Ascertaining the burden of invasive Salmonella disease in hospitalised febrile children aged under four years in Blantyre, Malawi

Abstract

Typhoid fever is endemic across sub-Saharan Africa. However, estimates of the burden of typhoid are undermined by insufficient blood volumes and lack of sensitivity of blood culture. Here, we aimed to address this limitation by exploiting pre-enrichment culture followed by PCR, alongside routine blood culture to improve typhoid case detection. We carried out a prospective diagnostic cohort study and enrolled children (aged 0–4 years) with non-specific febrile disease admitted to a tertiary hospital in Blantyre, Malawi from August 2014 to July 2016. Blood was collected for culture (BC) and real-time PCR after a pre-enrichment culture in tryptone soy broth and ox-bile. DNA was subjected to PCR for invA (Pan- Salmonella ), staG ( S . Typhi), and fliC ( S . Typhimurium) genes. A positive PCR was defined as invA plus either staG or fliC (CT<29). IgM and IgG ELISA against four S . Typhi antigens was also performed. In total, 643 children (median age 1.3 years) with nonspecific febrile disease were enrolled; 31 (4.8%) were BC positive for Salmonella (n = 13 S . Typhi, n = 16 S . Typhimurium, and n = 2 S . Enteritidis). Pre-enrichment culture of blood followed by PCR identified a further 8 S . Typhi and 15 S . Typhimurium positive children. IgM and IgG titres to the S. Typhi antigen STY1498 (haemolysin) were significantly higher in children that were PCR positive but blood culture negative compared to febrile children with all other non-typhoid illnesses. The addition of pre-enrichment culture and PCR increased the case ascertainment of invasive Salmonella disease in children by 62–94%. These data support recent burden estimates that highlight the insensitivity of blood cultures and support the targeting of pre-school children for typhoid vaccine prevention in Africa. Blood culture with real-time PCR following pre-enrichment should be used to further refine estimates of vaccine effectiveness in typhoid vaccine trials.

Intestinal Perforations Associated With a High Mortality and Frequent Complications During an Epidemic of Multidrug-resistant Typhoid Fever in Blantyre, Malawi

“…Elution was done using 30 μL elution buffer instead of 200 μL. Multiplex real-time polymerase chain reaction (PCR) tests were performed in a CFX96 thermal cycler (Bio-Rad) using the Quantifast Pathogen PC + IC Kit (Qiagen), targeting the pan- Salmonella invasion A gene, the S. Typhi fimbriae gene [ 15 ], and the kit’s internal control. The pan- Salmonella , S .…”

Performance of molecular methods for the detection of Salmonella in human stool specimens

“…Stool specimens collected from a longitudinal cohort of children aged 6 – 18 months recruited from Zingwangwa Health Centre (ZHC) in Blantyre, Malawi, were used to compare the performance of molecular and standard culture for detection of Salmonella in stool. The primary study started recruitment in August 2013, and follow-up was concluded in December 2014 17 . Group sensitisation of the study by well-trained study nurses was done to parents or guardians of six-month-old children attending a vaccination clinic at ZHC.…”

“…A previously-optimised in-house PCR protocol was used 17 . Briefly, the master mix for RT-PCR was prepared using pre-defined quantities.…”

Pathogenic signature of invasive non-typhoidal Salmonella in Africa: implications for vaccine development

“…6 By contrast, NTS species are often responsible for life-threatening systemic infections in low-income settings, where poor sanitation, lack of proper diagnostic tools, malnutrition and other comorbidities can further exacerbate the outcome of the disease. 7,8 According to current incidence data, invasive nontyphoidal Salmonella (iNTS) infections particularly haunt Africa. Of the estimated 535,000 cases of iNTS disease and 77,500 deaths that occurred globally in 2017, the highest incidence (34.5 cases per 100,000 person-years) was observed in Sub-Saharan Africa (sSA).…”

“… 6 By contrast, NTS species are often responsible for life-threatening systemic infections in low-income settings, where poor sanitation, lack of proper diagnostic tools, malnutrition and other comorbidities can further exacerbate the outcome of the disease. 7 , 8 …”