Safety, tolerability, and immunogenicity of an oral inactivated ETEC vaccine (ETVAX®) with dmLT adjuvant in healthy adults and children in Zambia: An age descending randomised, placebo-controlled trial.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is an important cause of moderate to severe diarrhoea in children for which there is no licensed vaccine. We evaluated ETVAX®, an oral, inactivated ETEC vaccine containing four E. coli strains over-expressing the major colonization factors CFA/I, CS3, CS5, and CS6, a toxoid (LCTBA) and double mutant heat-labile enterotoxin (dmLT) adjuvant for safety, tolerability, and immunogenicity. METHODS: A double-blind, placebo-controlled, age-descending, dose-finding trial was undertaken in 40 adults, 60 children aged 10-23 months, and 146 aged 6-9 months. Adults received one full dose of ETVAX® and children received 3 doses of either 1/4 or 1/8 dose. Safety was evaluated as solicited and unsolicited events for 7 days following vaccination. Immunogenicity was assessed by evaluation of plasma IgA antibody responses to CFA/I, CS3, CS5, CS6, and LTB, and IgG responses to LTB. RESULTS: Solicited adverse events were mostly mild or moderate with only 2 severe fever reports which were unrelated to the vaccine. The most common events were abdominal pain in adults (26.7 % in vaccinees vs 20 % in placebos), and fever in children aged 6-9 months (44 % vs 54  %). Dosage, number of vaccinations and decreasing age had no influence on severity or frequency of adverse events. The vaccine induced plasma IgA and IgG responses against LTB in 100 % of the adults and 80-90 % of the children. In the 6-23 months cohort, IgA responses to more than 3 vaccine antigens after 3 doses determined as ≥2-fold rise was significantly higher for 1/4 dose compared to placebo (56.7 % vs 27.2 %, p = 0.01). In the 6-9 months cohort, responses to the 1/4 dose were significantly higher than 1/8 dose after 3 rather than 2 doses. CONCLUSION: ETVAX® was safe, tolerable, and immunogenic in Zambian adults and children. The 1/4 dose induced significantly stronger IgA responses and is recommended for evaluation of protection in children. CLINICAL TRIALS REGISTRATION: The trial is registered with the Pan African Clinical Trials Registry (PACTR Ref. 201905764389804) and a description of this clinical trial is available on: https://pactr.samrc.ac.za/Trial Design.

Sensitivity and predictive value of dysentery in diagnosing shigellosis among under five children in Zambia.

BACKGROUND: Shigella is a leading cause of bacterial diarrhea morbidity and mortality affecting mainly children under five in the developing world. In Zambia, Shigella has a high prevalence of 34.7% in children with diarrhea and an attributable fraction of 6.7% in Zambian children with moderate to severe diarrhea. Zambian diarrhea management guidelines and the health ministry reporting tool Health Management Information System (HMIS) heavily rely on the WHO clinical classification of dysentery to potentially identify and estimate the burden of Shigella in children. This reliance on clinical dysentery as a proxy to shigellosis in under five children may be resulting in gross under-estimation of shigella disease burden in Zambia. METHODS: We used existing laboratory and clinical data to examine the sensitivity and predictive value of dysentery to correctly identify Shigella infection in under five children with PCR confirmed Shigella infection in Lusaka and Ndola districts, Zambia. RESULTS: Clinical dysentery had a sensitivity of 8.5% (34/401) in identifying under five children with Shigella by stool PCR. Dysentery was able to correctly classify Shigella in 34 of 68 bloody stool samples giving a corresponding positive predictive value of 50%. Of the 1087 with non-bloody diarrhea, 720 did not have Shigella giving a negative predictive value of 66.2%. CONCLUSIONS: Use of clinical dysentery as a screening symptom for Shigella infection in children under five presenting with moderate to severe diarrhea has low sensitivity and low positive predictive value respectively. Clinical dysentery as a screening symptom for Shigella contributes to gross under diagnosis and reporting of Shigella infection among under five children in Zambia. Further research is required to better inform practice on more accurate methods or tools to use in support of routine diagnosis, particularly in low middle-income settings where laboratory diagnosis remains a challenge.

Assessment of the influence of ABO blood groups on oral cholera vaccine immunogenicity in a cholera endemic area in Zambia.

