Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load.

The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ±â€¯1 °C for satellite-DNA and 78.1 °C ±â€¯1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10-3 parasite or 240 target copies, and for kDNA, 2 × 10-4 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.

Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic marker of active pulmonary tuberculosis.

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome.

Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil.

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

Single-round multiplex PCR with species-specific mitochondrial primers of P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum: Comparison with standard techniques.

A single-round multiplex PCR (mPCR) with species-specific primers (SSP) of three mitochondrial genes of Plasmodium, namely COX I, COX III and CYT B, was compared to microscopy and 18S rRNA semi-nested PCR, nested-PCR and Real Time PCRs (*PCRs). Each parasite has between 20 and 150 mitochondria and each mitochondria has one copy of each target gene, while 18S rRNA gene is repeated 4 to 8 times. The specificity of mPCR was assessed by testing Plasmodium from rodents and birds, parasites responsible for other endemic diseases in the country such as schistosomiasis, Chagas disease and leishmaniasis in addition to microorganisms that, like Plasmodium, can cause anemia (Bartonella henselae, Babesia vogeli, Rickettsia vini). No cross-reactions were detected. From a total of 149 specimens from suspected cases of malaria were tested, 97 were positive by microscopy (49 P. falciparum, 38 P. vivax, 6 P. malariae, 4 P. falciparum/P. vivax- mixed infections) and 52 were negative; 148 samples were positive by *PCRs (49 P. falciparum, 53 P. vivax, 7 P. malariae and 39 mixed infections) and one was negative; 146 were positive by mPCR (49 P. falciparum, 56 P. vivax, 9 P. malariae and 32 mixed infections) and three were negative. The comparison of groups found statistically significant differences between microscopy vs.*PCRs or vs. mPCR (p-values <0.0001), but no difference was found between mPCR vs. *PCRs (p=0.946). The agreement in the identification of Plasmodium species was only regular, with Kappa indices of 0.407 (microscopy vs. *PCRs), 0.433 (microscopy vs. mPCR) and 0.558 (*PCRs vs. mPCR). In conclusion, the diagnostic performance of mPCR was comparable to those of *PCRs, and superior to microscopy, although the identification of Plasmodium species showed many disagreements. In conclusion, a sensitive and specific one-round SSP multiplex PCR, capable of simultaneously detecting and identifying P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum may be useful in resource-constrained countries where quantitative amplifications are not yet fully accessible.

Silent circulation of Chikungunya virus among pregnant women and newborns in the Western Brazilian Amazon before the first outbreak of chikungunya fever.

The prevalence of immunity to Chikungunya virus (CHIKV) in pregnant women and newborns in the Western Brazilian Amazon was assessed at a time when previous studies did not report chikungunya fever in the area. In 435 asymptomatic pregnant women and 642 healthy unrelated newborns, the presence of IgM and IgG antibodies to CHIKV were determined by a commercial ELISA. All participants were negative to IgM anti-CHIKV. Anti-CHIKV IgG was identified in 41 (9.4%) pregnant women and 66 (10.3%) newborns. The presence of anti-CHIKV IgG was positively associated with the lowest socioeconomic status in pregnant women (OR 2.54, 95% CI 1.15-5.62, p=0.021) and in the newborns' mothers (OR 5.10, 95% CI 2.15-12.09, p< 0.001). Anti-CHIKV IgG was also associated with maternal age in both, the pregnant women (OR 1.06, 95% CI 1.00-1.11, p=0.037) and the newborns'mothers (OR 1.08, 95% CI 1.03-1.12, p=0.001). Pregnancy outcomes in which the mother or the newborn was anti-CHIKV IgG positive proceeded normally. Negative CHIKV serology was associated with being positive for DENV antibodies and having had malaria during pregnancy. These findings showed that there was already a silent circulation of CHIKV in this Amazon region before the first outbreak of chikungunya fever. Furthermore, seropositivity for CHIKV was surprisingly frequent (10%) in both, pregnant women and newborns, affecting mainly low-income women.

SARS-CoV-2 and rhinovirus infections: are there differences in clinical presentation, laboratory abnormalities, and outcomes in the pediatric population?

