Multiple SARS-CoV-2 immunizations of an unvaccinated population lead to complex immunity. A T cell reactivity study of blood donors in Antananarivo.

BACKGROUND: Madagascar has undergone multiple and robust COVID-19 waves. The resulting immune background developed by its poorly vaccinated population has however not been described. METHODS: In this study, serological analysis and specific T cell response descriptions were used to describe the history of exposures of the capital's blood donors to SARS-CoV-2 and its VOCs. Samples were collected early 2022, and pools of multiple immunogenic peptides of SARS-CoV-2 were used in an IFN-γ secretion ELISPOT assay to characterize the specific T-cell immunity developed against these potential epitopes. RESULTS: Multiple epidemic waves have led to 92.1% of donors having detectable antibodies, and 94.8% having developed T-cells against SARS-CoV-2. Heterogeneous reactivities to different strain-derived peptides suggested multiple immunological backgrounds in the population including 16.1% of individuals exposed at least once to a unique strain, 27.1% to two strains, 28.5% to three strains, and 23.1% to four distinct strains. CONCLUSIONS: Cross-reactivity increased with multiple exposures but did not decrease the risk of re-infection. These results describe the extremely complex immunological background developed following multiple natural immunizations.

Using a multiplex serological assay to estimate time since SARS-CoV-2 infection and past clinical presentation in malagasy patients.

Background: The world is facing a 2019 coronavirus (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this context, efficient serological assays are needed to accurately describe the humoral responses against the virus. These tools could potentially provide temporal and clinical characteristics and are thus paramount in developing-countries lacking sufficient ongoing COVID-19 epidemic descriptions. Methods: We developed and validated a Luminex xMAP® multiplex serological assay targeting specific IgM and IgG antibodies against the SARS-CoV-2 Spike subunit 1 (S1), Spike subunit 2 (S2), Spike Receptor Binding Domain (RBD) and the Nucleocapsid protein (N). Blood samples collected periodically for 12 months from 43 patients diagnosed with COVID-19 in Madagascar were tested for these antibodies. A random forest algorithm was used to build a predictive model of time since infection and symptom presentation. Findings: The performance of the multiplex serological assay was evaluated for the detection of SARS-CoV-2 anti-IgG and anti-IgM antibodies. Both sensitivity and specificity were equal to 100% (89.85-100) for S1, RBD and N (S2 had a lower specificity = 95%) for IgG at day 14 after enrolment. This multiplex assay compared with two commercialized ELISA kits, showed a higher sensitivity. Principal Component Analysis was performed on serologic data to group patients according to time of sample collection and clinical presentations. The random forest algorithm built by this approach predicted symptom presentation and time since infection with an accuracy of 87.1% (95% CI = 70.17-96.37, p-value = 0.0016), and 80% (95% CI = 61.43-92.29, p-value = 0.0001) respectively. Interpretation: This study demonstrates that the statistical model predicts time since infection and previous symptom presentation using IgM and IgG response to SARS-CoV2. This tool may be useful for global surveillance, discriminating recent- and past- SARS-CoV-2 infection, and assessing disease severity. Fundings: This study was funded by the French Ministry for Europe and Foreign Affairs through the REPAIR COVID-19-Africa project coordinated by the Pasteur International Network association. WANTAI reagents were provided by WHO AFRO as part of a Sero-epidemiological "Unity" Study Grant/Award Number: 2020/1,019,828-0 P·O 202546047 and Initiative 5% grant n°AP-5PC-2018-03-RO.

Characterization of Klebsiella pneumoniae isolated from patients suspected of pulmonary or bubonic plague during the Madagascar epidemic in 2017.

Klebsiella pneumoniae can lead to a wide range of diseases including pneumonia, bloodstream and urinary tract infections. During a short period of a pulmonary plague epidemic in October 2017 in Madagascar, 12 K. pneumoniae isolates were identified in ten sputum and two buboes aspirate samples. These isolates were from 12 patients suspected of plague, without epidemiological relationships, but were negative for Yersinia pestis in culture. Data were collected from the plague national surveillance system. The isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing. Real-time PCR was performed to confirm the presence of K. pneumoniae DNA in buboes. All isolates were identified as K. pneumoniae sensu stricto. Five isolates were extended-spectrum ß-lactamases producers; eleven different sequence types were identified. Five isolates belonged to known hypervirulent sequence types. Our results demonstrate community-acquired pneumonia caused by K. pneumoniae isolates in patients suspected of plague stressing the importance of bed-side differential diagnosis.

