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. FUNDING: Wellcome Trust, Bill & Melinda Gates Foundation, and National Institute for Health and Care Research (UK).

Efficacy of typhoid conjugate vaccine: final analysis of a 4-year, phase 3, randomised controlled trial in Malawian children.

BACKGROUND: Randomised controlled trials of typhoid conjugate vaccines among children in Africa and Asia have shown high short-term efficacy. Data on the durability of protection beyond 2 years are sparse. We present the final analysis of a randomised controlled trial in Malawi, encompassing more than 4 years of follow-up, with the aim of investigating vaccine efficacy over time and by age group. METHODS: In this phase 3, double-blind, randomised controlled efficacy trial in Blantyre, Malawi, healthy children aged 9 months to 12 years were randomly assigned (1:1) by an unmasked statistician to receive a single dose of Vi polysaccharide conjugated to tetanus toxoid vaccine (Vi-TT) or meningococcal capsular group A conjugate (MenA) vaccine. Children had to have no previous history of typhoid vaccination and reside in the study areas for inclusion and were recruited from government schools and health centres. Participants, their parents or guardians, and the study team were masked to vaccine allocation. Nurses administering vaccines were unmasked. We did surveillance for febrile illness from vaccination until follow-up completion. The primary outcome was first occurrence of blood culture-confirmed typhoid fever. Eligible children who were randomly assigned and vaccinated were included in the intention-to-treat analyses. This trial is registered at ClinicalTrials.gov, NCT03299426. FINDINGS: Between Feb 21, 2018, and Sept 27, 2018, 28 130 children were vaccinated; 14 069 were assigned to receive Vi-TT and 14 061 to receive MenA. After a median follow-up of 4·3 years (IQR 4·2-4·5), 24 (39·7 cases per 100 000 person-years) children in the Vi-TT group and 110 (182·7 cases per 100 000 person-years) children in the MenA group were diagnosed with a first episode of blood culture-confirmed typhoid fever. In the intention-to-treat population, efficacy of Vi-TT was 78·3% (95% CI 66·3-86·1), and 163 (129-222) children needed to be vaccinated to prevent one case. Efficacies by age group were 70·6% (6·4-93·0) for children aged 9 months to 2 years; 79·6% (45·8-93·9) for children aged 2-4 years; and 79·3% (63·5-89·0) for children aged 5-12 years. INTERPRETATION: A single dose of Vi-TT is durably efficacious for at least 4 years among children aged 9 months to 12 years and shows efficacy in all age groups, including children younger than 2 years. These results support current WHO recommendations in typhoid-endemic areas for mass campaigns among children aged 9 months to 15 years, followed by routine introduction in the first 2 years of life. FUNDING: Bill & Melinda Gates Foundation.

Typhoid conjugate vaccine effectiveness in Malawi: evaluation of a test-negative design using randomised, controlled clinical trial data.

BACKGROUND: Typhoid conjugate vaccines are being introduced in low-income and middle-income countries to prevent typhoid illness in children. Vaccine effectiveness studies assess vaccine performance after introduction. The test-negative design is a commonly used method to estimate vaccine effectiveness that has not been applied to typhoid vaccines because of concerns over blood culture insensitivity. The overall aim of the study was to evaluate the appropriateness of using a test-negative design to assess typhoid Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT) effectiveness using a gold standard randomised controlled trial database. METHODS: Using blood culture data from a randomised controlled trial of Vi-TT in Malawi, we simulated a test-negative design to derive vaccine effectiveness estimates using three different approaches and compared these to randomised trial efficacy results. In the randomised trial, 27 882 children aged 9 months to 12 years were randomly assigned (1:1) to receive a single dose of Vi-TT or meningococcal capsular group A conjugate vaccine between Feb 21 and Sept 27, 2018, and were followed up for blood culture-confirmed typhoid fever until Sept 30, 2021. FINDINGS: For all three test-negative design approaches, vaccine effectiveness estimates (test-negative design A, 80·3% [95% CI 66·2 to 88·5] vs test-negative design B, 80·5% [66·5 to 88·6] vs test-negative design C, 80·4% [66·9 to 88·4]) were almost identical to the randomised trial results (80·4% [95% CI 66·4 to 88·5]). Receipt of Vi-TT did not affect the risk of non-typhoid fever (vaccine efficacy against non-typhoid fever -0·4% [95% CI -4·9 to 3·9] vs -1% [-5·6 to 3·3] vs -2·5% [-6·4 to 1·3] for test-negative design A, test-negative design B, and test-negative design C, respectively). INTERPRETATION: This study validates the test-negative design core assumption for typhoid vaccine effectiveness estimation and shows the accuracy and precision of the estimates compared with the randomised controlled trial. These results show that the test-negative design is suitable for assessing typhoid conjugate vaccine effectiveness in post-introduction studies using blood culture surveillance. FUNDING: Bill & Melinda Gates Foundation.

