Background COVID-19 remains a global public health challenge due to new immune-evasive SARS-CoV-2 variants and heterogeneous immunity. Methods In this cross-sectional study, we evaluated the adaptive immune responses in US active duty personnel who completed a COVID-19 primary vaccine series and had heterogenous SARS-CoV-2 vaccination and infection histories to 3 previously dominant variants (ancestral, Delta, BA.5) and 3 circulating variants (XBB.1.5, EG.5, and BA.2.86) in late 2023. Analyses were based on the most recent exposure in terms of timing (within or beyond 12 months) and type (vaccine or infection). Results Significant reduction was observed in binding antibodies, neutralization antibodies, memory B cells, and CD8+ T cells against circulating variants when compared with previous variants. The reduction in antibody response was more pronounced in those whose most recent exposure was >12 months from enrollment. In contrast, the CD4+ T-cell response was largely consistent across all tested variants. The type of most recent exposure was not a significant factor in determining the magnitude of current immune responses. Conclusions Administration of the XBB.1.5-based booster is likely to enhance cross-reactive humoral responses against SARS-CoV-2 circulating lineages. Ongoing surveillance of immune responses to emerging variants is needed for informing vaccine composition and timing.

Background: Resistance to antimalarials in Plasmodium falciparum spreads if mutations increase parasite survival when encountering the drug. Because such mutations typically reduce parasite growth (metabolic costs), they are believed to spread only if drug pressure is sufficiently high, and drug sensitivity returns after drug use is discontinued, as observed with chloroquine resistance. However, this was not observed with sulfadoxine-pyrimethamine (SP) resistance. For instance, when SP was discontinued in Peru the resistant 50R/51I/108N replaced the highly resistant 51I/108N/164L Pfdhfr variant, after the slightly resistant 108N mutant became temporarily predominant.Methods: Prevalence data of SP resistant Pfdhfr mutations from Peru and results from published yeast expression systems for antifolate sensitivity are combined with an evolutionary-genetic model tailored towards malaria to explain the spread of the 51I/108N/164L and 50R/51II108N mutants.Findings: The increased parasite growth rate (metabolic advantage) of the C50R mutation, seen in yeast expression systems, explains empirical observations in Peru, and is consistent with findings in other countries. Drug pressure is necessary for the 50R/51I/108N mutant to emerge. However, it cannot become predominant in the presence of more resistant variants until drug pressure is lifted. Afterwards, the 50R/51I/108N mutant spreads and prevents the re-emergence of sensitive types.Interpretation: If the C50R mutation spreads after SP is discontinued, its associated increased growth rate can negatively affect the clinical pathogenesis of malaria infection or even lead to an increased likelihood of SP-unrelated treatment failures. Particularly, the mutation potentially excels the spread of artemisinin resistance. Also, novel mutations might be falsely associated with drug resistance if the spread of the advantageous C50R mutation is not accounted for. Therefore, surveillance of the often-disregarded C50R mutation must be reconsidered on a global scale in the context of antimalarial drug resistance.Funding: This study was funded by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch, ProMIS ID P0134 23 N6. This work was also supported by grants of the German Academic Exchange (DAAD; https://www. daad.de/de/; Project-ID 57417782, Project-ID: 57599539), the S ̈achsisches Staatsministerium f ̈ur Wis- senschaft, Kultur und Tourismus and S ̈achsische Aufbaubank – F ̈orderbank (SMWK-SAB; https://www. smwk.sachsen.de/; https://www.sab.sachsen.de/; project “Innovationsvorhaben zur Profilsch ̈arfung an Hochschulen f ̈ur angewandte Wissenschaften”, Project-ID 100257255; project “Innovationsvorhaben zur Profilsch ̈arfung 2022”, Project-ID: 100613388), the Federal Ministry of Education and Research (BMBF) and the DLR (Project-ID 01DQ20002; https://www.bmbf.de/; https://www.dlr.de/).Declaration of Interest: The authors have declared that no competing interests exist.Ethical Approval: The protocol for this study (NAMRU6.2019.0009) was reviewed and approved by the head of the Research Administration Program of the Naval Medical Research Unit-6 (NAMRU-6) and determined it to be a non-human subject research. This study was a secondary data analysis and informed consent was not required for this since it does not meet the definition of research involving human subjects per US Code of Federal Regulations, 32 CFR Part 219 - PROTECTION OF HUMAN SUBJECTS, section 219.104 Exempt research. For samples collected as part of previous NAMRU-6 protocols, we used sample codes from volunteers who provided consent for future use.