BACKGROUND: Histo-blood group antigens (HBGAs) which include the ABO and Lewis antigen systems have been known for determining predisposition to infections. For instance, blood group O individuals have a higher risk of severe illness due to V. cholerae compared to those with non-blood group O antigens. We set out to determine the influence that these HBGAs have on oral cholera vaccine immunogenicity and seroconversion in individuals residing within a cholera endemic area in Zambia. METHODOLOGY: We conducted a longitudinal study nested under a clinical trial in which samples from a cohort of 223 adults who were vaccinated with two doses of Shanchol™ and followed up over 4 years were used. We measured serum vibriocidal geometric mean titers (GMTs) at Baseline, Day 28, Months 6, 12, 24, 30, 36 and 48 in response to the vaccine. Saliva obtained at 1 year post vaccination was tested for HBGA phenotypes and secretor status using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the 133/223 participants included in the final analysis, the majority were above 34 years old (58%) and of these, 90% were males. Seroconversion rates to V. cholerae O1 Inaba with non-O (23%) and O (30%) blood types were comparable. The same pattern was observed against O1 Ogawa serotype between non-O (25%) and O (35%). This trend continued over the four-year follow-up period. Similarly, no significant differences were observed in seroconversion rates between the non-secretors (26%) and secretors (36%) against V. cholerae O1 Inaba. The same was observed for O1 Ogawa in non-secretors (22%) and the secretors (36%). CONCLUSION: Our results do not support the idea that ABO blood grouping influence vaccine uptake and responses against cholera.

Machine Learning Algorithms for understanding the determinants of under-five Mortality.

BACKGROUND: Under-five mortality is a matter of serious concern for child health as well as the social development of any country. The paper aimed to find the accuracy of machine learning models in predicting under-five mortality and identify the most significant factors associated with under-five mortality. METHOD: The data was taken from the National Family Health Survey (NFHS-IV) of Uttar Pradesh. First, we used multivariate logistic regression due to its capability for predicting the important factors, then we used machine learning techniques such as decision tree, random forest, Naïve Bayes, K- nearest neighbor (KNN), logistic regression, support vector machine (SVM), neural network, and ridge classifier. Each model's accuracy was checked by a confusion matrix, accuracy, precision, recall, F1 score, Cohen's Kappa, and area under the receiver operating characteristics curve (AUROC). Information gain rank was used to find the important factors for under-five mortality. Data analysis was performed using, STATA-16.0, Python 3.3, and IBM SPSS Statistics for Windows, Version 27.0 software. RESULT: By applying the machine learning models, results showed that the neural network model was the best predictive model for under-five mortality when compared with other predictive models, with model accuracy of (95.29% to 95.96%), recall (71.51% to 81.03%), precision (36.64% to 51.83%), F1 score (50.46% to 62.68%), Cohen's Kappa value (0.48 to 0.60), AUROC range (93.51% to 96.22%) and precision-recall curve range (99.52% to 99.73%). The neural network was the most efficient model, but logistic regression also shows well for predicting under-five mortality with accuracy (94% to 95%)., AUROC range (93.4% to 94.8%), and precision-recall curve (99.5% to 99.6%). The number of living children, survival time, wealth index, child size at birth, birth in the last five years, the total number of children ever born, mother's education level, and birth order were identified as important factors influencing under-five mortality. CONCLUSION: The neural network model was a better predictive model compared to other machine learning models in predicting under-five mortality, but logistic regression analysis also shows good results. These models may be helpful for the analysis of high-dimensional data for health research.

Development of a diarrhoea severity scoring scale in a passive health facility-based surveillance system.

BACKGROUND: Diarrhoeal disease remains a leading cause of death among children mostly in low and middle-income countries. Factors contributing to disease severity are complex and there is currently no consensus on a scoring tool for use in community-based studies. METHODS: Data were collected during a passive surveillance system in an outpatient health facility in Lusaka, Zambia from March 2019 to July 2019. Diarrhea episodes were assessed for severity using an in-house severity scoring tool (CIDRZ) and previously published scores (Vesikari, Clark, CODA, and DHAKA). The CIDRZ score was constructed using fieldworker-reported clinical signs and exploratory factor analysis. We used precision-recall curves measuring severe diarrhoea (i.e., requiring intravenous rehydration or referred for hospital admission) to determine the best performing scores. Then, we used Cronbach's alpha to assess the scale's internal consistency. Finally, we used Cohen's kappa to assess agreement between the scores. RESULTS: Of 110 diarrhea episodes, 3 (3%) required intravenous rehydration or were referred for hospital admission. The precision-recall area under the curve of each score as a predictor of severe diarrhoea requiring intravenous rehydration or hospital admission was 0.26 for Vesikari, 0.18 for CODA, 0.24 for Clark, 0.59 for DHAKA, and 0.59 for CIDRZ. The CIDRZ scale had substantial reliability and performed similarly to the DHAKA score. CONCLUSIONS: Diarrhoea severity scores focused on characteristics specific to dehydration status may better predict severe diarrhea among children in Lusaka. Aetiology-specific scoring tools may not be appropriate for use in community healthcare settings. Validation studies for the CIDRZ score in diverse settings and with larger sample sizes are warranted.

