Reinfection by the SARS-CoV-2 Gamma variant in blood donors in Manaus, Brazil.

BACKGROUND: The city of Manaus, north Brazil, was stricken by a second epidemic wave of SARS-CoV-2 despite high seroprevalence estimates, coinciding with the emergence of the Gamma (P.1) variant. Reinfections were postulated as a partial explanation for the second surge. However, accurate calculation of reinfection rates is difficult when stringent criteria as two time-separated RT-PCR tests and/or genome sequencing are required. To estimate the proportion of reinfections caused by Gamma during the second wave in Manaus and the protection conferred by previous infection, we identified anti-SARS-CoV-2 antibody boosting in repeat blood donors as a mean to infer reinfection. METHODS: We tested serial blood samples from unvaccinated repeat blood donors in Manaus for the presence of anti-SARS-CoV-2 IgG antibodies using two assays that display waning in early convalescence, enabling the detection of reinfection-induced boosting. Donors were required to have three or more donations, being at least one during each epidemic wave. We propose a strict serological definition of reinfection (reactivity boosting following waning like a V-shaped curve in both assays or three spaced boostings), probable (two separate boosting events) and possible (reinfection detected by only one assay) reinfections. The serial samples were used to divide donors into six groups defined based on the inferred sequence of infection and reinfection with non-Gamma and Gamma variants. RESULTS: From 3655 repeat blood donors, 238 met all inclusion criteria, and 223 had enough residual sample volume to perform both serological assays. We found 13.6% (95% CI 7.0-24.5%) of all presumed Gamma infections that were observed in 2021 were reinfections. If we also include cases of probable or possible reinfections, these percentages increase respectively to 22.7% (95% CI 14.3-34.2%) and 39.3% (95% CI 29.5-50.0%). Previous infection conferred a protection against reinfection of 85.3% (95% CI 71.3-92.7%), decreasing to respectively 72.5% (95% CI 54.7-83.6%) and 39.5% (95% CI 14.1-57.8%) if probable and possible reinfections are included. CONCLUSIONS: Reinfection by Gamma is common and may play a significant role in epidemics where Gamma is prevalent, highlighting the continued threat variants of concern pose even to settings previously hit by substantial epidemics.

Correction to: Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

An amendment to this paper has been published and can be accessed via the original article.

Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

BACKGROUND: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy. METHODS: In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite. RESULTS: Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM. CONCLUSION: The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Detection of Mansonella ozzardi among blood donors from highly endemic interior cities of Amazonas state, northern Brazil.

BACKGROUND: In the Brazilian Amazon, the filarial nematode Mansonella ozzardi co-exists with malaria parasites and thick blood smear microscopy is considered the diagnostic gold standard. Transfusion of M. ozzardi microfilariae does not establish new infections, however microfilariae can survive approximately 2 years in blood-recipients with unknown risk of pathology. Data on transfusion-transmitted filariasis are lacking. This study investigated M. ozzardi parasitemias in blood donors from decentralized centers of "Fundação Hematologia e Hemoterapia do Estado do Amazonas/HEMOAM," Northern Brazil. STUDY DESIGN AND METHODS: Cross-sectional investigation employing blood smear microscopy (n = 356) and qualitative nested-M. ozzardi-PCR (227 out of 356) in donor candidates from 19 hemocenters in interior/rural municipalities of Amazonas state. FINDINGS: Participants were mostly young males. Positivity by microscopy was 7.9% (28 out of 356) and 23.8% by M. ozzardi-PCR (54 out of 227). Parasitaemias were found in 16 out of 19 municipalities. In 54 M. ozzardi-positives, 24 were ineligible; among 30 that donated, 27 were interdicted by seropositivity (22 anti-HBc, 3 anti-HBc + HBsAg, 1 Chagas+malaria, 1 VDRL). Seropositivty was higher in M. ozzardi-PCR-positives vs M. ozzardi-PCR-negatives (OR = 15.8, 95% CI 4.5-56.1, p < 0.0001). Three M. ozzardi contaminated blood units were transfused, but no follow-up information on the recipients is available. MAIN CONCLUSIONS: Our study provides important baseline data on M. ozzardi among blood donors from the Brazilian Amazon. Further investigations in endemic areas are necessary to clarify possible association between M. ozzardi and other infections and also to elucidate whether there is any significant clinical effect upon transfusion of contaminated blood.

