Prevention of multiple whole blood donations by an individual at the same month through the creation of a national Deferred Donor Registry (DDR).

INTRODUCTION: The Colombian National Institute of Health administers the National Information System of Haemovigilance (SIHEVI-INS). Today, SIHEVI-INS constitutes a national blood donor and recipient database, which contains a national deferred donor registry (DDR), allowing blood banks to take acceptance or rejection decisions of a potential donor in real time. The study aimed to determine the rate of people who have made more than one whole blood donation monthly in Colombia, violating the national guideline of intervals between donations (three months for men and four for women), since DDR implementation. METHODS: We detected the unique personal identification number of people who, in 30 calendar days, made more than one whole blood donation at any of the 83 blood banks set up in Colombia. There were three comparison periods: 01/01/2018-08/31/2019 (launch of SIHEVI-INS and first national feedback); 09/01/2019-12/31/2020 (second feedback) and 01/01/2021-09/30/2022 (massive incorporation of web services). RESULTS: For the first period, blood banks accepted 18.0 donations per 1000 people. There was a rate of 28.8 people/10,000 donations who had donated whole blood twice within 30 days. In the second period, there were 17.0 donations/1000 people and a rate of 2.1 people/10,000 donations (OR:14.0 CI95 %:12.2-16.0). For the last period, there were 18.2 donations/1000 people and a rate of 0.9 individuals/10,000 donations (OR:31.3 CI95 %:26.6-36.9, p < 0.001). CONCLUSION: DDR reduced by 31 times the acceptance of blood donors who made more than one whole blood donation in the same month. It was necessary to provide periodic feedback and promote web service implementation to reduce this risky behavior.

SARS-CoV-2 BA.1 and BA.2 coinfection detected by genomic surveillance in Brazil, January 2022.

In January 2022, our genomic surveillance network identified a SARS-CoV-2 BA.1 and BA.2 coinfection in a sample from a patient residing in Brazil. Our results suggest that the true number of SARS-CoV-2 coinfections remains largely underestimated.

Local Transmission of SARS-CoV-2 Lineage B.1.1.7, Brazil, December 2020.

In December 2020, research surveillance detected the B.1.1.7 lineage of severe acute respiratory syndrome coronavirus 2 in São Paulo, Brazil. Rapid genomic sequencing and phylogenetic analysis revealed 2 distinct introductions of the lineage. One patient reported no international travel. There may be more infections with this lineage in Brazil than reported.

COL1A1, COL4A3, TIMP2 and TGFB1 polymorphisms in cervical insufficiency.

OBJECTIVES: To investigate the association between selected single nucleotide polymorphisms (SNPs) with cervical insufficiency and its relationship with obstetric history. METHODS: Twenty-eight women with cervical insufficiency (case group) and 29 non-pregnant women (control group) were included. The SNPs sequenced included rs2586490 in collagen type I alpha 1 chain (COL1A1), rs1882435 in collagen type IV alpha 3 chain (COL4A3), rs2277698 in metallopeptidase inhibitor 2 (TIMP2), and rs1800468 in transforming growth factor beta 1 (TGFB1). RESULTS: We found a higher frequency of the normal allele in the control group (65.5%) and the homozygous mutated genotype in the case group (64.3%) for rs2586490 in COL1A1 (p=0.023). An unplanned finding in the cervical insufficiency group was a higher gestational age of delivery (median≥38 weeks) in the mutated allele than in the wild-type genotype (median of 28.2 weeks) for rs2857396, which is also in the COL1A1 gene (p=0.011). CONCLUSIONS: The findings of the present study corroborate the hypothesis that cervical insufficiency has a genetic component and probably involves genes encoding proteins in the extracellular matrix, in addition to inflammatory processes.

Sequential measurement of Trypanosoma cruzi parasitic load in endomyocardial biopsies for early detection and follow-up of Chagas disease reactivation after heart transplantation.

