[Molecular biology analysis of 2 rare RhD variant individuals with RHD*DEL37].

The Rh blood grouping system is a critical standardized test in transfusion medicine, especially for the cases related to haemolytic transfusion reactions and neonatal haemolytic disease caused by clinical RhD blood group incompatibility. In the present case report, we presented two cases with the uncommon RHD gene variation RHD*DEL37. The blood samples of the two subjects were mistakenly identified as RhD-negative through conventional serological testing. Firstly, both blood samples were tested negative for the RhD antigen using traditional tube test and gel microcolumn methods. The phenotyping of RhCE were identified as ccEe and ccee for each sample, respectively. Secondly, genetic analysis was performed using polymerase chain reaction-sequence specific prime (PCR-SSP) which revealed that neither sample belonging to the several common RHD gene variants which was found in Asia. Moreover, they turned out to be positive for the RHD haplotype, which indicated that exons 1-10 on one of the RHD alleles were entirely absent. In addition, a T>C mutation was observed at bases 1154-31 in intron 8 of the other allele, which was located at the intron 8 breakpoint. This result was obtained after further Sanger sequencing of exons 1-10 of the RHD gene. The mutant allele was designated as RHD*DEL37 by the International Society of Blood Transfusion (ISBT) and was identified as D-elute(Del) by phenotype ana-lysis. Both samples were genotyped as RHD*DEL37 and showed positive results. In summary, the true genotype of the two blood samples, of which the screening results only using serological testing method was negative, were RHD*DEL37 /RHD-(RHD*01N.01). Notably, this kind of genotype was reported for the first time in Chinese population. Moreover, the two individuals did not have ties of consanguinity, indicating that some of the Chinese individuals could be carriers of the genetic mutation. Therefore, it might be necessary to further confirm the frequency of this mutation in the Chinese population and the possibility of homozygosity for this mutation. This report identifies infrequent RHD gene mutation samples by coupling molecular biology and serological methods to prevent misclassification of blood groups. Combining serological and molecular biology test results to determine blood group is critical in protecting patients during clinical transfusion procedures.

CYP21A2 Gene Analysis in Southern Iranian CAH Patients and a Brief Review of the Mutation Spectrum.

Background: CYP21A2 gene mutations are responsible for more than 95% of Congenital Adrenal Hyperplasia (CAH) disorders with autosomal recessive inheritance. Most of these pathogenic mutations originate from the CYP21A1P, a neighboring pseudogene with 98% homology, due to unequal crossing over or gene conversion events. Mutation identification of the gene could be beneficial for accurate diagnosis and outcome prediction. Methods: Twelve unrelated patients with CAH diagnosis were recruited for genetic counseling. To ensure distinct amplification of the CYP21A2 gene rather than its pseudogene, the complete sequence of the gene was amplified through two overlapping fragments by specific primers. The entire sequences were screened by direct Sanger sequencing using new sequencing primers. Results: Only two pathogenic point mutations were identified. The c.293-13C>G, also known as In2G, and the c.955C>T mutations were found in 37.5 and 33.3% of alleles, respectively. One patient showed homozygous gene deletion. We also reviewed recent reports on CYP21A2 gene mutations in Iran. Conclusion: Evaluating the ethnicity-specific gene mutation data is significant for populations with diverse ethnic groups including the Iranian population. Although several common mutations have been reported as causative mutations among CAH patients, identifying only two common point mutations in Fars province would help prioritize exon sequencing and reduce the cost and time of genotyping.

Apolipoprotein-E genotyping in formalin-fixed and paraffin-embedded post-mortem brain tissue.

Formalin-fixed paraffin-embedded (FFPE) brain tissue held in tissue banks constitutes a valuable research resource, especially when associated with clinical annotations and longitudinal psychometric testing. Apolipoprotein-E (APOE) genotyping is important to fully characterise this resource, however older FFPE tissue may not be suitable for genotyping. We performed polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays on DNA extracted from post-mortem FFPE brain tissue ranging from 2-19 years old. A maximum of three years in paraffin was determined for robust APOE genotyping of FFPE tissue using PCR-RFLP which may suggest prolonged storage of fixed tissue as FFPE blocks may have deleterious effects on DNA.

Diversity of picornaviruses detected in diarrheal samples from children in Belém, Brazilian Amazon (1982-2019).

