Evaluating the relationship between ciprofloxacin prescription and non-susceptibility in Salmonella Typhi in Blantyre, Malawi: an observational study

Background Ciprofloxacin is the first-line drug for treating typhoid fever in many countries in Africa with a high disease burden, but the emergence of non-susceptibility poses a challenge to public health programmes. Through enhanced surveillance as part of vaccine evaluation, we investigated the occurrence and potential determinants of ciprofloxacin non-susceptibility in Blantyre, Malawi. Methods We conducted systematic surveillance of typhoid fever cases and antibiotic prescription in two health centres in Blantyre, Malawi, between Oct 1, 2016, and Oct 31, 2019, as part of the STRATAA and TyVAC studies. In addition, blood cultures were taken from eligible patients presenting at Queen Elizabeth Central Hospital, Blantyre, as part of routine diagnosis. Inclusion criteria were measured or reported fever, or clinical suspicion of sepsis. Microbiologically, we identified Salmonella enterica serotype Typhi (S Typhi) isolates with a ciprofloxacin non-susceptible phenotype from blood cultures, and used whole-genome sequencing to identify drug-resistance mutations and phylogenetic relationships. We constructed generalised linear regression models to investigate associations between the number of ciprofloxacin prescriptions given per month to study participants and the proportion of S Typhi isolates with quinolone resistance-determining region (QRDR) mutations in the following month. Findings From 46 989 blood cultures from Queen Elizabeth Central Hospital, 502 S Typhi isolates were obtained, 30 (6%) of which had either decreased ciprofloxacin susceptibility, or ciprofloxacin resistance. From 11 295 blood cultures from STRATAA and TyVAC studies, 241 microbiologically confirmed cases of typhoid fever were identified, and 198 isolates from 195 participants sequenced (mean age 12·8 years [SD 10·2], 53% female, 47% male). Between Oct 1, 2016, and Aug 31, 2019, of 177 typhoid fever cases confirmed by whole-genome sequencing, four (2%) were caused by S Typhi with QRDR mutations, compared with six (33%) of 18 cases between Sept 1 and Oct 31, 2019. This increase was associated with a preceding spike in ciprofloxacin prescriptions. Every additional prescription of ciprofloxacin given to study participants in the preceding month was associated with a 4·2% increase (95% CI 1·8–7·0) in the relative risk of isolating S Typhi with a QRDR mutation (p=0·0008). Phylogenetic analysis showed that S Typhi isolates with QRDR mutations from September and October, 2019, belonged to two distinct subclades encoding two different QRDR mutations, and were closely related (4–10 single-nucleotide polymorphisms) to susceptible S Typhi endemic to Blantyre. Interpretation We postulate a causal relationship between increased ciprofloxacin prescriptions and an increase in fluoroquinolone non-susceptibility in S Typhi. Decreasing ciprofloxacin use by improving typhoid diagnostics, and reducing typhoid fever cases through the use of an efficacious vaccine, could help to limit the emergence of resistance

Regulation of naturally acquired mucosal immunity to Streptococcus pneumoniae in healthy Malawian adults and children.

Worldwide, invasive pneumococcal disease caused by Streptococcus pneumoniae is most common in young children. In adults, disease rates decline following intermittent colonization and the acquisition of naturally acquired immunity. We characterized mucosal and systemic pneumococcal-specific T-cell responses in African children and adults who contend with intense rates of colonization, up to 100% and 60% respectively. We find most Malawian children have high pneumococcal-specific T-cell responses in tonsil tissue and peripheral blood. In addition, frequent commensalism generates CD25(hi) (Tregs) which modulate mucosal pneumococcal-specific T-cell responses in some children and ≥50% of adults. We propose that immune regulation may prolong pneumococcal colonization and predispose vulnerable individuals to disease

Delayed reconstitution of B cell immunity to pneumococcus in HIV-infected Malawian children on antiretroviral therapy