Field evaluation of a novel, rapid diagnostic assay, and molecular epidemiology of enterotoxigenic E. coli among Zambian children presenting with diarrhea.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting. METHODS: We conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the "gold standard" to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC. RESULTS: The study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively. CONCLUSION: The results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings.

Effect of HIV status and retinol on immunogenicity to oral cholera vaccine in adult population living in an endemic area of Lukanga Swamps, Zambia.

BACKGROUND: We set out to assess the impact of human immunodeficiency virus (HIV) and micronutrient deficiency as indicated by serum retinol levels on the immune responses to Oral Cholera Vaccine (Shanchol™) in a cohort of participants in Lukanga Swamps, Zambia. Cholera remains endemic in Zambia with vaccines being the only effective preventive measures. However, the effect of these vaccines on populations living with HIV has not been widely documented. METHODS: HIV testing and confirmation was done using the Alere Determine™ HIV-1/2 and Uni-Gold™ kits while vibriocidal antibody assay was applied for vaccine immunogenicity. Serum retinol analysis was assessed by Shimadzu Prominence HCT-2010 High Performance Liquid Chromatography (HPLC). The primary outcome was log transformed geometric mean titre. RESULTS: From 47 participants screened for HIV, 51% (24) tested positive. There was a statistically significant reduction in Ogawa geometric mean ratio (GMR) by 67% (GMR = 0.33; 95% CI: -0.15, 0.76; p-value = 0.009) attributable to HIV positivity with a non-significant reduction in Inaba GMR by about 50% due to HIV positivity. When doubling of retinol levels modelled, GMR reduction against Ogawa were non-significant but that against Inaba resulted in a significant reduction in geometric mean titer (GMT) (GMT-0.33, C.I 0.16-0.66, p-value 0.002). At 1000copies/ml viral load cut off and 350 cells/µl CD4 counts, Ogawa GMT was two times higher 11.16 (95%CI: 8.20-15.19) versus 6.06 (95%CI: 4.04-9.10) in low viremia participants, and three times higher in above threshold CD4 count participants; 24.81 (95%CI: 18.94-32.50) versus 7.07 (95%CI: 5.22-9.58). CONCLUSION: Our results show that while Shanchol™ is immunogenic in both HIV+/- individuals, HIV + participants responded poorly. Viral load and CD4 count affected vaccine immunogenicity. More research is required for detailed understanding of this in order to appropriately inform policy and practice.

Shigella-specific antibodies in the first year of life among Zambian infants: A longitudinal cohort study.

INTRODUCTION: Shigellosis, is a leading cause of moderate-to-severe diarrhoea and related mortality in young children in low and middle income countries (LMICs). Knowledge on naturally acquired immunity can support the development of Shigella candidate vaccines mostly needed in LMICs. We aimed to quantify Shigella-specific antibodies of maternal origin and those naturally acquired in Zambian infants. METHODS: Plasma samples collected from infants at age 6, 14 and 52-weeks were tested for Shigella (S. sonnei and S. flexneri 2a) lipopolysaccharide (LPS) antigen specific immunoglobulin G (IgG) and A (IgA) by enzyme-linked immunosorbent assay. RESULTS: At 6 weeks infant age, the IgG geometric mean titres (GMT) against S. sonnei (N = 159) and S. flexneri 2a (N = 135) LPS were 311 (95% CI 259-372) and 446 (95% CI 343-580) respectively. By 14 weeks, a decline in IgG GMT was observed for both S. sonnei to 104 (95% CI 88-124), and S. flexneri 2a to 183 (95% CI 147-230). Both S. sonnei and S. flexneri 2a specific IgG GMT continued to decrease by 52 weeks infant age when compared to 6 weeks. In 27% and 8% of infants a significant rise in titre (4 fold and greater) against S. flexneri 2a and S. sonnei LPS, respectively, was detected between the ages of 14 and 52 weeks. IgA levels against both species LPS were very low at 6 and 14 weeks and raised significantly against S. flexneri 2a and S. sonnei LPS in 29% and 10% of the infants, respectively. CONCLUSION: In our setting, transplacental IgG anti-Shigella LPS is present at high levels in early infancy, and begins to decrease by age 14 weeks. Our results are consistent with early exposure to Shigella and indicate naturally acquired IgG and IgA antibodies to S. flexneri 2a and S. sonnei LPS in part of infants between 14 and 52 weeks of age. These results suggest that a potential timing of vaccination would be after 14 and before 52 weeks of age to ensure early infant protection against shigellosis.