This study tested the hypothesis that high frequencies of natural killer (NK) cells are protective against symptomatic SARS-CoV-2 infection. Samples were utilized from the COVID-19 Health Action Response for Marines study, a prospective, observational study of SARS-CoV-2 infection in which participants were enrolled prior to infection and then serially monitored for development of symptomatic or asymptomatic infection. Frequencies and phenotypes of NK cells (CD3−CD14−CD19−CD56+) were assessed by flow cytometry. Individuals that developed asymptomatic infections were found to have higher pre-infection frequencies of total NK cells compared to symptomatic individuals (10.61% [SD 4.5] vs 8.33% [SD 4.6], p = 0.011). Circulating total NK cells decreased over the course of infection, reaching a nadir at 4 weeks, while immature NK cells increased, a finding confirmed by multidimensional reduction analysis. These results indicate that NK cells likely play a key role in controlling the severity of clinical illness in individuals infected with SARS-CoV-2.

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

Adaptive immunity mediated by antibodies is important for controlling SARS-CoV-2 infection. While vaccines against COVID-19 are currently widely distributed, a high proportion of the global population is still unvaccinated.

Malaria is a major health problem in Peru despite substantial progress achieved by the ongoing malaria elimination program. This study explored the population genetics of 63 Plasmodium falciparum and 170 P. vivax cases collected in the Peruvian Amazon Basin between 2015 and 2019. Microscopy and PCR were used for malaria detection and positive samples were genotyped at neutral and drug resistance-associated regions. The P. falciparum population exhibited a low nucleotide diversity (π = 0.02) whereas the P. vivax population presented a higher genetic diversity (π = 0.34). All P. falciparum samples (n = 63) carried chloroquine (CQ) resistant mutations on Pfcrt . Most P. falciparum samples (53 out of 54) carried sulfadoxine (SD) resistant mutations on Pfdhfr and Pfdhps . No evidence was found of artemisinin resistance mutations on kelch13 . Population structure showed that a single cluster accounted for 93.4% of the P. falciparum samples whereas three clusters were found for P. vivax . Our study shows a low genetic diversity for both species with significant differences in genetic sub-structuring. The high prevalence of CQ-resistance mutations could be a result of indirect selection pressures driven by the P. vivax treatment scheme. These results could be useful for public health authorities to safeguard the progress that Peru has achieved towards malaria elimination.

BACKGROUND:Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort. METHODS:Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens. RESULTS:Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks. CONCLUSIONS:Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Young adults infected with SARS-CoV-2 are frequently asymptomatic or develop only mild disease. Because capturing representative mild and asymptomatic cases require active surveillance, they are less characterized than moderate or severe cases of COVID-19. However, a better understanding of SARS-CoV-2 asymptomatic infections might shed light into the immune mechanisms associated with the control of symptoms and protection. To this aim, we have determined the temporal dynamics of the humoral immune response, as well as the serum inflammatory profile, of mild and asymptomatic SARS-CoV-2 infections in a cohort of 172 initially seronegative prospectively studied United States Marine recruits, 149 of whom were subsequently found to be SARS-CoV-2 infected. The participants had blood samples taken, symptoms surveyed and PCR tests for SARS-CoV-2 performed periodically for up to 105 days. We found similar dynamics in the profiles of viral load and in the generation of specific antibody responses in asymptomatic and mild symptomatic participants. A proteomic analysis using an inflammatory panel including 92 analytes revealed a pattern of three temporal waves of inflammatory and immunoregulatory mediators, and a return to baseline for most of the inflammatory markers by 35 days post-infection. We found that 23 analytes were significantly higher in those participants that reported symptoms at the time of the first positive SARS-CoV-2 PCR compared with asymptomatic participants, including mostly chemokines and cytokines associated with inflammatory response or immune activation (i.e., TNF-α, TNF-β, CXCL10, IL-8). Notably, we detected 7 analytes (IL-17C, MMP-10, FGF-19, FGF-21, FGF-23, CXCL5 and CCL23) that were higher in asymptomatic participants than in participants with symptoms; these are known to be involved in tissue repair and may be related to the control of symptoms. Overall, we found a serum proteomic signature that differentiates asymptomatic and mild symptomatic infections in young adults, including potential targets for developing new therapies and prognostic tests.

Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex differ-ence, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infec-tions occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of anti-viral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infec-tion. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.

Peru was the first country where pfhrp2 and pfhrp3 gene deletions were detected despite the fact that rapid diagnostics tests are not commonly used for confirmatory malaria diagnosis. This context provides a unique scenario to study the dynamics of pfhrp2 and pfhrp3 gene deletions without apparent RDTs selection pressure. In this study we characterized the presence of pfhrp2 and pfhrp3 genes on 325 P. falciparum samples collected in Iquitos and surrounding communities between 2011 and 2018 in order to understand the dynamics of gene deletion prevalence, potential associations with clinical symptomatology and parasite genetic background. P. falciparum presence was confirmed by microscopy and PCR of 18 s rRNA, pfmsp1 and pfmsp2. Gene deletions were assessed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCRs. Confirmation of absence of HRP2 expression was assessed by ELISA of HRP2 and pLDH. Genotyping of 254 samples were performed using a panel of seven neutral microsatellite markers. Overall, pfhrp2 and pfhrp3 dual gene deletions were detected in 67% (217/324) parasite samples. Concordance between pfhrp2 deletion and negligible HRP2 protein levels was observed (Cohen's Kappa = 0.842). Prevalence of gene deletions was heterogeneous across study sites (adjusted p < 0.005) but there is an overall tendency towards increase through time in the prevalence of dual pfhrp2/3-deleted parasites between 2011 (14.3%) and 2016 (88.39%) stabilizing around 65% in 2018. Dual deletions increase was associated with dominance of a single new parasite haplotype (H8) which rapidly spread to all study sites during the 8 study years. Interestingly, participants infected with dual pfhrp2/3-deleted parasites had a significantly lower parasitemias than those without gene deletions in this cohort. Our study showed the increase of pfhrp2/3 deletions in the absence of RDTs pressure and a clonal replacement of circulating lines in the Peruvian Amazon basin. These results suggest that other factors linked to the pfhrp2/3 deletion provide a selective advantage over non-deleted strains and highlight the need for additional studies and continuing surveillance.

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses against S, N and M proteins in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were enrolled in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p = 0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic but not symptomatic subjects. Asymptomatic IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but not symptomatic participants. The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-γ and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

As NK cells are critical for control of viral infections, we tested the hypothesis that individuals with asymptomatic SARS-CoV-2 infection have greater NK cell frequencies prior to infection than individuals with mild, symptomatic infection. Marine recruits who elected to participate in the COVID-19 Health Action Response for Marines (CHARM) study were prospectively followed for two months twice weekly using PCR testing for SARS-CoV-2 infection and symptom questionnaires. Peripheral blood mononuclear cells obtained prior to infection during enrollment in the study and at various time points post-infection from 47 participants diagnosed with asymptomatic infection and 49 with symptomatic infection were analyzed by flow cytometry for frequencies of total NK cells, NK cell subsets, and surface expression of activating and inhibitory receptors. Consistent with our hypothesis, frequencies of total (CD3−CD14−CD19−CD56+) NK cells were significantly greater at baseline in asymptomatic compared to symptomatic participants (10.4% vs 8.3%, p = 0.015). This difference was due to increased frequencies of mature (CD56dimCD16+) NK cells in asymptomatic individuals (76.3% vs 69.8%, p = 0.049). No differences were observed in baseline frequencies of immature (CD56brightCD16−) or adaptive (CD56dimCD16+NKG2C+CD57+) NK cells, or in surface expression of activating (NKG2C, NKG2D) or inhibitory (NKG2A, KIR2DL1, KIR2Dl2/L3/S2, KIR3DL1) receptors. Frequencies of total and mature NK cells decreased significantly post-infection, reaching a nadir at 3–4 weeks. In contrast, frequencies of immature NK cells increased by 4 weeks. These results suggest that NK cells may play a protective role against symptomatic SARS-CoV-2 infection. This work was supported by a grant from the Defense Health Agency through the Naval Medical Research Center (work unit number 9700130).

