Enteric fever in Cambodia: A multi-disciplinary study

Enteric fever is a systemic infection caused by the bacteria Salmonella Typhi or Salmonella Paratyphi A. Humans are the only host and transmission occurs through food and water contaminated by stool from patients or chronic carriers. Most cases globally are caused by Salmonella Typhi. However, the proportion of Salmonella Paratyphi A infections has been increasing during the last 20 years for reasons that are yet unknown.As explained in Chapter 1, enteric fever is a major cause of morbidity and mortality in low resource settings where its spread is linked to poor water quality and sanitation. The disease is endemic in Cambodia and during 2013-2015 an outbreak of Salmonella Paratyphi A infections occurred, which also affected international travelers returning from Cambodia. Based on these observations and the associated research questions, a multi-disciplinary approach was adopted to study enteric fever in Cambodia, integrating studies across disciplines and with a particular focus on Salmonella Paratyphi A. Most data were collected at the Sihanouk Hospital Center of HOPE in Phnom Penh, Cambodia, where the presence of a surveillance system for bloodstream infections allowed inclusion of confirmed cases and specimens.It was suggested that the Salmonella Paratyphi A outbreak in Phnom Penh was caused by the introduction of a new clone with a possible genetic advantage compared to other clones already circulating. Classic typing methods, such as pulsed-field gel electrophoresis (PFGE), do not perform well on bacteria with limited genetic diversity such as Salmonella Paratyphi A. Therefore, whole-genome sequencing with genetic comparison and establishment of phylogenetic relationships was used for the retrospective investigation of the outbreak (Chapter 2). The outbreak isolates clustered together in one clade (C5) that had been present in Cambodia and Southeast Asia region since many years. Historical isolates, isolates from sporadic cases, and isolates from another Salmonella Paratyphi A outbreak in China revealed different clusters. The Chinese outbreak isolates belonged to a different clade within the same lineage (C4) and displayed antibiotic resistance, unlike the Cambodian isolates which were pan-susceptible.Next, the clinical and microbiological characteristics of Salmonella Paratyphi A versus Salmonella Typhi infections were compared in this setting (Chapter 3). Demographic, clinical and laboratory data were retrospectively collected from the clinical charts of 254 blood culture confirmed enteric fever patients. Antibiotic susceptibility testing was performed and the genomes of a selection of isolates were sequenced. Presenting symptoms of both serotypes were similar. Higher rates of inflammatory markers in Salmonella Typhi infections suggests more severe disease. With exception of one, all Salmonella Typhi isolates belonged to haplotype H58. This haplotype is associated with multidrug resistance (MDR) (i.e. resistance to the antibiotics ampicillin, chloramphenicol and co-trimoxazole) and increased virulence. Nearly half actually displayed the MDR phenotype, while none of the Salmonella Paratyphi A isolates did. Decreased ciprofloxacin susceptibility (DCS) was observed for nearly all Salmonella Typhi isolates versus a minority of Salmonella Paratyphi A isolates. From 2015 onwards however, DCS increased rapidly for Salmonella Paratyphi A. No resistance against azithromycin or ceftriaxone was detected.Diagnosing enteric fever remains challenging. Blood culture is used as the reference method but has a low sensitivity and takes days until diagnosis. Rapid diagnostic tests (RDTs) that can detect Salmonella antigens might shorten the time to diagnosis. In Chapter 4 we evaluated the performance of these RDTs when used on blood culture broth. The products, not yet marketed for application on blood culture broth, were first assessed using spiked blood culture samples. Of six RDT products evaluated four did not reliably detect Salmonella Typhi and/or Salmonella Paratyphi. The limits of detection of the two-best performing RDTs were established to be 107-108 CFU/ml. Next, these two RDTs were applied to 413 stored grown blood culture broths from Cambodia and the Democratic Republic of the Congo. The Standard Diagnostics Bioline RDT had a sensitivity of 97.9% and specificity of 98.5% for the combined detection of Salmonella Typhi and Salmonella Enteritidis. For the Creative Diagnostics RDT, sensitivity was 78.3% and specificity 91.0% for all Salmonella serotypes combined. The low sensitivity was a result of the test's inability to reliably detect Salmonella Paratyphi A.The main mechanism of Salmonella Typhi and Salmonella Paratyphi A transmission is fecal shedding of the bacteria. Fecal shedding occurs during acute infections and chronic carriage states. The current standard for tracing carriers is stool culture. This is challenging for Salmonella Paratyphi A as it does not produce hydrogen sulfide (H2S), a feature which makes colonies of other Salmonella serotypes