This study aims to assess COVID-19 and other respiratory viruses in pediatric patients. Between April 17 and September 30, 2020, we collected 1,566 respiratory samples from 1,044 symptomatic patients who were younger than 18 years old to assess SARS-CoV-2 infection. Of these, 919 were analyzed for other respiratory pathogens (ORP). Patients with laboratory-confirmed COVID-19 or ORP were included. We evaluated 76 pediatric COVID-19 infections and 157 other respiratory virus infections. Rhinovirus occurred in 132/157 (84%). COVID-19 patients who were significantly older, had more fevers, headaches and pneumonia than those with ORP. The median white blood cell count was lower in patients with SARS-CoV-2 than in those with ORP (6,470 versus 8,170; p=0.02). COVID-19 patients had significantly worse symptoms than those with ORP.

Technical performance of a lateral flow immunoassay for detection of anti-SARS-CoV-2 IgG in the outpatient follow-up of non-severe cases and at different times after vaccination: comparison with enzyme and chemiluminescent immunoassays.

This study assessed the technical performance of a rapid lateral flow immunochromatographic assay (LFIA) for the detection of anti-SARS-CoV-2 IgG and compared LFIA results with chemiluminescent immunoassay (CLIA) results and an in-house enzyme immunoassay (EIA). To this end, a total of 216 whole blood or serum samples from three groups were analyzed: the first group was composed of 68 true negative cases corresponding to blood bank donors, healthy young volunteers, and eight pediatric patients diagnosed with other coronavirus infections. The serum samples from these participants were obtained and stored in a pre-COVID-19 period, thus they were not expected to have COVID-19. In the second group of true positive cases, we chose to replace natural cases of COVID-19 by 96 participants who were expected to have produced anti-SARS-CoV-2 IgG antibodies 30-60 days after the vaccine booster dose. The serum samples were collected on the same day that LFIA were tested either by EIA or CLIA. The third study group was composed of 52 participants (12 adults and 40 children) who did or did not have anti-SARS-CoV-2 IgG antibodies due to specific clinical scenarios. The 12 adults had been vaccinated more than seven months before LFIA testing, and the 40 children had non-severe COVID-19 diagnosed using RT-PCR during the acute phase of infection. They were referred for outpatient follow-up and during this period the serum samples were collected and tested by CLIA and LFIA. All tests were performed by the same healthcare operator and there was no variation of LFIA results when tests were performed on finger prick whole blood or serum samples, so that results were grouped for analysis. LFIA's sensitivity in detecting anti-SARS-CoV-2 IgG antibodies was 90%, specificity 97.6%, efficiency 93%, PPV 98.3%, NPV 86.6%, and likelihood ratio for a positive or a negative result were 37.5 and 0.01 respectively. There was a good agreement (Kappa index of 0.677) between LFIA results and serological (EIA or CLIA) results. In conclusion, LFIA analyzed in this study showed a good technical performance and agreement with reference serological assays (EIA or CLIA), therefore it can be recommended for use in the outpatient follow-up of non-severe cases of COVID-19 and to assess anti-SARS-CoV-2 IgG antibody production induced by vaccination and the antibodies decrease over time. However, LFIAs should be confirmed by using reference serological assays whenever possible.

A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area.

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50 ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.

Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?

OBJECTIVES: We evaluated the performance of a nucleoprotein-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to SARS-CoV-2. METHODS: The ELISA was based on serum IgG reactivity to a 46-kDa protein derived from the recombinant SARS-CoV2 nucleoprotein. Assay sensitivity was assessed using serum samples from 134 COVID-19 confirmed cases obtained > 15 days after symptom onset. Specificity was determined by testing sera from 94 healthy controls. Cross-reactivity was evaluated with sera from 96 individuals with previous dengue or zika virus-confirmed infections, with 44 sera from individuals with confirmed infections to other respiratory viruses or with bacterial and fungal infections that cause pneumonia and with 40 sera negative for SARS-CoV-2 nucleoprotein by commercial ELISA kits. RESULTS: The majority of subjects were male and ≥ 60 years old. Assay sensitivity was 90.3 % (95 % confidence interval 84.1 %-94.2 %) and specificity was 97.9 % (92.6 %-99.4 %). There was no cross-reactivity with sera from individuals diagnosed with dengue, zika virus, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus, seasonal coronavirus, Mycobacterium tuberculosis, Staphylococcus (S. aureus and coagulase-negative), Streptococcus pneumoniae, Klebsiella pneumoniae and the fungus Aspergillus fumigatus. The level of concordance of our test with results from commercial ELISA kits was 100 %. CONCLUSION: The nucleoprotein-based ELISA was specific for detection of IgG anti-nucleoprotein antibodies to SARS-CoV-2. It utilizes a frequently employed low expense assay protocol and is easier to perform than other currently available commercial SARS-CoV2 antibody detection tests.