High rate of seromarkers for HIV, HBV and syphilis among blood donors using confidential unit exclusion, before and after HIV-NAT implementation at a major public blood bank in the Brazilian Amazon.

BACKGROUND: Confidential unit exclusion (CUE) was introduced in the 1980's as an additional layer to blood safety, before highly specific and sensitive nucleic acid tests (NAT) for HIV were implemented. The utility of CUE-use in settings that have implemented NAT should be evaluated over time. STUDY DESIGN, METHODS: Cross-sectional retrospective study carried out from June 2010-November 2015, at Manaus Hemocenter (HEMOAM), Amazonas, Brazil that implemented HIV-NAT in 2012. The HIV, HCV, HBV, HTLV, Chagas disease, and syphilis rates were compared among CUE and non-CUE blood donors, before and after HIV-NAT implementation. RESULTS: Among 287,588 donations, 2,154 (0.75%) were associated with CUE, mainly voluntary donations (64.2%), by repeat donors (58.4%) from young (median age = 31 years), males (84.4%), unmarried (63.1%). CUE-users compared to non-CUE donors (n = 285,434) had higher seropositivity rates to HIV (OR = 6.09, 95% CI: 3.68-10.07, p < 0.001), HBV (anti-HBc OR = 1.81 95% CI: 1.24-2.64, p = 0.004; HBsAg OR = 5.68, 95% CI: 1.78-18.07, p = 0.017), and syphilis (OR = 1.78, 95% CI: 1.05-3.04, p = 0.030). Most (97.2%) discarded blood units associated to CUE was seronegative for all pathogens. Most donations (73.4%) were tested by HIV-NAT and showed four window period donations, positive by HIV-NAT only among non-CUE donors. CONCLUSION: A high rate of transfusion transmissible infections/TTIs was observed at HEMOAM especially in CUE-users. CUE-use offered an additional layer of blood safety by its association with anti-HBc/HBsAg and syphilis that are not covered by NAT. For blood banks in highly endemic areas for HIV and TTI, as HEMOAM, the identification of at risk donors, and the orientation to be tested at proper sites remain a great challenge.

Performance of immunological assays for universal and differential diagnosis of HTLV-1/2 infection in candidates for blood donations from the Brazilian Amazon.

The present study compares the ability of distinct immunological assays (chemiluminescence immunoassay-CLIA, western blot-WB and flow cytometry-FC-Simplex and Duplex) to detect anti-HTLV (human T-lymphotropic virus) antibodies in candidates for blood donations at the Amazonas State Blood Center (Brazil) between January 2018 and December 2022. Overall, 257,942 samples from candidates for blood donations were screened using CLIA, which led to 0.15% seropositivity for HTLV (409 samples). A total of 151 candidates for blood donations were enrolled for retesting with CLIA followed by additional testing using WB and FC-Simplex and Duplex analysis. Our results demonstrated that 62% (93/151), 20% (30/151) and 17% (26/151) of the samples presented positive results with retesting using CLIA, WB and FC-Simplex analysis, respectively. Additional analysis of the CLIA, WB and FC-Simplex results revealed an overall agreement of 56% for CLIA and WB (22 co-negative; 30 co-positive samples), 48% for CLIA and FC-Simplex (21 co-negative; 24 co-positive samples) and 80% for WB and FC-Simplex (51 co-negative; 23 co-positive samples). Considering the WB as the reference standard for the diagnosis of infection with HTLV-1/2, we observed that the CLIA results of ≤3.0 RLU and >10.0 RLU in the retest can be used define a negative or positive result, respectively, and could be used as new specific cut-off values. The overall agreement between WB and FC-Duplex for accomplishing the differential diagnosis was evaluated and demonstrated 100% correspondence for the diagnosis of HTLV-1 (15/15) and HTLV-2 (7/7). Our findings demonstrate that gaps in the diagnosis of infection with HTLV-1/2 could be overcome by the simultaneous use of distinct immunological assays during retesting of candidates for blood donations.