In this investigation, fecal specimens from children with diarrhea were collected from four community studies conducted between 1982 and 2019 in Belém, Brazilian Amazon. A total of 234 samples were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect infections by picornaviruses of the Enterovirus (EV), Parechovirus (HPeV), Cosavirus (HCoSV), Kobuvirus (Aichivirus - AiV) and Salivirus (SalV) genera. The positive samples were subjected to different amplification protocols of the VP1 region of the genome, such as nested PCR or snPCR, and were subsequently genotyped by sequencing VP1 and VP3 of the viral genome. Positivity was observed in 76.5% (179/234) of the samples tested using RT-qPCR for at least one virus, and co-infection was observed in 37.4% (67/179) of the cases. EV was detected in 50.8% (119/234), HPeV in 29.9% (70/234), HCoSV in 27.3% (64/234), and AiV/SalV in 2.1% (5/234) of the specimens tested by RT-qPCR. Using nested PCR and/or snPCR techniques, the positivity rates were 94.11% (112/119) for EV, 72.85% (51/70) for HPeV, and 20.31% (13/64) for HCoSV. It was not possible to amplify the samples that were positive for AiV/SalV. Sequencing revealed 67.2% (80/119) EV, 51.4% (36/70) HPeV, and 20.31% (13/64) HCoSV. Forty-five different types of EV were found among species A, B, and C; HCoSV identified five species, including a possible recombinant strain; all HPeV were identified as belonging to species A, in two samples a possible recombination involving three different strains was verified. This study demonstrated the high circulation and diversity of different types of picornaviruses in fecal samples, including those collected more than 30 years ago. This endorsed the evaluation of important points in the epidemiology of these viruses, such as the presence of co-infection and the possibility of knowing more about these agents, considering that some were recently described; therefore, their detection in older samples can provide more data about their ancestry.

Longitudinal Assessment of Virological Failure and Antiretroviral Drug Resistance among Treatment-naive Subjects Living with HIV.

INTRODUCTION: Human immunodeficiency virus (HIV) infection, the etiological agent of acquired immunodeficiency syndrome (AIDS), is a serious public health issue. Therapeutic measures have been successful in increasing the survival and improving the quality of life. However, some treatment-naive subjects living with HIV present resistance-associated mutations as a result of late diagnosis and/or mutant strain infections. The objective of this study was to identify the virus genotype and assess the antiretroviral resistance profile based on the results of HIV genotyping in treatment-naive subjects living with HIV, after six months of taking antiretroviral therapy. METHODS: This was a prospective cohort study on treatment-naive adults living with HIV attending a specialized outpatient clinic in southern Santa Catarina State, Brazil. The participants were interviewed and had blood samples drawn. The genotypic antiretroviral drug resistance profile was examined in patients with detectable viral loads. RESULTS: 65 treatment-naive subjects living with HIV were recruited for this study. After six months of taking antiretroviral therapy, resistance-associated mutations were observed in 3 (4.6%) subjects living with HIV. CONCLUSION: Subtype C was identified as the circulating subtype in southern Santa Catarina State, and L10V, K103N, A98G, and Y179D were the most common mutations found in treatment-naive subjects.

Genetic linkage analysis of head and neck cancer in a Spanish family.

OBJECTIVES: To describe the genetic variants that may be associated with the development of head and neck cancer (HNC) and functionally validating the molecular implications. MATERIALS AND METHODS: A prospective observational study was carried out on a family of 3 generations in which 3 members had developed HNC. Peripheral blood sample was taken in a routine procedure for exome sequencing in one relative and genotyping in the remaining twelve relatives. For the functional analysis all-trans retinoic acid (atRA) was extracted from saliva and serum and measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The presence of HPV-DNA. RESULTS: None of the patients smoked or consumed alcohol. The presence of HPV DNA was not detected in any of the biopsied samples. A total amount of 6 members out of 13 (46.15%) carried out the same mutation of CYP26B1 (2p13.2; G>T). The mean plasma concentration of atRA was 3.3109 ± 1.4791 pg/mL for the study family and 4.7370 ± 1.5992 pg/mL for the controls (p = 0.042). CONCLUSION: Lower levels of atRA were confirmed in the study family, which may open the way to the possible relationship between the polymorphism CYP26B1 (2p13.2; G>T) and HNC.