SARS-CoV-2 infections with emphasis on pediatric patients: a narrative review.

This narrative review summarizes the main aspects underlying the new coronavirus SARS-CoV-2, its epidemiology, pathophysiology, pointing to differences of SARS-CoV-2 main receptors ACE2, in terms of expression and the amount of soluble ACE2 in the circulation of children, men and women, and also in those with risk factors such as the smokers and pregnant women or presenting with comorbidities (diabetes, obesity, hypertension and other cardiovascular diseases, renal and CNS pre-existing diseases). Clinical manifestations in adults and children were also described, emphasizing the particularities already seen in children, regarding signs, symptoms, viral excretion time and the involvement of all organs and systems. The COVID-19 in the pediatric population was divided into two sections: one dedicated to previously healthy children and adolescents with COVID-19, and the other to those who live with comorbidities and acquired COVID-19. A few paragraphs were reserved to the recently described severe multisystemic inflammatory syndrome associated with COVID-19 (MIS-C) that shares certain characteristics with Kawasaki disease. Some studies on the infection in pregnant and postpartum women, as well as neonates were shown. This review has also covered the laboratory diagnosis of COVID-19, passing through the imaging diagnosis made by the chest tomography revealing ground glass patching opacities, and results of non-specific exams such as the total blood with lymphopenia, the coagulation tests with increased prothrombin times, as well as marked increments of the D-dimer, troponin and proinflammatory cytokines. In the section devoted to the specific laboratory diagnosis of COVID-19, the most used RT-PCR protocols were described and some studies on the serological diagnosis with IgA, IgM and IgG detection were detailed, including the use of rapid immunochromatographic assays and discussing the ideal period after the onset of symptoms to perform each type of test. In the end, the management of pediatric patients with COVID-19 based mainly on supportive measures has been briefly commented.

A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene.

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/µL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.

Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections.

CONTEXT.­: Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. OBJECTIVE.­: To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. DESIGN.­: Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. RESULTS.­: Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. CONCLUSIONS.­: The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.

Severe clinical spectrum with high mortality in pediatric patients with COVID-19 and multisystem inflammatory syndrome.

OBJECTIVES: To assess the outcomes of pediatric patients with laboratory-confirmed coronavirus disease (COVID-19) with or without multisystem inflammatory syndrome in children (MIS-C). METHODS: This cross-sectional study included 471 samples collected from 371 patients (age<18 years) suspected of having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The study group comprised 66/371 (18%) laboratory-confirmed pediatric COVID-19 patients: 61 (92.5%) patients tested positive on real-time reverse transcription-polymerase chain reaction tests for SARS-CoV-2, and 5 (7.5%) patients tested positive on serological tests. MIS-C was diagnosed according to the criteria of the Center for Disease Control. RESULTS: MIS-C was diagnosed in 6/66 (9%) patients. The frequencies of diarrhea, vomiting, and/or abdominal pain (67% vs. 22%, p=0.034); pediatric SARS (67% vs. 13%, p=0.008); hypoxemia (83% vs. 23%, p=0.006); and arterial hypotension (50% vs. 3%, p=0.004) were significantly higher in patients with MIS-C than in those without MIS-C. The frequencies of C-reactive protein levels >50 mg/L (83% vs. 25%, p=0.008) and D-dimer levels >1000 ng/mL (100% vs. 40%, p=0.007) and the median D-dimer, troponin T, and ferritin levels (p<0.05) were significantly higher in patients with MIS-C. The frequencies of pediatric intensive care unit admission (100% vs. 60%, p=0.003), mechanical ventilation (83% vs. 7%, p<0.001), vasoactive agent use (83% vs. 3%, p<0.001), shock (83% vs. 5%, p<0.001), cardiac abnormalities (100% vs. 2%, p<0.001), and death (67% vs. 3%, p<0.001) were also significantly higher in patients with MIS-C. Similarly, the frequencies of oxygen therapy (100% vs. 33%, p=0.003), intravenous immunoglobulin therapy (67% vs. 2%, p<0.001), aspirin therapy (50% vs. 0%, p<0.001), and current acute renal replacement therapy (50% vs. 2%, p=0.002) were also significantly higher in patients with MIS-C. Logistic regression analysis showed that the presence of MIS-C was significantly associated with gastrointestinal manifestations [odds ratio (OR)=10.98; 95%CI (95% confidence interval)=1.20-100.86; p=0.034] and hypoxemia [OR=16.85; 95%CI=1.34-211.80; p=0.029]. Further univariate analysis showed a positive association between MIS-C and death [OR=58.00; 95%CI=6.39-526.79; p<0.0001]. CONCLUSIONS: Pediatric patients with laboratory-confirmed COVID-19 with MIS-C had a severe clinical spectrum with a high mortality rate. Our study emphasizes the importance of investigating MIS-C in pediatric patients with COVID-19 presenting with gastrointestinal involvement and hypoxemia.