BACKGROUND: Reactivation of Chagas disease after heart transplantation is characterized by proliferation and dissemination of Trypanosoma cruzi parasites to several organs. Reactivation affecting the allograft can simulate acute cellular rejection, from which it should be distinguished through the analysis of endomyocardial biopsies (EMB). METHODS: We evaluated retrospectively 100 EMB collected in the first year of follow-up from 13 heart-transplanted, chagasic patients who presented reactivation and were successfully treated. Additionally, 37 EMB from 8 patients who did not present reactivation constituted the control group. We reviewed histopathology and performed a real-time PCR-based assay in order to evaluate the T cruzi parasitic load of each EMB. RESULTS: The parasitic load of the EMB at the time of reactivation ranged from 22.80 to 190 000/106 cells (median: 1555). In 6 patients, none of the EMB obtained prior to reactivation amplified T cruzi DNA. On the other hand, 10 EMB from 7 patients, obtained 9-105 days before reactivation (median: 26 days), showed parasitic load ranging from 8.25 to 625/106 cells (median: 167.55). In all patients, the parasitic load increased at the time of reactivation, usually sharply. After initiation of treatment, all patients showed negative PCR or a dramatic reduction of the parasitic load in the following EMB. None of the EMB from the control group amplified T cruzi DNA. CONCLUSIONS: Sequential measurement of T cruzi parasitic load in EMB is useful for monitoring Chagas disease reactivation after heart transplantation. Its increase suggests imminent reactivation and its decrease after treatment indicates favorable evolution for cure of the episode of reactivation.

SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil.

We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is needed to adequately estimate risk for Zika virus-associated disease and determine patient care.

Geographic heterogeneity in the prevalence of human papillomavirus in head and neck cancer.

Human papillomavirus (HPV) causes oropharyngeal squamous cell carcinoma (OPSCC), although strongly divergent results have been reported regarding the prevalence of HPV16 in different countries, whether this represents important differences in etiology remains unclear. Applying rigorous protocols for sample processing, we centrally evaluated 1,420 head and neck tumors (533 oropharynx, 395 oral cavity and 482 larynx) from studies conducted in the US, Europe and Brazil for mucosal HPV DNA and p16INK4a expression to evaluate regional heterogeneity in the proportion of HPV16-associated OPSCC and other head and neck cancer, and to assess covariates associated with the risk of HPV16-positive OPSCC. While majority of OPSCC in the US (60%) were HPV16-positive, this proportion was 31% in Europe and only 4% in Brazil (p < 0.01). Similar differences were observed for other head and neck tumors, ranging from 7% in the US and 5% in Europe, to 0% in South America. The odds of HPV16-positive OPSCC declined with increasing pack years of smoking (OR: 0.75; 95% CI: 0.64-0.87) and drink years of alcohol use (OR: 0.64; 95% CI: 0.54-0.76). These results suggest that while the contribution of HPV16 is substantial for the oropharynx, it remains limited for oral cavity and laryngeal cancers.

RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients.

BACKGROUND: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants. AIMS: To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support. METHODS: Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared. RESULTS: Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion. CONCLUSION: The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.

Cross reactivity of commercial anti-dengue immunoassays in patients with acute Zika virus infection.

Several countries have local transmission of multiple arboviruses, in particular, dengue and Zika viruses, which have recently spread through many American countries. Cross reactivity among Flaviviruses is high and present a challenge for accurate identification of the infecting agent. Thus, we evaluated the level of cross reactivity of anti-dengue IgM/G Enzyme-Linked Immunosorbent Assays (ELISA) from three manufacturers against 122 serum samples obtained at two time-points from 61 patients with non-dengue confirmed Zika virus infection. All anti-dengue ELISAs cross reacted with serum from patients with acute Zika infection at some level and a worrisome number of seroconversion for dengue IgG and IgM was observed. These findings may impact the interpretation of currently standard criteria for dengue diagnosis in endemic regions.

Determination of Fetal RHD Genotype Including the RHD Pseudogene in Maternal Plasma.