Clinical presentation of congenital syphilis in a rotavirus vaccine cohort study in Lusaka: a case series.

BACKGROUND: Despite an otherwise robust national antenatal clinic program, maternal and congenital syphilis remains an important public health issue in Zambia. This case series reports the clinical presentation of seven infants diagnosed with congenital syphilis in Lusaka, Zambia. CASE PRESENTATIONS: The cases in this series were incidental findings from a cohort of infants enrolled in a rotavirus vaccine immunogenicity study recruiting infants at 6 weeks of age. As part of clinical care for enrolled participants, we screened mothers of children who presented with adverse events of (i) repeated upper respiratory tract infections/coryza, (ii) skin lesions, and (iii) poor weight gain, for syphilis using rapid plasma reagin test. From a cohort of 214 mother-infant pairs enrolled between September and December 2018, a total of 115 (44.4%) of the mothers reported to have not been screened during antenatal care. Of these, four (3.5%) reported to have tested positive; and only two received treatment. Seven out of 57 (26.6%) children meeting the screening criteria had a positive rapid plasma reagin test result. The mean age at diagnosis was 4.5 months (1.3 months standard deviation), and the common presenting features included coryza (6/7), skin lesions (4/7), conjunctivitis (3/7), pallor/anemia (5/7), wasting (2/7), and underweight (5/7). Three of the seven infants were exposed to human immunodeficiency virus. Following diagnosis, all seven cases received standard treatment according to national treatment guidelines. That is, 6/7 cases received inpatient care with benzylpenicillin for 10 days, while 1/7 was treated as an outpatient and received daily procaine penicillin for 10 days. CONCLUSION: These findings suggest that, though screening for syphilis is part of the standard antenatal care in Zambia, it is not offered optimally. There is urgent need to address programmatic shortcomings in syphilis screening and treatment to avoid long-term sequelae. Additionally, clinicians need to raise their index of suspicion and rule out syphilis when confronted with these clinical symptoms, regardless of the mother's human immunodeficiency virus status.

Sero-prevalence of arthropod-borne viral infections among Lukanga swamp residents in Zambia.

INTRODUCTION: The re-emergence of vector borne diseases affecting millions of people in recent years has drawn attention to arboviruses globally. Here, we report on the sero-prevalence of chikungunya virus (CHIKV), dengue virus (DENV), mayaro virus (MAYV) and zika virus (ZIKV) in a swamp community in Zambia. METHODS: We collected blood and saliva samples from residents of Lukanga swamps in 2016 during a mass-cholera vaccination campaign. Over 10,000 residents were vaccinated with two doses of Shanchol™ during this period. The biological samples were collected prior to vaccination (baseline) and at specified time points after vaccination. We tested a total of 214 baseline stored serum samples for IgG antibodies against NS1 of DENV and ZIKV and E2 of CHIKV and MAYV on ELISA. We defined sero-prevalence as the proportion of participants with optical density (OD) values above a defined cut-off value, determined using a finite mixture model. RESULTS: Of the 214 participants, 79 (36.9%; 95% CI 30.5-43.8) were sero-positive for Chikungunya; 23 (10.8%; 95% CI 6.9-15.7) for Zika, 36 (16.8%; 95% CI 12.1-22.5) for Dengue and 42 (19.6%; 95% CI 14.5-25.6) for Mayaro. Older participants were more likely to have Zika virus whilst those involved with fishing activities were at greater risk of contracting Chikungunya virus. Among all the antigens tested, we also found that Chikungunya saliva antibody titres correlated with baseline serum titres (Spearman's correlation coefficient = 0.222; p = 0.03). CONCLUSION: Arbovirus transmission is occurring in Zambia. This requires proper screening tools as well as surveillance data to accurately report on disease burden in Zambia.

Patients' Satisfaction with HIV Care Providers in Public Health Facilities in Lusaka: A Study of Patients who were Lost-to-Follow-Up from HIV Care and Treatment.

Prognosis among those who are HIV infected has improved but long-term retention is challenging. Health systems may benefit from routinely measuring patient satisfaction which is a potential driver of engagement in HIV care, but it is not often measured in Africa, and Zambia in particular. This study aims to internally validate a patient satisfaction tool, assess satisfaction among patients previously lost-to-follow up (LTFU) from HIV care in Lusaka province and to measure association between patient satisfaction with their original clinic and re-engagement in HIV care. A cross-sectional assessment of satisfaction was conducted by tracing sampled patients drawn from public health facilities. Our findings suggest that satisfaction tool, previously validated in USA, exhibits high internal consistency for measuring patient satisfaction in the Zambian health system. Patient satisfaction with healthcare providers is associated with re-engagement in HIV care. Future interventions on patient-centred care are likely to optimize and support retention in care.