Leveraging serology to titrate immunisation programme functionality for diphtheria in Madagascar.

Diphtheria is a potentially devastating disease whose epidemiology remains poorly described in many settings, including Madagascar. Diphtheria vaccination is delivered in combination with pertussis and tetanus antigens and coverage of this vaccine is often used as a core measure of health system functioning. However, coverage is challenging to estimate due to the difficulty in translating numbers of doses delivered into numbers of children effectively immunised. Serology provides an alternative lens onto immunisation, but is complicated by challenges in discriminating between natural and vaccine-derived seropositivity. Here, we leverage known features of the serological profile of diphtheria to bound expectations for vaccine coverage for diphtheria, and further refine these using serology for pertussis. We measured diphtheria antibody titres in 185 children aged 6-11 months and 362 children aged 8-15 years and analysed them with pertussis antibody titres previously measured for each individual. Levels of diphtheria seronegativity varied among age groups (18.9% of children aged 6-11 months old and 11.3% of children aged 8-15 years old were seronegative) and also among the districts. We also find surprisingly elevated levels of individuals seropositive to diphtheria but not pertussis in the 6-11 month old age group suggesting that vaccination coverage or efficacy of the pertussis component of the DTP vaccine remains low or that natural infection of diphtheria may be playing a significant role in seropositivity in Madagascar.

Seroprevalence of pertussis in Madagascar and implications for vaccination.

Pertussis is a highly contagious infectious disease and remains an important cause of mortality and morbidity worldwide. Over the last decade, vaccination has greatly reduced the burden of pertussis. Yet, uncertainty in individual vaccination coverage and ineffective case surveillance systems make it difficult to estimate burden and the related quantity of population-level susceptibility, which determines population risk. These issues are more pronounced in low-income settings where coverage is often overestimated, and case numbers are under-reported. Serological data provide a direct characterisation of the landscape of susceptibility to infection; and can be combined with vaccination coverage and basic theory to estimate rates of exposure to natural infection. Here, we analysed cross-sectional data on seropositivity against pertussis to identify spatial and age patterns of susceptibility in children in Madagascar. A large proportion of individuals surveyed were seronegative; however, there were patterns suggestive of natural infection in all the regions analysed. Improvements in vaccination coverage are needed to help prevent additional burden of pertussis in the country.

Assessment of tuberculosis spatial hotspot areas in Antananarivo, Madagascar, by combining spatial analysis and genotyping.

BACKGROUND: Tuberculosis (TB) remains a public health problem in Madagascar. A crucial element of TB control is the development of an easy and rapid method for the orientation of TB control strategies in the country. Our main objective was to develop a TB spatial hotspot identification method by combining spatial analysis and TB genotyping method in Antananarivo. METHODS: Sputa of new pulmonary TB cases from 20 TB diagnosis and treatment centers (DTCs) in Antananarivo were collected from August 2013 to May 2014 for culture. Mycobacterium tuberculosis complex (MTBC) clinical isolates were typed by spoligotyping on a Luminex® 200 platform. All TB patients were respectively localized according to their neighborhood residence and the spatial distribution of all pulmonary TB patients and patients with genotypic clustered isolates were scanned respectively by the Kulldorff spatial scanning method for identification of significant spatial clustering. Areas exhibiting spatial clustering of patients with genotypic clustered isolates were considered as hotspot TB areas for transmission. RESULTS: Overall, 467 new cases were included in the study, and 394 spoligotypes were obtained (84.4%). New TB cases were distributed in 133 of the 192 Fokontany (administrative neighborhoods) of Antananarivo (1 to 15 clinical patients per Fokontany) and patients with genotypic clustered isolates were distributed in 127 of the 192 Fokontany (1 to 13 per Fokontany). A single spatial focal point of epidemics was detected when ignoring genotypic data (p = 0.039). One Fokontany of this focal point and three additional ones were detected to be spatially clustered when taking genotypes into account (p < 0.05). These four areas were declared potential TB transmission hotspots in Antananarivo and will be considered as priority targets for surveillance in the future. CONCLUSION: This method, combining spatial analysis and TB genotyping will now be used for further focused clinical and epidemiological studies in Madagascar and will allow better TB control strategies by public health authorities.