We investigated serological responses following a SARS-CoV-2 outbreak in spring 2020 on a US Marine recruit training base. 147 participants that were isolated during an outbreak of respiratory illness were enrolled in this study, with visits approximately 6 and 10 weeks post-outbreak (PO). This cohort is comprised of young healthy adults, ages 18-26, with a high rate of asymptomatic infection or mild symptoms, and therefore differs from previously reported longitudinal studies on humoral responses to SARS-CoV-2, which often focus on more diverse age populations and worse clinical presentation. 80.9% (119/147) of the participants presented with circulating IgG antibodies against SARS-CoV-2 spike (S) receptor-binding domain (RBD) at 6 weeks PO, of whom 97.3% (111/114) remained positive, with significantly decreased levels, at 10 weeks PO. Neutralizing activity was detected in all sera from SARS-CoV-2 IgG positive participants tested (n=38) at 6 and 10 weeks PO, without significant loss between time points. IgG and IgA antibodies against SARS-CoV-2 RBD, S1, S2, and the nucleocapsid (N) protein, as well neutralization activity, were generally comparable between those participants that had asymptomatic infection or mild disease. A multiplex assay including S proteins from SARS-CoV-2 and related zoonotic and human endemic betacoronaviruses revealed a positive correlation for polyclonal cross-reactivity to S after SARS-CoV-2 infection. Overall, young adults that experienced asymptomatic or mild SARS-CoV-2 infection developed comparable humoral responses, with no decrease in neutralizing activity at least up to 10 weeks after infection.

In a study of US Marine recruits, seroprevalence of severe acute respiratory syndrome coronavirus 2 IgG was 9.0%. Hispanic and non-Hispanic Black participants and participants from states affected earlier in the pandemic had higher seropositivity rates. These results suggest the need for targeted public health strategies among young adults at increased risk for infection.

Previous studies have shown that P. falciparum parasites in South America have undergone population bottlenecks resulting in clonal lineages that are differentially distributed and that have been responsible for several outbreaks different endemic regions. In this study, we explored the genomic profile of 18 P. falciparum samples collected in the Peruvian Amazon Basin (Loreto) and 6 from the Peruvian North Coast (Tumbes). Our results showed the presence of three subpopulations that matched previously typed lineages in Peru: Bv1 (n = 17), Clonet D (n = 4) and Acre-Loreto type (n = 3). Gene coverage analysis showed that none of the Bv1 samples presented coverage for pfhrp2 and pfhrp3. Genotyping of drug resistance markers showed a high prevalence of Chloroquine resistance mutations S1034C/N1042D/D1246Y in pfmdr1 (62.5%) and K45T in pfcrt (87.5%). Mutations associated with sulfadoxine and pyrimethamine treatment failure were found on 88.8% of the Bv1 samples which were triple mutants for pfdhfr (50R/51I/108N) and pfdhps (437G/540E/581G). Analysis of the pfS47 gene that allows P. falciparum to evade mosquito immune responses showed that the Bv1 lineage presented one pfS47 haplotype exclusive to Loreto and another haplotype that was present in both Loreto and Tumbes. Furthermore, a possible expansion of Bv1 was detected since 2011 in Loreto. This replacement could be a result of the high prevalence of CQ resistance polymorphisms in Bv1, which could have provided a selective advantage to the indirect selection pressures driven by the use of CQ for P. vivax treatment.