stand out based on colony color. Salmonella Paratyphi A may resemble other common gut bacteria, and this results in time-consuming additional testing. In Chapter 5, the performance of chromogenic media for the detection of Salmonella Paratyphi A in stool is assessed. These media use a combination of chromogenic substrates and are highly specific. They were shown to detect Salmonella Paratyphi A in concentrations as low as 103 CFU/ml of stool and resulted in few false positive results. This suggests that chromogenic media may be used for the detection of Salmonella Paratyphi A carriage.As the results of Chapter 2 suggest, human or environmental factors rather than bacterial factors probably played a key role in the recent emergence of Salmonella Paratyphi A in Phnom Penh. Disease transmission is facilitated by a lack of safe water and (food) hygiene, inadequate disposal of human excreta, ineffective treatment and lack of health literacy. In Chapter 6 illness interpretations and perceptions on treatment were assessed through in-depth interviews among both lay-persons and healthcare providers in Phnom Penh. In addition, first line treatment practices and health seeking behavior associated with enteric fever were identified. Results showed that lay people did not link the disease to the bacterial contamination of foods. For initial and mild symptoms, patients tried home treatments. If symptoms persisted, or were severe, patients visit a range of health care providers, including drug sellers and private practitioners prescribing a cocktail of pills including antibiotics without a prescription. The diagnosis was rarely confirmed. During recovery patients adhered to dietary restrictions and took traditional medicine thinking it prevented disease recurrence.Some of the barriers to proper diagnosis and treatment of enteric fever should be seen in the context of Cambodia's recent history. As a post-conflict country health infrastructure is still developing, which gives rise to a set of questionable and potentially harmful health care practices across all health sectors. In the Appendix, it is argued that these practices have been become standard due to local realities and need to be addressed in order to improve health provision and outcomes.In Chapter 7 it is concluded that several measures need to be taken to curb the transmission of enteric fever and to improve its diagnosis and treatment in both the hospital and community setting. The topic of antibiotic resistance is touched upon and actions for its containment are suggested.Enteric fever is a systemic infection caused by the bacteria Salmonella Typhi or Salmonella Paratyphi A. Humans are the only host and transmission occurs through food and water contaminated by stool from patients or chronic carriers. Most cases globally are caused by Salmonella Typhi. However, the proportion of Salmonella Paratyphi A infections has been increasing during the last 20 years for reasons that are yet unknown.As explained in Chapter 1, enteric fever is a major cause of morbidity and mortality in low resource settings where its spread is linked to poor water quality and sanitation. The disease is endemic in Cambodia and during 2013-2015 an outbreak of Salmonella Paratyphi A infections occurred, which also affected international travelers returning from Cambodia. Based on these observations and the associated research questions, a multi-disciplinary approach was adopted to study enteric fever in Cambodia, integrating studies across disciplines and with a particular focus on Salmonella Paratyphi A. Most data were collected at the Sihanouk Hospital Center of HOPE in Phnom Penh, Cambodia, where the presence of a surveillance system for bloodstream infections allowed inclusion of confirmed cases and specimens.It was suggested that the Salmonella Paratyphi A outbreak in Phnom Penh was caused by the introduction of a new clone with a possible genetic advantage compared to other clones already circulating. Classic typing methods, such as pulsed-field gel electrophoresis (PFGE), do not perform well on bacteria with limited genetic diversity such as Salmonella Paratyphi A. Therefore, whole-genome sequencing with genetic comparison and establishment of phylogenetic relationships was used for the retrospective investigation of the outbreak (Chapter 2). The outbreak isolates clustered together in one clade (C5) that had been present in Cambodia and Southeast Asia region since many years. Historical isolates, isolates from sporadic cases, and isolates from another Salmonella Paratyphi A outbreak in China revealed different clusters. The Chinese outbreak isolates belonged to a different clade within the same lineage (C4) and displayed antibiotic resistance, unlike the Cambodian isolates which were pan-susceptible.Next, the clinical and microbiological characteristics of Salmonella Paratyphi A versus Salmonella Typhi infections were compared in this setting (Chapter 3). Demographic, clinical and laboratory data were retrospectively collected from the clinical charts of 254 blood culture confirmed