SummaryObjectiveDespite CD4+ count restoration and viral load suppression with antiretroviral therapy (ART), HIV-infected children remain at increased risk of life-threatening infections including invasive pneumococcal disease (IPD). We therefore investigated whether persistent susceptibility to IPD following ART is associated with incomplete recovery of B-cell function.Methods41 HIV-infected Malawian children commencing ART were followed-up for a 1 year period during which time blood samples were collected at 0, 3, 6 and 12 months for comprehensive immunophenotyping and pneumomococcal-specific Memory B-cell Enzyme-Linked Immunospot assays. In addition, nasopharyngeal swab samples were cultured to determine pneumococcal carriage rates.ResultsNormalization of major lymphocyte subsets such as CD4+ percentages was evident following 3 months of ART. The proportions of mature naïve B cells (CD19+ CD10− CD27− CD21hi) and resting memory B cells (CD19+ CD27+ CD21hi) increased and apoptosis-prone mature activated B cells (CD19+ CD21lo CD10−) decreased markedly by 12 months. However, in the context of high nasopharyngeal pneumococcal carriage rates (83%), restoration of pneumococcal protein antigen-specific B-cell memory was more delayed.ConclusionsThese data show that, in chronically HIV-infected children receiving ART, improvement in B-cell memory profiles and function is slower than CD4+ T-cells. This supports early initiation of ART and informs research into optimal timing of immunization with pneumococcal vaccines

Inhibition of pneumococcal, influenza and MTB responses by regulatory T cells in young children and onwards.

<p>Mucosal CD4 T cell responses to pneumoCCS (<b>a</b>) and PlyCCS (<b>b</b>) influenza (<b>c</b>) and MTB PPD (<b>d</b>) by CD25^hi regulatory T cells in subjects above 3 years old as indicated by increased cell proliferation following depletion of CD25^hi cells from tonsillar MNC population. Subjects (n = 22) were grouped into those aged 3–7 yrs, 8 to 17 yrs and >18 yrs. Individual subject’s proliferative response pre (<b>circle</b>) and post CD25^hi cell depletion (<b>square</b>) are shown with a connecting lines. Data was analyzed using Wilcoxon signed rank test, * = p<0.05. Representative flow cytometric plot of CD25 undepleted and depleted tonsillar mononuclear cells (TMNC) (<b>e</b>).</p

Mucosal and peripheral blood CD4 T cell responses to pneumococcal, influenza and MTB-PPD antigens in Malawian children and adults.

<p>Mucosal CD4 T cell proliferation in response to pneumoCCS n = 26 (<b>a</b>) and Ply-CCS n = 24 (<b>b</b>) influenza n = 25 (<b>c</b>) and MTB n = 25 (<b>d</b>) in children and adults. Circulating CD4 T cell proliferation in response to pneumoCCS n = 66 (<b>e</b>), Ply-CCS n = 67 (<b>f</b>), influenza n = 65 (<b>g</b>) and MTB-PPD n = 58 (<b>h</b>) in children and adults. Data was analyzed by nonlinear regression modeling, fitted with second order polynomial.</p

Domestic river water use and risk of typhoid fever:results from a case-control study in Blantyre, Malawi