BackgroundWhether young adults who are infected with SARS-CoV-2 are at risk of subsequent infection is uncertain. We investigated the risk of subsequent SARS-CoV-2 infection among young adults seropositive for a previous infection.MethodsThis analysis was performed as part of the prospective COVID-19 Health Action Response for Marines study (CHARM). CHARM included predominantly male US Marine recruits, aged 18–20 years, following a 2-week unsupervised quarantine at home. After the home quarantine period, upon arrival at a Marine-supervised 2-week quarantine facility (college campus or hotel), participants were enrolled and were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a dilution of 1:150 or more on receptor-binding domain and full-length spike protein ELISA. Participants also completed a questionnaire consisting of demographic information, risk factors, reporting of 14 specific COVID-19-related symptoms or any other unspecified symptom, and brief medical history. SARS-CoV-2 infection was assessed by PCR at weeks 0, 1, and 2 of quarantine and participants completed a follow-up questionnaire, which included questions about the same COVID-19-related symptoms since the last study visit. Participants were excluded at this stage if they had a positive PCR test during quarantine. Participants who had three negative swab PCR results during quarantine and a baseline serum serology test at the beginning of the supervised quarantine that identified them as seronegative or seropositive for SARS-CoV-2 then went on to basic training at Marine Corps Recruit Depot—Parris Island. Three PCR tests were done at weeks 2, 4, and 6 in both seropositive and seronegative groups, along with the follow-up symptom questionnaire and baseline neutralising antibody titres on all subsequently infected seropositive and selected seropositive uninfected participants (prospective study period).FindingsBetween May 11, 2020, and Nov 2, 2020, we enrolled 3249 participants, of whom 3168 (98%) continued into the 2-week quarantine period. 3076 (95%) participants, 2825 (92%) of whom were men, were then followed up during the prospective study period after quarantine for 6 weeks. Among 189 seropositive participants, 19 (10%) had at least one positive PCR test for SARS-CoV-2 during the 6-week follow-up (1·1 cases per person-year). In contrast, 1079 (48%) of 2247 seronegative participants tested positive (6·2 cases per person-year). The incidence rate ratio was 0·18 (95% CI 0·11–0·28; p<0·001). Among seropositive recruits, infection was more likely with lower baseline full-length spike protein IgG titres than in those with higher baseline full-length spike protein IgG titres (hazard ratio 0·45 [95% CI 0·32–0·65]; p<0·001). Infected seropositive participants had viral loads that were about 10-times lower than those of infected seronegative participants (ORF1ab gene cycle threshold difference 3·95 [95% CI 1·23–6·67]; p=0·004). Among seropositive participants, baseline neutralising titres were detected in 45 (83%) of 54 uninfected and in six (32%) of 19 infected participants during the 6 weeks of observation (ID50 difference p<0·0001).InterpretationSeropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies.FundingDefense Health Agency and Defense Advanced Research Projects Agency.