Estimating the economic burden of typhoid in children and adults in Blantyre, Malawi: A costing cohort study.

BACKGROUND: Typhoid causes preventable death and disease. The World Health Organization recommends Typhoid Conjugate Vaccine for endemic countries, but introduction decisions depend on cost-effectiveness. We estimated household and healthcare economic burdens of typhoid in Blantyre, Malawi. METHODS: In a prospective cohort of culture-confirmed typhoid cases at two primary- and a referral-level health facility, we collected direct medical, non-medical costs (2020 U.S. dollars) to healthcare provider, plus indirect costs to households. RESULTS: From July 2019-March 2020, of 109 cases, 63 (58%) were <15 years old, 44 (40%) were inpatients. Mean hospitalization length was 7.7 days (SD 4.1). For inpatients, mean total household and provider costs were $93.85 (95%CI: 68.87-118.84) and $296.52 (95%CI: 225.79-367.25), respectively. For outpatients, these costs were $19.05 (95%CI: 4.38-33.71) and $39.65 (95%CI: 33.93-45.39), respectively. Household costs were due mainly to direct non-medical and indirect costs, medical care was free. Catastrophic illness cost, defined as cost >40% of non-food monthly household expenditure, occurred in 48 (44%) households. CONCLUSIONS: Typhoid can be economically catastrophic for families, despite accessible free medical care. Typhoid is costly for government healthcare provision. These data make an economic case for TCV introduction in Malawi and the region and will be used to derive vaccine cost-effectiveness.

Safety and immunogenicity of a typhoid conjugate vaccine among children aged 9 months to 12 years in Malawi: a nested substudy of a double-blind, randomised controlled trial.

BACKGROUND: Typhoid fever is a substantial public health problem in Africa, yet there are few clinical trials of typhoid conjugate vaccine (TCV). We assessed immunogenicity and safety of Typbar TCV in Malawi. METHODS: This substudy was nested within a phase 3, double-blind, parallel design, randomised controlled trial of TCV in children from Ndirande Health Centre in Ndirande township, Blantyre, Malawi. To be eligible, participants had to be aged between 9 months and 12 years with no known immunosuppression or chronic health conditions, including HIV or severe malnutrition; eligible participants were enrolled into three strata of approximately 200 children (9-11 months, 1-5 years, and 6-12 years), randomly assigned (1:1) to receive TCV or control (meningococcal serogroup A conjugate vaccine [MCV-A]) intramuscularly. Serum was collected before vaccination and at 28 days and 730-1035 days after vaccination to measure anti-Vi antibodies by ELISA. Because of COVID-19, day 730 visits were extended up to 1035 days. This nested substudy evaluated reactogenicity, safety, and immunogenicity by age stratum. Safety outcomes, analysed in the intention-to-treat population, included solicited adverse events within 7 days of vaccination (assessed on 3 separate days) and unsolicited adverse events within 28 days of vaccination. This trial is registered with ClinicalTrials.gov, NCT03299426. FINDINGS: Between Feb 22 and Sept 6, 2018, 664 participants were screened, and 631 participants were enrolled and randomly assigned (320 to the TCV group and 311 to the MCV-A group). 305 participants in the TCV group and 297 participants in the MCV-A group were vaccinated. Among TCV recipients, anti-Vi IgG geometric mean titres increased more than 500 times from 4·2 ELISA units (EU)/mL (95% CI 4·0-4·4) at baseline to 2383·7 EU/mL (2087·2-2722·3) at day 28, then decreased to 48·0 EU/mL (39·9-57·8) at day 730-1035, remaining more than 11 times higher than baseline. Among MCV-A recipients, anti-Vi IgG titres remained unchanged: 4·3 EU/mL (4·0-4·5) at baseline, 4·4 EU/mL (4·0-4·7) on day 28, and 4·6 EU/mL (4·2-5·0) on day 730-1035. TCV and MCV-A recipients had similar solicited local (eight [3%] of 304, 95% CI 1·3-5·1 and three [1%] of 293, 0·4-3·0) and systemic (27 [9%] of 304, 6·2-12·6 and 27 [9%] of 293, 6·4-13·1) reactogenicity. Related unsolicited adverse events occurred similarly in TCV and MCV-A recipients in eight (3%) of 304 (1·3-5·1) and eight (3%) of 293 (1·4-5·3). INTERPRETATION: This study provides evidence of TCV safety, tolerability, and immunogenicity up to 730-1035 days in Malawian children aged 9 months to 12 years. FUNDING: Bill & Melinda Gates Foundation.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