OBJECTIVE: To examine the accuracy of fetal RHD genotype and RHD pseudogene determination in a multiethnical population. METHODS: Prospective study involving D-negative pregnant women. Cell-free DNA was extracted from 1 ml of maternal plasma by an automated system (MagNA Pure Compact, Roche) and real-time PCR was performed in triplicate targeting the RHD gene exons 5 and 7. Inconclusive samples underwent RHD pseudogene testing by real-time PCR analysis employing novel primers and probe. RESULTS: A positive result was observed in 128/185 (69.2%) samples and negative in 50 (27.0%). Umbilical cord blood phenotype confirmed all cases with a positive or negative PCR result. Seven (3.8%) cases were found inconclusive (exon 7 amplification only) and RHD pseudogene testing with both conventional and real-time PCR demonstrated a positive result in five of them, while two samples were also RHD pseudogene negative. CONCLUSION: Real-time PCR targeting RHD exons 5 and 7 simultaneously in maternal plasma is an accurate method for the diagnosis of fetal D genotype in our population. The RHD pseudogene real-time PCR assay is feasible and is particularly useful in populations with a high prevalence of this allele.

HPV genotype distribution in Brazilian women with and without cervical lesions: correlation to cytological data.

BACKGROUND: Human Papillomavirus (HPV) genotype distribution varies according to the method of assessment and population groups. This study analyzed type-specific HPV infections among women ranging from 14-95 years old, displaying normal and abnormal cytology, from São Paulo and Barretos cities, Brazil. METHODS: Women found positive for High Risk-HPVs DNA by either the Hybrid Capture 2 (HC2) or Cobas HPV Test (n = 431) plus a random sample of 223 negative by both assays and 11 samples with indeterminate results, totalizing 665 samples, were submitted to HPV detection by the PapilloCheck test. Cytological distribution included 499 women with a cytological result of Negative for Intraepithelial Lesion or Malignancy and 166 with some abnormality as follows: 54 Atypical Squamous Cells of Undetermined Significance; 66 Low-Grade Squamous Intraepithelial Lesion; 43 High-Grade Squamous Intraepithelial Lesion and 3 (0.5 %) Invasive Cervical Cancer. RESULTS: From the 323 samples (48.6 %) that had detectable HPV-DNA by the PapilloCheck assay, 31 were HPV negative by the cobas HPV and HC2 assays. Out of these 31 samples, 14 were associated with HR-HPVs types while the remaining 17 harbored exclusively low-risk HPVs. In contrast, 49 samples positive by cobas HPV and HC 2 methods tested negative by the PapilloCheck assay (19.8 %). Overall, the most frequent HR-HPV type was HPV 16 (23.2 %), followed by 56 (21.0 %), 52 (8.7 %) and 31 (7.7 %) and the most frequent LR-HPV type was HPV 42 (12.1 %) followed by 6 (6.2 %). Among the HR-HPV types, HPV 56 and 16 were the most frequent types in NILM, found in 19.1 and 17.7 % of the patients respectively while in HSIL and ICC cases, HPV 16 was the predominant type, detected in 37.2 and 66.7 % of these samples. CONCLUSIONS: In the population studied, HPV 16 and 56 were the most frequently detected HR-HPV types. HPV 56 was found mainly in LSIL and NILM suggesting a low oncogenic potential. HPV 16 continues to be the most prevalent type in high-grade lesions whereas HPV 18 was found in a low frequency both in NILM and abnormal smears. Surveillance of HPV infections by molecular methods is an important tool for the development and improvement of prevention strategies.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA.

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Clinical characteristics of women diagnosed with carcinoma who tested positive for cervical and anal high-risk human papillomavirus DNA and E6 RNA.