The reinfection risk in individuals previously infected with SARS-CoV-2 is about a fifth of those never infected.1Hansen CH Michlmayr D Gubbels SM Mølbak K Ethelberg S Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Summary Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar Whether reinfected individuals shed viable virus has been identified as an important question relevant to pandemic control.3Ledford H Coronavirus reinfections: three questions scientists are asking.Nature. 2020; 585: 168-169Crossref PubMed Scopus (39) Google Scholar In a prospective cohort study in The Lancet Respiratory Medicine,2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar we identified 19 cases of reinfection in people who at study entry were seropositive for both SARS-CoV-2 receptor binding domain and full-length spike protein, tested negative on three nasal swab PCR tests over a 2-week quarantine period, and subsequently developed a positive PCR test at least 2 weeks after leaving quarantine.2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar We have now investigated whether these SARS-CoV-2-reinfected individuals shed viable virus. Viral transport media was available from the first and some subsequent PCR-positive tests from 16 (84%) of 19 reinfected participants. Samples were cultured for SARS-CoV-2 in Vero E6 cells expressing TMPRSS24Matsuyama S Nao N Shirato K et al.Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.Proc Natl Acad Sci USA. 2020; 117: 7001-7003Crossref PubMed Scopus (852) Google Scholar in T25 cm2 flasks and monitored for cytopathic effect for 4 days followed by a second passage onto fresh cells to allow additional time for virus amplification. Samples were simultaneously titred by plaque assay in Vero E6/TMPRSS2 cells to quantify the level of detectable infectious virus. Viable virus was detected in only four (25%) of 16 participants, and only once in each, with titres ranging from 1·7 to 5·5 log10 plaque-forming units per mL. Serology and PCR cycle threshold (Ct) values using the US Food and Drug Administration-authorised Thermo Fisher TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA, USA) were compared in those with viable virus detected (shedders) and those without viable virus detected (non-shedders; table; appendix p 2). At the time of positive viral culture, samples from shedders had lower Ct values. Although all participants had detectable SARS-CoV-2 spike IgG at study enrolment, none of the four shedders and only four (33%) of the 12 non-shedders had detectable serum neutralisation activity (limit of detection was 50% inhibitory dilution of 20) in serum at the time of enrolment into the study (methods as described2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Only one (25%) of the four who shed virus was symptomatic when viable virus was detected.TableBaseline serology, clinical features, and SARS-CoV-2 Ct levels in reinfected participants with samples cultured for viable virusVirus culture titreDifference*Difference in mean for the continuous variables and difference in percentage for binary variables.Positive (n=4)Negative (n=12)Baseline IgG S titre (log10)2·8 (0·2)2·9 (0·7)−0·08 (−0·88 to 0·72), p=0·834Baseline ID50 detected (>20)04 (33%)−33% (−87 to 21), p=0·207PCR positive >7 days3 (75%)4 (33%)42% (−20 to 100), p=0·166Symptomatic1 (25%)3 (25%)−0% (−57 to 57), p=1·000N gene Ct16·2 (4·0)31·9 (6·1)−15·64 (−22·68 to −8·60), p=0·0003S gene Ct17·8 (4·1)32·3 (6·2)−14·52 (−21·76 to −7·28), p=0·0007ORF1ab gene Ct16·8 (3·9)31·6 (6·1)−14·83 (−21·88 to −7·78), p=0·0005Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution.* Difference in mean for the continuous variables and difference in percentage for binary variables. Open table in a new tab Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution. All reinfections occurred before December, 2020, when the B.1.1.7 variant was first reported in the USA. Full-length viral genome was recovered from seven (44%) of 16 participants studied by viral transport medium culturing, including all four culture-positive individuals (methods previously described5Letizia AG Ramos I Obla A et al.SARS-CoV-2 transmission among marine recruits during quarantine.N Engl J Med. 2020; 383: 2407-2416Crossref PubMed Scopus (64) Google Scholar). Although the strains isolated all had the Asp614Gly spike protein mutation and two of the isolates had the Leu18Phe spike protein mutation, they did not have mutations associated with the B.1.1.7, P.1, B.1.351, or other variants of concern as currently defined by the US Centers for Disease Control and Prevention (appendix p 3). Limitations of our study are the use of virus culture as a proxy for capacity for transmission without direct study of transmission, the small sample size, the absence of specimens available for culturing from all reinfected participants, and the inability to compare potential for transmissibility between those who are infected for the first time and those who are reinfected. Overall, our findings suggest that about a quarter of young healthy individuals with subsequent SARS-CoV-2 reinfection shed viable virus. Some of these individuals were asymptomatic and could unknowingly transmit SARS-CoV-2 to others. All authors declare no competing interests. This study was supported by the Defense Health Agency and Defense Advanced Research Projects Agency. AGL, DRS, CG, DLW, HWC, RAL, SEL, JM, EN, CKP, ESA, MS, VAS, PS, and MT are military service members or government service employees. This work was prepared as part of their official duties. Title 17, US Code §105 provides that copyright protection under this title is not available for any work of the US Government. Title 17, US code §101 defines a US Government work as a work prepared by a military service member or employee of the US Government as part of that person's official duties. The views expressed in the article are those of the authors and do not necessarily express the official policy and position of the US Navy, the Department of Defense, the US Government, or the institutions affiliated with the authors. Download .pdf (.23 MB) Help with pdf files Supplementary appendix SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort studySeropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies. Full-Text PDF