Comprehensive laboratory diagnosis of Fanconi anaemia: comparison of cellular and molecular analysis.

BACKGROUND: Fanconi anaemia (FA) is a rare inherited bone marrow failure disease caused by germline pathogenic variants in any of the 22 genes involved in the FA-DNA interstrand crosslink (ICL) repair pathway. Accurate laboratory investigations are required for FA diagnosis for the clinical management of the patients. We performed chromosome breakage analysis (CBA), FANCD2 ubiquitination (FANCD2-Ub) analysis and exome sequencing of 142 Indian patients with FA and evaluated the efficiencies of these methods in FA diagnosis. METHODS: We performed CBA and FANCD2-Ub analysis in the blood cells and fibroblasts of patients with FA. Exome sequencing with improved bioinformatics to detect the single number variants and CNV was carried out for all the patients. Functional validation of the variants with unknown significance was done by lentiviral complementation assay. RESULTS: Our study showed that FANCD2-Ub analysis and CBA on peripheral blood cells could diagnose 97% and 91.5% of FA cases, respectively. Exome sequencing identified the FA genotypes consisting of 45 novel variants in 95.7% of the patients with FA. FANCA (60.2%), FANCL (19.8%) and FANCG (11.7%) were the most frequently mutated genes in the Indian population. A FANCL founder mutation c.1092G>A; p.K364=was identified at a very high frequency (~19%) in our patients. CONCLUSION: We performed a comprehensive analysis of the cellular and molecular tests for the accurate diagnosis of FA. A new algorithm for rapid and cost-effective molecular diagnosis for~90% of FA cases has been established.

Serotype Distribution and Multi Locus Sequence Type (MLST) of Erythromycin-Resistant Streptococcus Pneumoniae Isolates in Tehran, Iran.

Background: The number of erythromycin-resistant Streptococcus pneumoniae has significantly increased around the world. The present study aimed to determine the serotype distribution and molecular epidemiology of the erythromycin-resistant Streptococcus pneumoniae (ERSP) isolated from patients with invasive disease. Methods: A total of 44 Streptococcus pneumoniae isolates were tested for susceptibility to several antimicrobial agents. Additionally, the polymerase chain reaction (PCR) was applied to evaluate ERSP isolates in terms of the presence of erythromycin resistance genes (e.g., ermB and mefA). The isolates were serotyped using the sequential multiplex-PCR method, and molecular epidemiology was assessed through the multilocus sequence typing (MLST) analysis. Results: The results represented multidrug resistance (MDR) in approximately half of the pneumococcal isolates. Among 22 ERSP isolates, 20 (90.9%) and 12 (56%) ones contained ermB and mefA, respectively. Further, 14 (31.8%), 3 (22.7%), and 19A (18.1%) were the common serotypes among the isolates. No significant correlation was observed between serotypes and erythromycin resistance genes. Furthermore, the MLST results revealed 18 different sequence types (STs), the top ones of which were ST3130 (3 isolates) and ST166 (3 isolates). Population genetic analysis disclosed that CC63 (32%), CC156 (18%), and CC320 (18%) were identified as the predominant clonal complexes. Conclusions: The ERSP isolates exhibited high genetic diversity. The large frequency of MDR isolates suggests the emergence of high resistant strains, as well as the need to implement vaccination in the immunization schedule of Iran. These accumulating evidences indicate that 13-valent pneumococcal conjugate vaccines provided higher serotype coverage in the ERSP isolates.

The Accuracy of Sequence-Specific Oligonucleotide and Real-Time Polymerase Chain Reaction HLA Typing in Determining the Presence of Pre-Transplant Donor-Specific Anti-HLA Antibodies and Total Eplet Mismatches for Deceased Donor Kidney Transplantation.