Evaluating the costs of cholera illness and cost-effectiveness of a single dose oral vaccination campaign in Lusaka, Zambia.

INTRODUCTION: In 2016, for the very first time, the Ministry of Health in Zambia implemented a reactive outbreak response to control the spread of cholera and vaccinated at-risk populations with a single dose of Shancol-an oral cholera vaccine (OCV). This study aimed to assess the costs of cholera illness and determine the cost-effectiveness of the 2016 vaccination campaign. METHODOLOGY: From April to June 2017, we conducted a retrospective cost and cost-effectiveness analysis in three peri-urban areas of Lusaka. To estimate costs of illness from a household perspective, a systematic random sample of 189 in-patients confirmed with V. cholera were identified from Cholera Treatment Centre registers and interviewed for out-of-pocket costs. Vaccine delivery and health systems costs were extracted from financial records at the District Health Office and health facilities. The cost of cholera treatment was derived by multiplying the subsidized cost of drugs by the quantity administered to patients during hospitalisation. The cost-effectiveness analysis measured incremental cost-effectiveness ratio-cost per case averted, cost per life saved and cost per DALY averted-for a single dose OCV. RESULTS: The mean cost per administered vaccine was US$1.72. Treatment costs per hospitalized episode were US$14.49-US$18.03 for patients ≤15 years old and US$17.66-US$35.16 for older patients. Whereas households incurred costs on non-medical items such as communication, beverages, food and transport during illness, a large proportion of medical costs were borne by the health system. Assuming vaccine effectiveness of 88.9% and 63%, a life expectancy of 62 years and Gross Domestic Product (GDP) per capita of US$1,500, the costs per case averted were estimated US$369-US$532. Costs per life year saved ranged from US$18,515-US$27,976. The total cost per DALY averted was estimated between US$698-US$1,006 for patients ≤15 years old and US$666-US$1,000 for older patients. CONCLUSION: Our study determined that reactive vaccination campaign with a single dose of Shancol for cholera control in densely populated areas of Lusaka was cost-effective.

Early linear growth retardation: results of a prospective study of Zambian infants.

BACKGROUND: Linear growth retardation is the most dominant nutritional problem globally. We aimed to describe linear growth trajectory among infants under 2 years of age using the WHO growth velocity standards. METHOD: This was a prospective cohort study of infants enrolled at 6 weeks of age and followed up for up to 24 months in Kamwala Urban Health Centre, Lusaka, Zambia. The study was conducted between April 2013 and March 2015. Infants were enrolled if they were 6-12 weeks of age and the mother was willing to participate voluntarily and provided informed consent. Anthropometric data were collected at scheduled clinic visits at 1 month, 2 months, 3 months, then quarterly until the infant was 24 months old. We defined linear growth velocity as the rate of change in height. We estimated linear growth velocity as the first derivative of the penalized cubic spline mixed effects model. RESULTS: A total of 338 children were included in the analysis. Of these, 185 (54.7%) were female, 115 (34.1%) were born to HIV positive mothers and thus classified as HIV Exposed (HE). The mean age of children at enrollment was 1.6 months (SD = 0.15). On average, the growth velocity for 3-month length increments conditional on age were 0-3 months = 2.97 cm/3mo (95%CI = 2.69, 3.25); 3-6 months = 2.62 cm/3mo (95%CI = 2.38, 2.87); 6-9 months = 1.57 cm/3mo (95%CI = 1.43, 1.71); 9-12 months = 1.18 cm/3mo (95%CI = 1.08, 1.28); 12-15 month = 1.14 cm/3mo (95%CI = 1.02, 1.27); 15-18 months = 0.87 cm/3mo (95%CI = 0.79, 0.96); 18-21 months = 0.80 cm/3mo (95%CI = 0.72, 0.89); and 21-24 months = 0.86 cm/3mo (95%CI = 0.77, 0.96). For both boys and girls, the growth velocity in our cohort were consistently below the 3rd percentile of the WHO linear growth velocity standard. The estimated mean height and the age at which growth begins to falter were 68.6 cm (95%CI = 68.0, 69.2) and 13.6 months (95%CI = 13.2, 14.1) respectively. CONCLUSION: We found slower rate of growth among otherwise healthy Zambian infants. The data suggests that growth retardation is universal and profound in this cohort and may have already been occurring in utero.