High-risk human papillomavirus (hrHPV) is an essential cause of cervical carcinoma and is also strongly related to anal cancer development. The hrHPV E6 oncoprotein plays a major role in carcinogenesis. We aimed to evaluate the frequency of hrHPV DNA and E6 oncoprotein in the anuses of women with cervical carcinoma. We analyzed 117 women with cervical cancer and 103 controls for hrHPV and the E6 oncogene. Positive test results for a cervical carcinoma included 66.7 % with hrHPV-16 and 7.7 % with hrHPV-18. One case tested positive for both HPV variants (0.9 %). The samples from the anal canal were positive for HPV-16 in 59.8 % of the cases. Simultaneous presence of HPV in the cervix and anal canal was found in 53.8 % of the cases. Regarding expression of E6 RNA, positivity for HPV-16 in the anal canal was found in 21.2 % of the cases, positivity for HPV-16 in the cervix was found in 75.0 %, and positivity for HPV-18 in the cervix was found in 1.9 %. E6 expression in both the cervix and anal canal was found in 19.2 % of the cases. In the controls, 1 % tested positive for HPV-16 and 0 % for HPV-18. Anal samples from the controls showed a hrHPV frequency of 4.9 % (only HPV16). The presence of hrHPV in the anal canal of women with cervical cancer was detected at a high frequency. We also detected E6 RNA expression in the anal canal of women with cervical cancer, suggesting that these women are at risk for anal hrHPV infection.

A cross-sectional study of high-risk human papillomavirus clustering and cervical outcomes in HIV-infected women in Rio de Janeiro, Brazil.

BACKGROUND: In Brazil, the rate of cervical cancer remains high despite the availability of screening programs. With ongoing vaccine development and implementation, information on the prevalence of specific HPV types is needed, particularly among high-risk populations, such as HIV-infected women. METHODS: We performed a study of HIV-infected women in Rio de Janeiro, Brazil, who underwent cervical HPV genotype testing between 2005-2013. We examined the prevalence of high-risk HPV types and the patterns of high-risk HPV type clustering. Using logarithmic binomial regression, we estimated the risk of abnormal cytology by HPV genotype result. RESULTS: Of the 562 women included, 498 (89 %) had at least one HPV type detected. 364 women (65 %) had at least one high-risk HPV type detected and 181 (32 %) had more than one high-risk type detected. HPV 58 was the most frequent HPV type detected overall (prevalence 19.8 % [95 % confidence interval 16.4-23.1]), followed by HPV 53 (prevalence 15.5 % [12.5-18.5]) and HPV 16 (prevalence 13 % [10.2-15.8]). Women infected with more than one high-risk HPV type were younger, had lower CD4+ lymphocyte counts, and were more likely to be infected with HPV 16 or 18. In adjusted analyses, presence of more than one high-risk HPV type was associated with a two-fold increased risk of abnormal cytology after adjusting for presence of individual high-risk type, age, and CD4+ lymphocyte count (adjusted prevalence ratios 1.88-2.07, all p <0.001). No single high-risk HPV type was statistically associated with abnormal cytology after adjusting for the presence of more than one high-risk HPV type. CONCLUSIONS: In the largest study of cervical HPV genotypes among HIV-infected women in Latin America, infection by high-risk HPV types other than 16 or 18 and infection by more than one high-risk HPV types were common. Infection by more than one high-risk type was more strongly associated with abnormal cervical cytology than any individual high-risk HPV type, highlighting the need for multi-valent HPV vaccines.

Real-time symptomatic case of transfusion-transmitted dengue.

BACKGROUND: Dengue virus transmission by blood transfusion is a rarely reported event. CASE REPORT: During a dengue outbreak in São Paulo city, a regular plateletpheresis donor informed the blood bank of being diagnosed a few days after donation. The recipient was hospitalized and displayed symptoms and laboratory evidence of dengue after transfusion. RESULTS: The donor was immunoglobulin (Ig)G, IgM, and polymerase chain reaction nonreactive on the index sample, seroconverting 20 days later. The platelet units were transfused into two patients. One of them developed fever 3 days after transfusion, with high viral load. His pretransfusion sample was negative for IgG, IgM, and dengue RNA, while the second recipient did not show any symptoms nor laboratory evidence of dengue infection. CONCLUSIONS: This case brings additional evidence that dengue is indeed transmissible by blood transfusion and clinical manifestations, although rare, do occur.