BACKGROUND: Typhoid fever remains a major cause of morbidity and mortality in low- and middle-income settings. In the last 10 years, several reports have described the reemergence of typhoid fever in southern and eastern Africa, associated with multidrug-resistant H58 Salmonella Typhi. Here, we identify risk factors for pediatric typhoid fever in a large epidemic in Blantyre, Malawi. METHODS: A case-control study was conducted between April 2015 and November 2016. Cases were recruited at a large teaching hospital, and controls were recruited from the community, matched by residential ward. Stepwise variable selection and likelihood ratio testing were used to select candidate risk factors for a final logistic regression model. RESULTS: Use of river water for cooking and cleaning was highly associated with risk of typhoid fever (odds ratio [OR], 4.6 [95% confidence interval {CI}, 1.7-12.5]). Additional risk factors included protective effects of soap in the household (OR, 0.6 [95% CI, .4-.98]) and >1 water source used in the previous 3 weeks (OR, 3.2 [95% CI, 1.6-6.2]). Attendance at school or other daycare was also identified as a risk factor (OR, 2.7 [95% CI, 1.4-5.3]) and was associated with the highest attributable risk (51.3%). CONCLUSIONS: These results highlight diverse risk factors for typhoid fever in Malawi, with implications for control in addition to the provision of safe drinking water. There is an urgent need to improve our understanding of transmission pathways of typhoid fever, both to develop tools for detecting S. Typhi in the environment and to inform water, sanitation, and hygiene interventions

What happens to patients on antiretroviral therapy who transfer out to another facility?

BACKGROUND: Long term retention of patients on antiretroviral therapy (ART) in Africa's rapidly expanding programmes is said to be 60% at 2 years. Many reports from African ART programmes make little mention of patients who are transferred out to another facility, yet Malawi's national figures show a transfer out of 9%. There is no published information about what happens to patients who transfer-out, but this is important because if they transfer-in and stay alive in these other facilities then national retention figures will be better than previously reported. METHODOLOGY/PRINCIPAL FINDINGS: Of all patients started on ART over a three year period in Mzuzu Central Hospital, North Region, Malawi, those who transferred out were identified from the ART register and master cards. Clinic staff attempted to trace these patients to determine whether they had transferred in to a new ART facility and their outcome status. There were 805 patients (19% of the total cohort) who transferred out, of whom 737 (92%) were traced as having transferred in to a new ART facility, with a median time of 1.3 months between transferring-out and transferring-in. Survival probability was superior and deaths were lower in the transfer-out patients compared with those who did not transfer. CONCLUSION/SIGNIFICANCE: In Mzuzu Central Hospital, patients who transfer-out constitute a large proportion of patients not retained on ART at their original clinic of registration. Good documentation of transfer-outs and transfer-ins are needed to keep track of national outcomes. Furthermore, the current practice of regarding transfer-outs as being double counted in national cohorts and subtracting this number from the total national registrations to get the number of new patients started on ART is correct

Spatial and genomic data to characterize endemic typhoid transmission

Background Diverse environmental exposures and risk factors have been implicated in the transmission of Salmonella Typhi, however, the dominant transmission pathways through the environment to susceptible humans remain unknown. Here, we utilize spatial, bacterial genomic, and hydrological data to refine our view of Typhoid transmission in an endemic setting. Methods 546 patients presenting to Queen Elizabeth Central Hospital in Blantyre, Malawi with blood culture-confirmed typhoid fever between April 2015 and January 2017 were recruited to a cohort study. The households of a subset of these patients were geolocated, and 256 S. Typhi isolates were whole genome sequenced. Pairwise single nucleotide variant (SNV) distances were incorporated into a geostatistical modeling framework using multidimensional scaling. Results Typhoid fever was not evenly distributed across Blantyre, with estimated minimum incidence ranging across the city from less than 15 to over 100 cases/100,000/year. Pairwise SNV distance and physical household distances were significantly correlated (p=0.001). We evaluated the ability of river catchment to explain the spatial patterns of genomics observed, finding that it significantly improved the fit of the model (p=0.003). We also found spatial correlation at a smaller spatial scale, of households living <192 meters apart. Conclusions These findings reinforce the emerging view that hydrological systems play a key role in the transmission of typhoid fever. By combining genomic and spatial data, we show how multi-faceted data can be used to identify high incidence areas, understand the connections between them, and inform targeted environmental surveillance, all of which will be critical to shape local and regional typhoid control strategies

Probability of Survival in Patients on ART who Transfer Out and who do not Transfer Out.

<p>Probability of Survival in Patients on ART who Transfer Out and who do not Transfer Out.</p