High resolution human leukocyte antigen (HLA) typing is important in establishing eplet compatibility and the specificity of donor-specific anti-HLA antibodies (DSA). In deceased donor kidney transplantation, high resolution donor HLA typing may not be immediately available, leading to inaccuracies during the organ allocation process. We aimed to determine the concordance and agreement of HLA-Class I and II eplet mismatches calculated using population frequency based allelic haplotype association (linkage disequilibrium, LD) from sequence-specific oligonucleotide (SSO) and real-time polymerase chain reaction (rtPCR) donor HLA typing (available at time of donor kidney allocation) compared to high-resolution Next Generation Sequencing (NGS) donor typing. NGS high resolution HLA typing were available for all recipients prior to donor kidney allocation. A cohort of 94 deceased donor-recipient pairs from a single Western Australian center were included (77 individual donors typed, 55 local and 22 interstate). The number of class I (HLA-A+B+C) and class II (HLA-DRB1+DRB3/4/5+DQB1+DQA1+DPB1+DPA1) eplet mismatches were calculated using HLAMatchmaker, comparing LD- and NGS-HLA typing. The accuracy in assigning pre-transplant DSA was compared between methods. The concordance correlation coefficient (95%CI) for HLA-class I and II eplet mismatches were 0.994 (0.992 to 0.996) and 0.991 (0.986 to 0.993), respectively. The 95% limits of agreement for class I were -1.3 (-1.6 to -1.1) to 1.4 (1.2 to 1.7) and -4.8 (-5.7 to -3.9) to 5.0 (4.1 to 5.9) for Class II. Disagreement between the two methods were present for 11 and 37 of the Class I and II donor/recipient pairs. Of which, 5 had a difference of ≥5 class II eplet mismatches. There were 34 (36%) recipients with potential pre-transplant DSA, of which 8 (24% of recipients with DSA) had indeterminate and ultimately false positive DSA assigned by donor LD-typing. While the concordance between NGS- and LD-typing was high, the limits of agreement suggest meaningful differences between these two techniques. The inaccurate assignment of DSA from donor LD-typing may result in associated HLA being considered unacceptable mismatches, inappropriately precluding candidates' access to transplantation. Accurate imputation of two-field HLA alleles based on LD from SSO and rtPCR HLA typing remains a substantial challenge in clinical practice in-lieu of widely available, rapid, high-resolution methods.

Identification and molecular characterization of penicillin-nonsusceptible Streptococcus pneumoniae isolates recovered from invasive infections in a pre-pneumococcal vaccine era.

BACKGROUND: Given the significant role of penicillin-nonsusceptible Streptococcus pneumoniae in inducing severe infectious diseases, identifying serotypes and genotypes that can mediate antimicrobial resistance has become a pillar of treatment strategies. This study aims to determine the correlation between the minimum inhibitory concentration of antimicrobial agents and amino acid mutations in penicillin-binding proteins. Moreover, molecular serotyping and multiple-locus variable number tandem repeat analysis typing were first-ever performed to characterize the invasive penicillin-nonsusceptible S. pneumoniae isolates in Iran. METHODS: Of 149 isolates, antimicrobial susceptibility tests were performed against penicillin, ceftriaxone, and cefotaxime by the MIC Test Strip, and sequence analysis of the pbp genes was performed through PCR-sequencing method. All penicillin-nonsusceptible S. pneumoniae isolates were serotyped and genotyped by sequential multiplex PCR and multiple-locus variable-number tandem repeat analysis, respectively. RESULTS: Among pneumococcal isolates, 53 isolates were classified as penicillin-nonsusceptible S. pneumoniae, of which 38 (71.7%) and 15 (28.3%) were resistant and intermediate to penicillin, respectively. Furthermore, ceftriaxone- and cefotaxime-nonsusceptible pneumococci constituted 33 (62.2%) and 29 cases (54.7%), respectively. Of note, there were 8 and 41 different serotypes and multiple-locus variable-number tandem repeat analysis types, respectively. CONCLUSIONS: Due to the increasing resistance to antimicrobial agents, the most efficient approach to preventing pneumococcal infection mortality as vaccine-preventable diseases is focusing on wide-spectrum vaccination. Based on our findings, the 13-valent pneumococcal conjugate vaccine could considerably reduce the incidence of invasive pneumococcal diseases due to the high rate of serotype coverage.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Association of single nucleotide polymorphisms in microRNAs with the risk of chronic spontaneous urticaria / 中华皮肤科杂志