Three-quarters attack rate of SARS-CoV-2 in the Brazilian Amazon during a largely unmitigated epidemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly in Manaus, the capital of Amazonas state in northern Brazil. The attack rate there is an estimate of the final size of the largely unmitigated epidemic that occurred in Manaus. We use a convenience sample of blood donors to show that by June 2020, 1 month after the epidemic peak in Manaus, 44% of the population had detectable immunoglobulin G (IgG) antibodies. Correcting for cases without a detectable antibody response and for antibody waning, we estimate a 66% attack rate in June, rising to 76% in October. This is higher than in São Paulo, in southeastern Brazil, where the estimated attack rate in October was 29%. These results confirm that when poorly controlled, COVID-19 can infect a large proportion of the population, causing high mortality.

Homogenous HIV-1 subtype B from the Brazilian Amazon with infrequent diverse BF1 recombinants, subtypes F1 and C among blood donors.

In the last decade a growing HIV/AIDS epidemic with increased incidence and AIDS-related mortality has been reported in Northern Brazil from which molecular data are scarce. Also, apparently healthy, adult blood donors, recently diagnosed with HIV-1 represent important sentinel populations for molecular studies. This cross-sectional study describes HIV-1 subtypes in blood donors from three reference public blood centers located in three States in Northern Brazil. HIV-1 pol sequencing (protease/PR, reverse transcriptase/RT) was performed on plasma samples of HIV-1 positive donors from HEMOAM, Manaus, Amazonas (n = 198), HEMERON, Porto Velho, Rondônia (n = 20) and HEMORAIMA, Boa Vista, Roraima (n = 9) collected from 2011-2017. HIV-1 subtypes were identified by REGA, phylogenetic inference; recombinant viruses were characterized by SIMPLOT. Young, single, males predominated, around half was first-time donors. Syphilis co-infection was detected in 17% (39 out of 227), 8% (18 out of 227) was anti-HBc positive. Subtype B represented ≥ 90% in Amazonas, Rondônia and Roraima, subtype C (3.1%) was found in Amazonas and Rondônia; subtype F1 (0.9%) and BF1 recombinants (5.3%) were only detected in Amazonas. Subtype B sequences from Amazonas (n = 179), Rondônia (n = 18) and Roraima (n = 9) were combined with viral strains representative of the BPANDEMIC (n = 300) and BCARIBBEAN/BCAR (n = 200) lineages. The BPANDEMIC lineage predominated (78%) although BCAR lineages were frequent in Roraima (56%) and Amazonas (22%). Subtype C and subtype F1 sequences identified here clustered within Brazilian CBR and F1BR lineages, respectively. Twelve BF1 mosaics showed 11 different recombination profiles: six were singleton unique-recombinant-forms/URFs, one displays a CRF28/29_BF-like recombinant pattern and the remaining four BF1 isolates branched with other Brazilian BF1 viruses previously described and may represent putative new CRF_BF1 from Northern Brazil. Our study shows a highly homogeneous molecular pattern with prevalent subtype B, followed by BF1, and sporadic subtype C and F1 in blood donors from the Northern region. Surveillance studies are important to monitor HIV-1 diversity which can reveal patterns of viral dissemination, especially in a highly endemic, remote and geographically isolated region as Northern Brazil.

Atypical Mansonella ozzardi Microfilariae from an Endemic Area of Brazilian Amazonia.

Mansonellosis is endemic in several regions of Africa, the Caribbean, and Latin America. Mansonella ozzardi and Mansonella perstans have been reported in Latin America, including the Amazon region. A morphological and molecular microfilariae study was performed in Pauini (Brazil). Blood samples were collected from 40 individuals, and were analyzed by Giemsa-stained blood film and by two different nested polymerase chain reactions which detect internal transcribed spacer-1 and the major sperm protein gene. By microscopy, 14 of 40 were positive: 11 as M. ozzardi and three as M. perstans-like infections. Both molecular methods detected 19 positive cases as M. ozzardi, including those 14 individuals detected by microscopy, without detectable genetic differences among any of the 19 positive samples. Molecular techniques showed an improvement of mansonellosis diagnosis and may become an effective tool to evaluate the present status of M. ozzardi and M. perstans in Latin America.