Assessment and comparison of bacterial load levels determined by quantitative amplifications in blood culture-positive and negative neonatal sepsis.

Bacterial sepsis remains a major cause of mortality and blood cultures are the gold standard of laboratory diagnosis even though they lack sensitivity in neonates. Culturenegative sepsis, also known as clinical sepsis, has long been considered a diagnosis in neonatal intensive care units because, as well as culture-positive infants, culture-negative neonates have worse prognosis in comparison with non-infected ones. Quantitative amplifications are used to detect bacterial infections in neonates but results are considered only in a qualitative way (positive or negative). The aim of the present study was to determine and compare bacterial load levels in blood culture-positive and culture-negative neonatal sepsis. Seventy neonates with clinical and laboratory evidence of infection admitted at three neonatal intensive care units were classified as blood culture-positive or culture-negative. Blood samples obtained at the same time of blood cultures had bacterial load levels assessed through a 16S rDNA qPCR. Blood cultures were positive in 29 cases (41.4%) and qPCR in 64 (91.4%). In the 29 culture-positive cases, 100% were also positive by qPCR, while in the 41 culture-negative cases, 35 (85.4%) were positive by qPCR. Bacterial load levels were in general < 50 CFU/mL, but were significantly higher in culture-positive cases (Mann-Whitney, p = 0.013), although clinical and laboratory findings were similar, excepting for deaths. In conclusion, the present study has shown that blood culture-negative neonates have lower bacteria load levels in their bloodstream when compared to blood culture-positive infants.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome

ABSTRACT Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

Association of Parasite Load Levels in Amniotic Fluid With Clinical Outcome in Congenital Toxoplasmosis.

OBJECTIVE: To correlate neonatal and infant clinical outcome with parasite load in amniotic fluid (AF). METHODS: We conducted a retrospective cohort study of 122 children whose mothers had toxoplasmosis during pregnancy. The children were monitored from birth to 12 months old. Stored AF samples were obtained at maternal diagnosis and tested by quantitative polymerase chain reaction. Gestational age at maternal infection, quantitative polymerase chain reaction results, neonatal anti-Toxoplasma gondii immunoglobulin (Ig) M, and clinical outcome at 12 months were correlated. RESULTS: Maternal infection occurred in 18 of 122 (14.7%) and 104 of 122 (85.2%) women in the first and second trimesters, respectively. At birth, IgM was present in 107 of 122 (87.7%) neonates and 36 (29.5%) were symptomatic. Of these, half occurred in the first and the other half in the second trimester and 6 of 36 had severe infections (16.7% of symptomatic, 4.9% of total), all infected in the first trimester. Parasite load levels were highly variable (median 35 parasites/mL, range 2-30,473). Logistic regression correlated symptomatic infection with gestational age (odds ratio [OR] 0.47, CI 0.31-0.73) and parasite load (OR 2.04, CI 1.23-3.37), but not with positive IgM (OR 6.81, CI 0.86-53.9). Negative correlations were found between gestational age and parasite load (rs -0.780, CI -0.843 to -0.696), gestational age and symptoms (rs -0.664, CI -0.755 to -0.547), but not gestational age and IgM (rs -0.136, CI -0.311 to 0.048). Parasite load levels distributed by percentile showed that all symptomatic patients appeared from the 75th percentile and all severe infections from the 95th percentile. Load rankings showed doubled the OR for each 20 parasite/mL increment. Parasite load was associated with symptomatic infections (area under the curve 0.959, CI 0.908-0.987) as well as gestational age (area under the curve 0.918, CI 0.855-0.960) and both parameters combined (area under the curve 0.969, CI 0.920-0.992). CONCLUSION: Parasite load in AF is associated with the clinical outcome in congenital toxoplasmosis, irrespective of gestational age at maternal infection.