A clinical and molecular epidemiological survey of hepatitis C in Blantyre, Malawi, suggests a historic mechanism of transmission.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. There are no previous representative community HCV prevalence studies from Southern Africa, and limited genotypic data. Epidemiological data are required to inform an effective public health response. We conducted a household census-based random sampling serological survey, and a prospective hospital-based study of patients with cirrhosis and hepatocellular carcinoma (HCC) in Blantyre, Malawi. We tested participants with an HCV antigen/antibody ELISA (Monolisa, Bio-Rad), confirmed with PCR (GeneXpert, Cepheid) and used line immunoassay (Inno-LIA, Fujiribio) for RNA-negative participants. We did target-enrichment whole-genome HCV sequencing (NextSeq, Illumina). Among 96,386 censused individuals, we randomly selected 1661 people aged ≥16 years. Population-standardized HCV RNA prevalence was 0.2% (95% CI 0.1-0.5). Among 236 patients with cirrhosis and HCC, HCV RNA prevalence was 1.9% and 5.0%, respectively. Mapping showed that HCV RNA+ patients were from peri-urban areas surrounding Blantyre. Community and hospital HCV RNA+ participants were older than comparator HCV RNA-negative populations (median 53 vs 30 years for community, p = 0.01 and 68 vs 40 years for cirrhosis/HCC, p < 0.001). Endemic HCV genotypes (n = 10) were 4v (50%), 4r (30%) and 4w (10%). In this first census-based community serological study in Southern Africa, HCV was uncommon in the general population, was centred on peri-urban regions and was attributable for <5% of liver disease. HCV infection was observed only among older people, suggesting a historic mechanism of transmission. Genotype 4r, which has been associated with treatment failure with ledipasvir and daclatasvir, is endemic.

Safety and Efficacy of a Typhoid Conjugate Vaccine in Malawian Children.