Malaria elimination efforts in Peru have dramatically reduced the incidence of cases in the Amazon Basin. To achieve the elimination, the detection of asymptomatic and submicroscopic carriers becomes a priority. Therefore, efforts should focus on tests sensitive enough to detect low-density parasitemia, deployable to resource-limited areas and affordable for large screening purposes. In this study, we assessed the performance of the Malachite-Green LAMP (MG-LAMP) using heat-treated DNA extraction (Boil & Spin; B&S MG-LAMP) on 283 whole blood samples collected from 9 different sites in Loreto, Peru and compared its performance to expert and field microscopy. A real-time PCR assay was used to quantify the parasite density. In addition, we explored a modified version of the B&S MG-LAMP for detection of submicroscopic infection in 500 samples and compared the turnaround time and cost of the MG-LAMP with microscopy. Compared to expert microscopy, the genus B&S MGLAMP had a sensitivity of 99.4% (95%CI: 96.9%-100%) and specificity of 97.1% (95%CI: 91.9%-99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%-100%) and specificity of 99.2% (95.5%-100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%-100%) and specificity of 99.3% (95%CI: 97.3%-99.8%). The modified genus B&S MG-LAMP assay detected eight submicroscopic malaria cases (1.6%) which the species-specific assays did not identify. The turnaround time of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. The modified B&S MG-LAMP offers a simple and sensitive molecular test of choice for the detection of submicroscopic infections that can be used for mass screening in resources limited facilities in endemic settings nearing elimination and where a deployable test is required.

The reinfection risk in individuals previously infected with SARS-CoV-2 is about a fifth of those never infected.1Hansen CH Michlmayr D Gubbels SM Mølbak K Ethelberg S Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Summary Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar Whether reinfected individuals shed viable virus has been identified as an important question relevant to pandemic control.3Ledford H Coronavirus reinfections: three questions scientists are asking.Nature. 2020; 585: 168-169Crossref PubMed Scopus (44) Google Scholar In a prospective cohort study in The Lancet Respiratory Medicine,2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar we identified 19 cases of reinfection in people who at study entry were seropositive for both SARS-CoV-2 receptor binding domain and full-length spike protein, tested negative on three nasal swab PCR tests over a 2-week quarantine period, and subsequently developed a positive PCR test at least 2 weeks after leaving quarantine.2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar We have now investigated whether these SARS-CoV-2-reinfected individuals shed viable virus. Viral transport media was available from the first and some subsequent PCR-positive tests from 16 (84%) of 19 reinfected participants. Samples were cultured for SARS-CoV-2 in Vero E6 cells expressing TMPRSS24Matsuyama S Nao N Shirato K et al.Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.Proc Natl Acad Sci USA. 2020; 117: 7001-7003Crossref PubMed Scopus (947) Google Scholar in T25 cm2 flasks and monitored for cytopathic effect for 4 days followed by a second passage onto fresh cells to allow additional time for virus amplification. Samples were simultaneously titred by plaque assay in Vero E6/TMPRSS2 cells to quantify the level of detectable infectious virus. Viable virus was detected in only four (25%) of 16 participants, and only once in each, with titres ranging from 1·7 to 5·5 log10 plaque-forming units per mL. Serology and PCR cycle threshold (Ct) values using the US Food and Drug Administration-authorised Thermo Fisher TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA, USA) were compared in those with viable virus detected (shedders) and those without viable virus detected (non-shedders; table; appendix p 2). At the time of positive viral culture, samples from shedders had lower Ct values. Although all participants had detectable SARS-CoV-2 spike IgG at study enrolment, none of the four shedders and only four (33%) of the 12 non-shedders had detectable serum neutralisation activity (limit of detection was 50% inhibitory dilution of 20) in serum at the time of enrolment into the study (methods as described2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Only one (25%) of the four who shed virus was symptomatic when viable virus was detected.TableBaseline serology, clinical features, and SARS-CoV-2 Ct levels in reinfected participants with samples cultured for viable virusVirus culture titreDifference*Difference in mean for the continuous variables and difference in percentage for binary variables.Positive (n=4)Negative (n=12)Baseline IgG S titre (log10)2·8 (0·2)2·9 (0·7)−0·08 (−0·88 to 0·72), p=0·834Baseline ID50 detected (>20)04 (33%)−33% (−87 to 21), p=0·207PCR positive >7 days3 (75%)4 (33%)42% (−20 to 100), p=0·166Symptomatic1 (25%)3 (25%)−0% (−57 to 57), p=1·000N gene Ct16·2 (4·0)31·9 (6·1)−15·64 (−22·68 to −8·60), p=0·0003S gene Ct17·8 (4·1)32·3 (6·2)−14·52 (−21·76 to −7·28), p=0·0007ORF1ab gene Ct16·8 (3·9)31·6 (6·1)−14·83 (−21·88 to −7·78), p=0·0005Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution.* Difference in mean for the continuous variables and difference in percentage for binary variables. Open table in a new tab Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution. All reinfections occurred before December, 2020, when the B.1.1.7 variant was first reported in the USA. Full-length viral genome was recovered from seven (44%) of 16 participants studied by viral transport medium culturing, including all four culture-positive individuals (methods previously described5Letizia AG Ramos I Obla A et al.SARS-CoV-2 transmission among marine recruits during quarantine.N Engl J Med. 2020; 383: 2407-2416Crossref PubMed Scopus (72) Google Scholar). Although the strains isolated all had the Asp614Gly spike protein mutation and two of the isolates had the Leu18Phe spike protein mutation, they did not have mutations associated with the B.1.1.7, P.1, B.1.351, or other variants of concern as currently defined by the US Centers for Disease Control and Prevention (appendix p 3). Limitations of our study are the use of virus culture as a proxy for capacity for transmission without direct study of transmission, the small sample size, the absence of specimens available for culturing from all reinfected participants, and the inability to compare potential for transmissibility between those who are infected for the first time and those who are reinfected. Overall, our findings suggest that about a quarter of young healthy individuals with subsequent SARS-CoV-2 reinfection shed viable virus. Some of these individuals were asymptomatic and could unknowingly transmit SARS-CoV-2 to others. All authors declare no competing interests. This study was supported by the Defense Health Agency and Defense Advanced Research Projects Agency. AGL, DRS, CG, DLW, HWC, RAL, SEL, JM, EN, CKP, ESA, MS, VAS, PS, and MT are military service members or government service employees. This work was prepared as part of their official duties. Title 17, US Code §105 provides that copyright protection under this title is not available for any work of the US Government. Title 17, US code §101 defines a US Government work as a work prepared by a military service member or employee of the US Government as part of that person's official duties. The views expressed in the article are those of the authors and do not necessarily express the official policy and position of the US Navy, the Department of Defense, the US Government, or the institutions affiliated with the authors. Download .pdf (.23 MB) Help with pdf files Supplementary appendix SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort studySeropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies. Full-Text PDF