Objective:To investigate the association between single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) and the risk of chronic spontaneous urticaria (CSU) .Methods:A case-control study was conducted. A total of 98 patients with CSU (CSU group) were collected from Department of Dermatology, Affiliated Hospital of Jining Medical University from January to June in 2019, and 148 health checkup examinees (control group) were collected at the same time, all of whom were of Han nationality from Shandong province. Genomic DNA was extracted from venous blood samples, and polymorphic sites rs2431697 (miRNA-146a) , rs57095329 (miRNA-146a) , rs3746444 (miRNA-499) , rs11614913 (miRNA-196a2) and rs895819 (miRNA-27a) were analyzed for SNP genotyping by multiplex PCR amplification and single-base extension. Chi-square test was used to analyze differences in the distribution of alleles, genotypes and genetic models between the two groups, and unconditional logistic regression to analyze the relationship between gene SNPs and the risk of CSU.Results:All samples were successfully genotyped by analysis of the 5 polymorphic sites. The alleles of the miRNA-196a2 SNP rs11614913 were T/C, and the absolute frequency of T allele was 110 (56.1%) in the CSU group and 131 (44.3%) in the control group; there was a significant difference in the T/C allele frequency distribution between the two groups ( χ2 = 6.64, P = 0.010) , and the T allele might be a risk factor for CSU ( OR=1.61, 95% CI: 1.12-2.32) . In addition, the absolute frequencies of CC, CT and TT genotypes of rs11614913 were 16 (16.3%) , 54 (55.1%) , 28 (28.6%) in the CSU group, and 48 (32.4%) , 69 (46.6%) , 31 (20.9%) in the control group respectively, and there was a significant difference in the genotype distribution between the two groups ( χ2 = 8.16, P = 0.017) ; the distribution of the dominant genetic model (TT + CT vs. CC) also significantly differed between the two groups ( χ2 = 7.95, P = 0.005) , which might increase the risk of CSU ( OR=2.46, 95% CI: 1.30-4.65) . Conclusion:The miRNA-196a2 SNPs may be associated with the risk of CSU in the Han population in Shandong, China, and the rs11614913 polymorphism may increase the risk of CSU.

Comparative analysis of routine parasitological methods for recovery of cysts, molecular detection, and genotyping of Giardia duodenalis.

In order to improve the diagnosis of giardiasis, fecal samples (high/medium/low concentration of cysts) were processed by the parasitological methods used in the routine: Faust, Lutz e Ritchie modified (replacement of formaldehyde by distilled water). The cysts were quantified; the DNA was extracted and amplified by semi-nested PCR (GDH gene). Fifteen clinical samples were analyzed to validate the study by PCR-RFLP. The results showed that the parasite was only detected and genotyped correctly when samples from children with high, medium, and low parasitic load, belonging to genotype AII, were processed by the modified Ritchie method, different from what was observed for the other methods used in laboratory routine (Faust and Lutz). The modified Ritchie method proved to be more suitable, recovering a greater number of cysts from samples, regardless of parasitic load, which reduces the chance of false negative results and has epidemiological repercussions since individuals with low parasite load are usually asymptomatic and the main disseminators of this infection.

Hydatidiform mole diagnostics using circulating gestational trophoblasts isolated from maternal blood.

BACKGROUND: In gestational trophoblastic disease, the prognosis is related to the genetic constitution. In some cases, taking a biopsy is contraindicated. METHODS: In a pregnant woman, ultrasound scanning suggested hydatidiform mole. To explore if the genetic constitution can be established without taking a biopsy (or terminating the pregnancy), cell-free DNA and circulating gestational trophoblasts were isolated from maternal blood before evacuation of the uterus. The evacuated tissue showed the morphology of a complete hydatidiform mole. Without prior whole-genome amplification, short tandem repeat analysis of 24 DNA markers was performed on the samples, and on DNA isolated from evacuated tissue, and from the blood of the patient and her partner. RESULTS: Identical genetic results were obtained in each of three circulating gestational trophoblasts and the evacuated tissue, showing that this conceptus had a diploid androgenetic nuclear genome. In contrast, analysis of cell-free DNA was less informative and less specific due to the inherent presence of cell-free DNA from the patient. CONCLUSION: Our results show that it is possible to isolate and analyze circulating gestational trophoblasts originating in a pregnancy without maternal nuclear genome. For diagnosing gestational trophoblastic diseases, genotyping circulating gestational trophoblasts appears to be superior to analysis of cell-free DNA.

Partial hydatidiform moles with unclear histopathological profile - importance of genetic diagnostics.

