and increasing cause of life-threatening disease in hospitalized neonates. Third generation cephalosporin resistance (3GC-R) is frequently a marker of multi-drug resistance, and can complicate management of infections. 3GC-R K. pneumoniae is hyper-endemic in many developing country settings, but its epidemiology is poorly understood and prospective studies of endemic transmission are lacking. We aimed to determine the transmission dynamics of 3GC-R K. pneumoniae in a newly opened neonatal unit (NU) in Cambodia and to address the following questions: what is the diversity of 3GC-R K. pneumoniae both within- and between-host; to what extent is high carriage prevalence driven by ward-based transmission; and to what extent can environmental contamination explain patterns of patient acquisition. Methods: We performed a prospective longitudinal study between September and November 2013. Rectal swabs from consented patients were collected upon NU admission and every 3 days thereafter. Morphologically different colonies from swabs growing cefpodoxime-resistant K. pneumoniae were selected for whole-genome sequencing (WGS). Results: One hundred and fifty-eight samples from 37 patients and 7 environmental sites were collected. 32/37 (86%) patients screened positive for 3GC-R K. pneumoniae and 93 colonies from 119 swabs were successfully sequenced. Isolates were resistant to a median of six (range 3-9) antimicrobials. WGS revealed high diversity; pairwise distances between isolates from the same patient were either 0-1 SNV or >1,000 SNVs; 19/32 colonized patients harbored K. pneumoniae colonies differing by >1000 SNVs. Diverse lineages accounted for 18 probable importations to the NU and nine probable transmission clusters involving 19/37 (51%) of screened patients. Median cluster size was five patients (range 3-9). Seven out of 46 environmental swabs (15%) were positive for 3GC-R K. pneumoniae. Environmental sources were plausible sources for acquisitions in 2/9 transmission clusters, though in both cases other patients were also plausible sources. Conclusion: The epidemiology of 3GC-R K. pneumoniae was characterized by multiple introductions, high within- and between host diversity and a dense network of cross-infection, with half of screened neonates part of a transmission cluster. We found no evidence to suggest that environmental contamination was playing a dominant role in transmission.

If you come from a well-known organisation, I will trust you: Exploring and understanding the community's attitudes towards healthcare research in Cambodia.

OBJECTIVE: To explore Cambodian community members' understanding of and attitudes towards healthcare research. DESIGN: This qualitative study generated data from semi-structured interviews and focus group discussions. This study was conducted at a non-governmental paediatric hospital and in nearby villages in Siem Reap province, Cambodia. A total of ten semi-structured interviews and four focus group discussions were conducted, involving 27 participants. Iterative data collection and analysis were performed concurrently. Data were analysed by thematic content analysis and the coding structure was developed using relevant literature. RESULTS: Participants did not have a clear understanding of what activities related to research compared with those for routine healthcare. Key attitudes towards research were responsibility and trust: personal (trust of the researcher directly) and institutional (trust of the institution as a whole). Villagers believe the village headman holds responsibility for community activities, while the village headman believes that this responsibility should be shared across all levels of the government system. CONCLUSIONS: It is essential for researchers to understand the structure and relationship within the community they wish to work with in order to develop trust among community participants. This aids effective communication and understanding among all parties, enabling high quality ethical research to be conducted.

Utilization of a clinical microbiology service at a Cambodian paediatric hospital and its impact on appropriate antimicrobial prescribing.

Background: Antimicrobial resistance threatens human health worldwide. Antimicrobial misuse is a major driver of resistance. Promoting appropriate antimicrobial use requires an understanding of how clinical microbiology services are utilized, particularly in resource-limited settings. Objectives: To assess the appropriateness of antimicrobial prescribing and the factors affecting utilization of the established clinical microbiology service (CMS). The CMS comprises the microbiology laboratory, clinical microbiologists (infection doctors) and antimicrobial treatment guidelines. Methods: This mixed-methods study was conducted at a non-governmental Cambodian paediatric hospital. Empirical and post-culture antimicrobial prescriptions were reviewed from medical records. The random sample included 10 outpatients per week in 2016 (retrospective) and 20 inpatients per week for 4 weeks in the medical, neonatal and intensive care wards (prospective). Post-culture prescriptions were assessed in patients with positive blood and cerebrospinal fluid cultures from 1 January 2014 to 31 December 2016. Focus group discussions and semi-structured interviews with clinicians explored barriers and facilitators to use of the CMS. Results: Only 31% of outpatients were prescribed empirical antimicrobials. Post-culture prescriptions (394/443, 89%) were more likely to be appropriate than empirical prescriptions (447/535, 84%), based on treatment guidelines, microbiology advice and antimicrobial susceptibility test results (P = 0.015). Being comprehensive, accessible and trusted enabled CMS utilization. Clinical microbiologists provided a crucial human interface between the CMS and physicians. The main barriers were a strong clinical hierarchy and occasional communication difficulties. Conclusions: Antimicrobial prescribing in this hospital was largely appropriate. A culturally appropriate human interface linking the laboratory and physicians is essential in providing effective microbiology services and ensuring appropriate antimicrobial prescribing in resource-limited settings.

"Know your audience": A hospital community engagement programme in a non-profit paediatric hospital in Cambodia.