BackgroundThe efficacy of public health measures to control the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been well studied in young adults.MethodsWe investigated SARS-CoV-2 infections among U.S. Marine Corps recruits who underwent a 2-week quarantine at home followed by a second supervised 2-week quarantine at a closed college campus that involved mask wearing, social distancing, and daily temperature and symptom monitoring. Study volunteers were tested for SARS-CoV-2 by means of quantitative polymerase-chain-reaction (qPCR) assay of nares swab specimens obtained between the time of arrival and the second day of supervised quarantine and on days 7 and 14. Recruits who did not volunteer for the study underwent qPCR testing only on day 14, at the end of the quarantine period. We performed phylogenetic analysis of viral genomes obtained from infected study volunteers to identify clusters and to assess the epidemiologic features of infections.ResultsA total of 1848 recruits volunteered to participate in the study; within 2 days after arrival on campus, 16 (0.9%) tested positive for SARS-CoV-2, 15 of whom were asymptomatic. An additional 35 participants (1.9%) tested positive on day 7 or on day 14. Five of the 51 participants (9.8%) who tested positive at any time had symptoms in the week before a positive qPCR test. Of the recruits who declined to participate in the study, 26 (1.7%) of the 1554 recruits with available qPCR results tested positive on day 14. No SARS-CoV-2 infections were identified through clinical qPCR testing performed as a result of daily symptom monitoring. Analysis of 36 SARS-CoV-2 genomes obtained from 32 participants revealed six transmission clusters among 18 participants. Epidemiologic analysis supported multiple local transmission events, including transmission between roommates and among recruits within the same platoon.ConclusionsAmong Marine Corps recruits, approximately 2% who had previously had negative results for SARS-CoV-2 at the beginning of supervised quarantine, and less than 2% of recruits with unknown previous status, tested positive by day 14. Most recruits who tested positive were asymptomatic, and no infections were detected through daily symptom monitoring. Transmission clusters occurred within platoons. (Funded by the Defense Health Agency and others.)