BACKGROUND: Typhoid fever caused by multidrug-resistant H58 Salmonella Typhi is an increasing public health threat in sub-Saharan Africa. METHODS: We conducted a phase 3, double-blind trial in Blantyre, Malawi, to assess the efficacy of Vi polysaccharide typhoid conjugate vaccine (Vi-TCV). We randomly assigned children who were between 9 months and 12 years of age, in a 1:1 ratio, to receive a single dose of Vi-TCV or meningococcal capsular group A conjugate (MenA) vaccine. The primary outcome was typhoid fever confirmed by blood culture. We report vaccine efficacy and safety outcomes after 18 to 24 months of follow-up. RESULTS: The intention-to-treat analysis included 28,130 children, of whom 14,069 were assigned to receive Vi-TCV and 14,061 were assigned to receive the MenA vaccine. Blood culture-confirmed typhoid fever occurred in 12 children in the Vi-TCV group (46.9 cases per 100,000 person-years) and in 62 children in the MenA group (243.2 cases per 100,000 person-years). Overall, the efficacy of Vi-TCV was 80.7% (95% confidence interval [CI], 64.2 to 89.6) in the intention-to-treat analysis and 83.7% (95% CI, 68.1 to 91.6) in the per-protocol analysis. In total, 130 serious adverse events occurred in the first 6 months after vaccination (52 in the Vi-TCV group and 78 in the MenA group), including 6 deaths (all in the MenA group). No serious adverse events were considered by the investigators to be related to vaccination. CONCLUSIONS: Among Malawian children 9 months to 12 years of age, administration of Vi-TCV resulted in a lower incidence of blood culture-confirmed typhoid fever than the MenA vaccine. (Funded by the Bill and Melinda Gates Foundation; ClinicalTrials.gov number, NCT03299426.).

Early Insights From Clinical Trials of Typhoid Conjugate Vaccine.

Clinical trials of typhoid conjugate vaccine (TCV) are ongoing in 4 countries. Early data confirm safety, tolerability, and immunogenicity of typhoid conjugate vaccine, and early efficacy results are promising. These data support World Health Organization recommendations and planned country introductions. Forthcoming trial data will continue to inform programmatic use of typhoid conjugate vaccine.

Compounding Benefits of Sentinel Lymph Node Biopsy for Perineal Melanoma: A Population-Based Retrospective Cohort Analysis.

INTRODUCTION: Sentinel lymph node biopsy (SLNB) in the treatment of melanoma is known to provide valuable prognostic information. However, there is no literature describing an overall or disease-specific survival (DDS) benefit of SLNB. In the perineum, melanoma is often more advanced at presentation with current treatment guidelines translated from nonanatomic specific melanoma. As a result, there is little understanding surrounding the role of SLNB in melanoma of the perineum. Our objective is to better understand the therapeutic benefits of SLNB in perineal melanoma. METHODS: The Surveillance, Epidemiology, and End Results program is a large population-based cancer registry including survival data from millions of patients in the United States. The registry was used to generate patient data for analysis from 2004 to 2016. Inclusion criteria included melanoma of the perineum; Breslow depth of 0.80 mm or greater and less than 0.80 mm with ulceration; SLNB or no intervention; clinically negative nodal disease; and available overall survival data. RESULTS: For 879 patients from 2004 to 2016 with perineal melanoma, significant predictors of reduced survival include older than 75 years, Clark level IV-V, Breslow depth of greater than 4.00 mm, positive ulceration status, regional and distant nodal micrometastases, and clinically positive nodes on presentation. Aggregates for overall survival (OS) and disease-specific survival (DSS) were improved with implementation of SLNB. The 5-year survival rates with SLNB versus no SLNB were 54.0% and 43.0% for OS (P = 0.001) and 57.8% and 53.1% for DSS (P = 0.044). Stratification by Breslow depth yielded significant OS and DSS advantage for greater than 1.00 to 2.00 mm (21.3% benefit, P =0.021, and 16.8% benefit, P = 0.044) and greater than 4.00 mm (30.3% benefit, P = 0.005, and 21.0% benefit, P = 0.007) Breslow depths. CONCLUSIONS AND RELEVANCE: Sentinel lymph node biopsy may provide therapeutic benefits in addition to prognostic information for melanoma of the perineum through an increase in 5-year OS.

Pediatric Laser Therapy in Pigmented Conditions.

Advances in laser therapy have led to novel therapeutic approaches to common pediatric skin conditions. As a non-invasive alternative to surgical options, laser therapy is efficacious in treating a broad range of conditions, from vascular and pigmented lesions to tattoo and hair removal. This paper reviews the basic mechanics of laser therapy, its role in common pigmented pediatric dermatoses, and special considerations for this unique age group.