Technical performance of a lateral flow immunoassay for detection of anti-SARS-CoV-2 IgG in the outpatient follow-up of non-severe cases and at different times after vaccination: comparison with enzyme and chemiluminescent immunoassays

ABSTRACT This study assessed the technical performance of a rapid lateral flow immunochromatographic assay (LFIA) for the detection of anti-SARS-CoV-2 IgG and compared LFIA results with chemiluminescent immunoassay (CLIA) results and an in-house enzyme immunoassay (EIA). To this end, a total of 216 whole blood or serum samples from three groups were analyzed: the first group was composed of 68 true negative cases corresponding to blood bank donors, healthy young volunteers, and eight pediatric patients diagnosed with other coronavirus infections. The serum samples from these participants were obtained and stored in a pre-COVID-19 period, thus they were not expected to have COVID-19. In the second group of true positive cases, we chose to replace natural cases of COVID-19 by 96 participants who were expected to have produced anti-SARS-CoV-2 IgG antibodies 30-60 days after the vaccine booster dose. The serum samples were collected on the same day that LFIA were tested either by EIA or CLIA. The third study group was composed of 52 participants (12 adults and 40 children) who did or did not have anti-SARS-CoV-2 IgG antibodies due to specific clinical scenarios. The 12 adults had been vaccinated more than seven months before LFIA testing, and the 40 children had non-severe COVID-19 diagnosed using RT-PCR during the acute phase of infection. They were referred for outpatient follow-up and during this period the serum samples were collected and tested by CLIA and LFIA. All tests were performed by the same healthcare operator and there was no variation of LFIA results when tests were performed on finger prick whole blood or serum samples, so that results were grouped for analysis. LFIA's sensitivity in detecting anti-SARS-CoV-2 IgG antibodies was 90%, specificity 97.6%, efficiency 93%, PPV 98.3%, NPV 86.6%, and likelihood ratio for a positive or a negative result were 37.5 and 0.01 respectively. There was a good agreement (Kappa index of 0.677) between LFIA results and serological (EIA or CLIA) results. In conclusion, LFIA analyzed in this study showed a good technical performance and agreement with reference serological assays (EIA or CLIA), therefore it can be recommended for use in the outpatient follow-up of non-severe cases of COVID-19 and to assess anti-SARS-CoV-2 IgG antibody production induced by vaccination and the antibodies decrease over time. However, LFIAs should be confirmed by using reference serological assays whenever possible.

HLA-DQA1/B1 alleles as putative susceptibility markers in congenital toxoplasmosis.

Host and parasite genotypes are among the factors associated with congenital toxoplasmosis pathogenesis. As HLA class II molecules play a key role in the immune system regulation, the aim of this study was to investigate whether HLA-DQA1/B1 alleles are associated with susceptibility or protection to congenital toxoplasmosis. One hundred and twenty-two fetuses with and 103 without toxoplasmosis were studied. The two study groups were comparable according to a number of socio-demographic and genetic variables. HLA alleles were typed by PCR-SSP. In the HLA-DQA1 region, the allele frequencies showed that *01:03 and *03:02 alleles could confer susceptibility (OR= 3.06, p = 0.0002 and OR= 9.60, p= 0.0001, respectively) as they were more frequent among infected fetuses. Regarding the HLA-DQB1 region, the *05:04 allele could confer susceptibility (OR = 6.95, p < 0.0001). Of the 122 infected fetuses, 10 presented susceptibility haplotypes contrasting with only one in the non-infected group. This difference was not statistically significant after correction for multiple comparison (OR = 9.37, p=0.011). In the casuistic, there were two severely damaged fetuses with high parasite loads determined in amniotic fluid samples and HLA-DQA1 susceptibility alleles. In the present study, a discriminatory potential of HLA-DQA1/B1 alleles to identify susceptibility to congenital toxoplasmosis and the most severe cases has been shown.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS.

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.