Human papillomavirus (HPV) 16 and the prognosis of head and neck cancer in a geographical region with a low prevalence of HPV infection.

BACKGROUND: The role of human papillomavirus (HPV) on head and neck squamous cell carcinoma (HNSCC) survival in regions with low HPV prevalence is not yet clear. We evaluated the HPV16 infection on survival of HNSCC Brazilian patient series. METHODS: This cohort comprised 1,093 HNSCC cases recruited from 1998 to 2008 in four Brazilian cities and followed up until June 2009. HPV16 antibodies were analyzed by multiplex Luminex assay. In a subset of 398 fresh frozen or paraffin blocks of HNSCC specimens, we analyzed for HPV16 DNA by L1 generic primer polymerase chain reaction. HNSCC survival according to HPV16 antibodies was evaluated through Kaplan-Meier method and Cox regression. RESULTS: Prevalence of HPV16 E6 and E6/E7 antibodies was higher in oropharyngeal cancer than in other head and neck tumor sites. HPV16 DNA positive in tumor tissue was also higher in the oropharynx. Seropositivity for HPV16 E6 antibodies was correlated with improved HNSCC survival and oropharyngeal cancer. The presence of HPV16 E6/E7 antibodies was correlated with improved HNSCC survival and oropharyngeal cancer survival. The death risk of oropharyngeal squamous cell carcinoma patients HPV16 E6/E7 antibodies positive was 78 % lower than to those who test negative. CONCLUSION: Oropharyngeal squamous cell carcinoma is less aggressive in the HPV16 E6/E7 positive serology patients. HPV16 E6/E7 antibody is a clinically sensible surrogate prognostic marker of oropharyngeal squamous cell carcinoma.

Unexpected detection of Plasmodium vivax and Plasmodium falciparum DNA in asymptomatic blood donors: fact or artifact?

A study searching for Plasmodium vivax and Plasmodium falciparum DNA among blood donors from the non-endemic area in Brazil reported a rate of 7.41%. This number is at least three times higher than what has been observed in blood donors from the Amazon, an endemic area concentrating >99% of all malaria cases in Brazil. Moreover, the majority of the donors were supposedly infected by P. falciparum, a rare finding both in men and anophelines from the Atlantic forest. These findings shall be taken with caution since they disagree with several publications in the literature and possibly overestimate the actual risk of malaria transmission by blood transfusion in São Paulo city.

Toxoplasmosis outbreak in São Paulo, Brazil: Epidemiology and visual outcome.

PURPOSE: To describe a 2019 acute toxoplasmosis outbreak in the city of São Paulo, Brazil, and to evaluate the laboratory serological profile for toxoplasmosis for three consecutive years. The ophthalmological manifestations of the patients involved in the outbreak were also studied. METHODS: A cross-sectional descriptive study of a toxoplasmosis outbreak in São Paulo, Brazil, between February and May 2019. Epidemiological data were described, as were the observed ocular manifestations. As part of this study the number of patients with positive IgM toxoplasmosis serology was obtained from a large laboratory network (DASA) for three consecutive years, including the year of the outbreak (2018, 2019, 2020). RESULTS: Eighty-three individuals were identified in the outbreak and two clusters were studied. The clinical picture of at least 77% of the patients, the epidemiological analysis, and the short incubation period (5-8 days) suggested contamination by oocysts. Serological laboratory data analysis revealed an increase of positive toxoplasmosis IgM in 2019 of 73% compared to the previous year. Ophthalmological examination revealed that at least 4.8% of the patients developed toxoplasmic retinochoroiditis, none of whom had been treated during the acute systemic disease. CONCLUSION: Our findings indicate vegetable contamination as the possible source of this outbreak, a high prevalence of toxoplasmosis in São Paulo during the outbreak period, and a drop in the number of tests during the COVID-19 pandemic. Retinochoroiditis was observed in at least 4.8% of the cases. We confirm the need to implement effective means for the prevention, diagnosis, and treatment of the disease. This may involve raising awareness among the population of the importance of vegetable hygiene, and improved quality control of food and water.