OBJECTIVE: The purpose of this evaluation is to explore the impact of the new hospital community engagement programme (comprised of a Young Persons Advisory Group and a Science Café) on community members and other stakeholders, with regard to their attitudes, skills and degree of engagement in a paediatric hospital in Cambodia. DESIGN: Data collection included feedback questionnaires and reflections produced after each YPAG and Science Café event. Further questionnaires and reflective interviews were conducted to gather the views of key stakeholders. Data were analysed by thematic content analysis and numerical data were expressed using descriptive statistics. RESULTS: The vast majority of participants expressed their enjoyment and satisfaction of the hospital community engagement programme. Delivering the programme in the right manner for the target audiences, by prioritising their needs was key to this. Participants valued the programmes in terms of the knowledge delivered around good health practices, the skills developed such as confidence and responsibility for their health, and the provision of opportunities to voice their opinions. All stakeholders recognised the importance of the programme in improving the quality of the healthcare service provided at the hospital. CONCLUSIONS: In order to have a successful hospital community engagement programme, understanding the target audience is essential. The engagement programme must be delivered in the right way to meet the needs of community members, including right communication, right setting, right people and right timing. This will ultimately result in a meaningful programme that is able to empower community members, potentially resulting in lasting change in healthcare practices. In conclusion, the gap between hospitals and the community could narrow, allowing everyone to interact and learn from each other.

Beliefs and practices during pregnancy, post-partum and in the first days of an infant's life in rural Cambodia.

BACKGROUND: The aim of this study was to record the beliefs, practices during pregnancy, post-partum and in the first few days of an infant's life, held by a cross section of the community in rural Cambodia to determine beneficial community interventions to improve early neonatal health. METHODS: Qualitative study design with data generated from semi structured interviews (SSI) and focus group discussions (FGD). Data were analysed by thematic content analysis, with an a priori coding structure developed using available relevant literature. Further reading of the transcripts permitted additional coding to be performed in vivo. This study was conducted in two locations, firstly the Angkor Hospital for Children and secondarily in five villages in Sotnikum, Siem Reap Province, Cambodia. RESULTS: A total of 20 participants underwent a SSIs (15 in hospital and five in the community) and six (three in hospital and three in the community; a total of 58 participants) FGDs were conducted. Harmful practices that occurred in the past (for example: discarding colostrum and putting mud on the umbilical stump) were not described as being practiced. Village elders did not enforce traditional views. Parents could describe signs of illness and felt responsible to seek care for their child even if other family members disagreed, however participants were unaware of the signs or danger of neonatal jaundice. Cost of transportation was the major barrier to healthcare that was identified. CONCLUSIONS: In the population examined, traditional practices in late pregnancy and the post-partum period were no longer commonly performed. However, jaundice, a potentially serious neonatal condition, was not recognised. Community neonatal interventions should be tailored to the populations existing practice and knowledge.

High Prevalence of Antimicrobial-resistant Gram-negative Colonization in Hospitalized Cambodian Infants.

BACKGROUND: Antimicrobial-resistant Gram-negative infections are a significant cause of mortality in young infants. We aimed to determine characteristics of, and risk factors for, colonization and invasive infection caused by 3rd generation cephalosporin (3GC) or carbapenem-resistant organisms in outborn infants admitted to a neonatal unit (NU) in Cambodia. METHODS: During the first year of operation, patients admitted to the Angkor Hospital for Children NU, Siem Reap, Cambodia, underwent rectal swabbing on admission and twice weekly until discharge. Swabs were taken also from 7 environmental sites. Swabs were cultured to identify 3GC or carbapenem-resistant Acinetobacter sp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. RESULTS: The study included 333 infants with a median age at NU admission of 10 days (range, 0-43). Colonization by ≥1 3GC-resistant organism was detected in 85.9% (286/333). Admission swabs were collected in 289 infants: 61.9% were colonized by a 3GC-resistant organism at the time of admission, and a further 23.2% were colonized during hospitalization, at a median of 4 days [95% confidence interval: 3-5]. Probiotic treatment (hazard ratio: 0.58; 95% confidence interval: 0.35-0.98) was associated with delayed colonization. Colonization by a carbapenem-resistant organism occurred in 25 (7.5%) infants. Six infants had NU-associated K. pneumoniae bacteremia; phenotypically identical colonizing strains were found in 3 infants. Environmental colonization occurred early. CONCLUSIONS: Colonization by antimicrobial-resistant Gram-negative organisms occurred early in hospitalized Cambodian infants and was associated with subsequent invasive infection. Trials of potential interventions such as probiotics are needed.

Urinary antibiotic activity in paediatric patients attending an outpatient department in north-western Cambodia.

OBJECTIVE: Antibiotic resistance is a prominent public and global health concern. We investigated antibiotic use in children by determining the proportion of unselected children with antibacterial activity in their urine attending a paediatric outpatient department in Siem Reap, Cambodia. METHODS: Caregiver reports of medication history and presence of possible infection symptoms were collected in addition to urine samples. Urine antibiotic activity was estimated by exposing bacteria to urine specimens, including assessment against multiresistant bacteria previously isolated from patients in the hospital (a methicillin-resistant Staphylococcus aureus (MRSA), a multiresistant Salmonella typhi and an extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolate). RESULTS: Medication information and urine were collected from 775 children. Caregivers reported medication use in 69.0% of children in the preceding 48 h. 31.7% samples showed antibacterial activity; 16.3% showed activity against a local multiresistant organism. No specimens demonstrated activity against an ESBL-producing E. coli. CONCLUSIONS: Antibiotics are widely used in the community setting in Cambodia. Parents are often ill-informed about drugs given to treat their children. Increasing the regulation and training of private pharmacies in Cambodia may be necessary. Regional surveillance of antibiotic use and resistance is also essential in devising preventive strategies against further development of antibiotic resistance, which would have both local and global consequences.