enteric fever patients. Antibiotic susceptibility testing was performed and the genomes of a selection of isolates were sequenced. Presenting symptoms of both serotypes were similar. Higher rates of inflammatory markers in Salmonella Typhi infections suggests more severe disease. With exception of one, all Salmonella Typhi isolates belonged to haplotype H58. This haplotype is associated with multidrug resistance (MDR) (i.e. resistance to the antibiotics ampicillin, chloramphenicol and co-trimoxazole) and increased virulence. Nearly half actually displayed the MDR phenotype, while none of the Salmonella Paratyphi A isolates did. Decreased ciprofloxacin susceptibility (DCS) was observed for nearly all Salmonella Typhi isolates versus a minority of Salmonella Paratyphi A isolates. From 2015 onwards however, DCS increased rapidly for Salmonella Paratyphi A. No resistance against azithromycin or ceftriaxone was detected.Diagnosing enteric fever remains challenging. Blood culture is used as the reference method but has a low sensitivity and takes days until diagnosis. Rapid diagnostic tests (RDTs) that can detect Salmonella antigens might shorten the time to diagnosis. In Chapter 4 we evaluated the performance of these RDTs when used on blood culture broth. The products, not yet marketed for application on blood culture broth, were first assessed using spiked blood culture samples. Of six RDT products evaluated four did not reliably detect Salmonella Typhi and/or Salmonella Paratyphi. The limits of detection of the two-best performing RDTs were established to be 107-108 CFU/ml. Next, these two RDTs were applied to 413 stored grown blood culture broths from Cambodia and the Democratic Republic of the Congo. The Standard Diagnostics Bioline RDT had a sensitivity of 97.9% and specificity of 98.5% for the combined detection of Salmonella Typhi and Salmonella Enteritidis. For the Creative Diagnostics RDT, sensitivity was 78.3% and specificity 91.0% for all Salmonella serotypes combined. The low sensitivity was a result of the test's inability to reliably detect Salmonella Paratyphi A.The main mechanism of Salmonella Typhi and Salmonella Paratyphi A transmission is fecal shedding of the bacteria. Fecal shedding occurs during acute infections and chronic carriage states. The current standard for tracing carriers is stool culture. This is challenging for Salmonella Paratyphi A as it does not produce hydrogen sulfide (H2S), a feature which makes colonies of other Salmonella serotypes stand out based on colony color. Salmonella Paratyphi A may resemble other common gut bacteria, and this results in time-consuming additional testing. In Chapter 5, the performance of chromogenic media for the detection of Salmonella Paratyphi A in stool is assessed. These media use a combination of chromogenic substrates and are highly specific. They were shown to detect Salmonella Paratyphi A in concentrations as low as 103 CFU/ml of stool and resulted in few false positive results. This suggests that chromogenic media may be used for the detection of Salmonella Paratyphi A carriage.As the results of Chapter 2 suggest, human or environmental factors rather than bacterial factors probably played a key role in the recent emergence of Salmonella Paratyphi A in Phnom Penh. Disease transmission is facilitated by a lack of safe water and (food) hygiene, inadequate disposal of human excreta, ineffective treatment and lack of health literacy. In Chapter 6 illness interpretations and perceptions on treatment were assessed through in-depth interviews among both lay-persons and healthcare providers in Phnom Penh. In addition, first line treatment practices and health seeking behavior associated with enteric fever were identified. Results showed that lay people did not link the disease to the bacterial contamination of foods. For initial and mild symptoms, patients tried home treatments. If symptoms persisted, or were severe, patients visit a range of health care providers, including drug sellers and private practitioners prescribing a cocktail of pills including antibiotics without a prescription. The diagnosis was rarely confirmed. During recovery patients adhered to dietary restrictions and took traditional medicine thinking it prevented disease recurrence.Some of the barriers to proper diagnosis and treatment of enteric fever should be seen in the context of Cambodia's recent history. As a post-conflict country health infrastructure is still developing, which gives rise to a set of questionable and potentially harmful health care practices across all health sectors. In the Appendix, it is argued that these practices have been become standard due to local realities and need to be addressed in order to improve health provision and outcomes.In Chapter 7 it is concluded that several measures need to be taken to curb the transmission of enteric fever and to improve its diagnosis and treatment in both the hospital and community setting. The topic of antibiotic resistance is touched upon and actions for its containment are suggested.

Jaar van publicatie:2019