OBJECTIVE: The aim of the study was the genetic characterization of a set of cases with an unclear morphological profile of the placental tissue suspected of a partial hydatidiform mole. PATIENTS AND METHODS: This work presents the results of a genetic analysis of a group of 10 patients with various clinical manifestations of reproductive loss, where a partial hydatidiform mole was suspected on the basis of a histopathological examination. The composition of the genome of the products of conception was determined by short tandem repeats (STR) genotyping using a commercial kit;Devyser Compact v3 (Devyser). RESULTS AND CONCLUSIONS: Out of 10 analyzed cases, five had diandric monogynic triploid genome, characteristic for a partial mole. Aneuploidies of chromosomes 13, 18, 21, X and Y were excluded in four cases and Pataus syndrome was dia-gnosed in one case. In the case of an unclear histopathological profile, consultative DNA analysis (ideally STR genotyping) can significantly help the pathologist in the differential dia-gnosis of a partial mole. The histopathological profile of a partial hydatidiform mole may be in some cases incomplete and unclear, especially in the early weeks of gestation, which can lead to false negativity of the examination. On the other hand, other pathologies, for example aneuploides or digynic triploidy, may produce a histopathological profile similar to a partial mole, which leads to false positivity. Accurate dia-gnosis of a partial hydatidiform mole using molecular genetic methods contributes to the determination of adequate dispensary care for patients.

A Genotyping Study in Benin Comparing the Carriage of Plasmodium falciparum Infections Before Pregnancy and in Early Pregnancy: Story of a Persistent Infection.

BACKGROUND: Malaria infections in the first trimester of pregnancy are frequent and deleterious for both mother and child health. To investigate if these early infections are newly acquired or already present in the host, we assessed whether parasites detected before pregnancy and those detected in early pregnancy are the same infection. METHODS: We used data from the preconceptional "RECIPAL" study (Benin, 2014-2017). Sixty-three pregnant women of 411 included who had a malaria infection detected by quantitative polymerase chain reaction both before pregnancy and at the first antenatal care (ANC) visit were selected for this study. Two highly polymorphic markers, msp-2 and glurp, and a fragment-analysis method were used to enumerate the Plasmodium falciparum genotypes and to quantify their proportions within isolates. An infection was considered as persistent when identical msp-2 and glurp genotypes were found in the corresponding prepregnancy and early-pregnancy samples. RESULTS: The median time between the 2 malaria screenings was 3 months. The median gestational age at the first ANC visit was 6.4 weeks. Most infections before pregnancy were submicroscopic infections. Based on both msp-2 and glurp genotyping, the infection was similar before and in early pregnancy in 46% (29/63) of cases. CONCLUSIONS: Almost half of P. falciparum infections detected in the first trimester originate before pregnancy. Protecting young women from malaria infection before pregnancy might reduce the prevalence of malaria in early pregnancy and its related poor maternal and birth outcomes.

Precision diagnosis of a full-term pregnancy with hydatidiform mole based on postnatal short tandem repeat DNA genotyping: a case report / 中华围产医学杂志

We describe a case of spontaneous conception following ovarian stimulation, in which a singleton pregnancy was revealed by ultrasound at 17 gestational weeks, with a multi-cystic "honeycomb" pattern in part of the placenta. With close monitoring, the patient delivered a healthy male neonate through cesarean section at 38 gestational weeks. The clinical findings, combined with ultrasound, laboratory, pathological, and immunohistochemistry examination, and short tandem repeat genotyping, confirmed a twin pregnancy consisting of a complete mole and coexisting fetus. No obvious abnormalities were found in the mother or the boy during a four-and-a-half-year's follow-up.

Update on and future perspectives for the diagnosis of alpha-1 antitrypsin deficiency in Brazil / Atualização e perspectivas futuras para o diagnóstico da deficiência de alfa-1 antitripsina no Brasil

ABSTRACT Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by a mutation in the SERPINA1 gene, which encodes the protease inhibitor alpha-1 antitrypsin (AAT). Severe AATD predisposes individuals to COPD and liver disease. Early diagnosis is essential for implementing preventive measures and limiting the disease burden. Although national and international guidelines for the diagnosis and management of AATD have been available for 20 years, more than 85% of cases go undiagnosed and therefore untreated. In Brazil, reasons for the underdiagnosis of AATD include a lack of awareness of the condition among physicians, a racially diverse population, serum AAT levels being assessed in a limited number of individuals, and lack of convenient diagnostic tools. The diagnosis of AATD is based on laboratory test results. The standard diagnostic approach involves the assessment of serum AAT levels, followed by phenotyping, genotyping, gene sequencing, or combinations of those, to detect the specific mutation. Over the past 10 years, new techniques have been developed, offering a rapid, minimally invasive, reliable alternative to traditional testing methods. One such test available in Brazil is the A1AT Genotyping Test, which simultaneously analyzes the 14 most prevalent AATD mutations, using DNA extracted from a buccal swab or dried blood spot. Such advances may contribute to overcoming the problem of underdiagnosis in Brazil and elsewhere, as well as being likely to increase the rate detection of AATD and therefore mitigate the harmful effects of delayed diagnosis.

RESUMO A deficiência de alfa-1 antitripsina (DAAT) é um distúrbio genético raro causado por uma mutação no gene SERPINA1, que codifica o inibidor de protease alfa-1 antitripsina (AAT). A DAAT predispõe os indivíduos a DPOC e doença hepática. O diagnóstico precoce é essencial para a implementação de medidas preventivas e para limitar a carga da doença. Embora diretrizes nacionais e internacionais para o diagnóstico e manejo da DAAT estejam disponíveis há 20 anos, mais de 85% dos casos não são diagnosticados e, portanto, não são tratados. No Brasil, os motivos para o subdiagnóstico da DAAT incluem o desconhecimento dos médicos sobre a condição, a diversidade racial da população, o fato de os níveis séricos de AAT serem avaliados em um número limitado de indivíduos e a falta de ferramentas diagnósticas convenientes. O diagnóstico da DAAT baseia-se em resultados de exames laboratoriais. A abordagem diagnóstica padrão envolve a avaliação dos níveis séricos de AAT, seguida de fenotipagem, genotipagem, sequenciamento gênico ou suas combinações para detecção da mutação específica. Nos últimos 10 anos, novas técnicas foram desenvolvidas, oferecendo uma alternativa rápida, minimamente invasiva e confiável aos métodos tradicionais de teste. Um desses testes disponíveis no Brasil é o teste de genotipagem A1AT, que analisa simultaneamente as 14 mutações mais prevalentes da DAAT usando DNA extraído de swab bucal ou de sangue em papel-filtro. Esses avanços podem contribuir para a superação do problema do subdiagnóstico no Brasil e em outros países, bem como podem aumentar a taxa de detecção da DAAT e, portanto, mitigar os malefícios do diagnóstico tardio.

Genetic Diversity of ctxB Gene Among Classical O1 and El Tor Strains of Vibrio cholerae using High-Resolution Melting Curve Analysis.

BACKGROUND & OBJECTIVE: Vibrio cholerae is a natural inhabitant of the environment and causes severe diarrhea ailments (cholera) that affects thousands of people each year worldwide. The most important virulence factors of this pathogen are cholera toxin (cholera toxin CT) and Type IV pili (toxin co-regulated pili TCP), which are encoded within the genome of the filamentous bacteriophage CTXφ. In the present study, according to researchers' report on genotypic variations of cholera toxin, we evaluated the sequence of ctxB subunit obtained from 100 strains of patients infected with cholera in Iran. METHODS: The evaluation of genotype variations of cholera toxin was made by high-resolution melting curve analysis illustrating a single nucleotide change. Then, ctxB gene sequencing was performed. Through this analysis and the sequencing process, two standard samples were studied. RESULTS: Using serologic tests, all the strains analyzed in this study were identified to be in O1 serotype. However, there have been differences in sequences of ctxB as some were similar to V . cholerae O1 biovar El Tor str. N16961 while others were similar to the genotype of V . cholerae ATCC 14035. We did not observe any particular pattern within the process of mutation. CONCLUSION: The analysis of the new samples of ctxB showed that they were potentially different. It seems that these complicated species were affected by a new genetic exchange of El Tor and classic genotypes.

Targeted antenatal anti-D prophylaxis for RhD-negative pregnant women: a systematic review.

BACKGROUND: All non-sensitized Rhesus D (RhD)-negative pregnant women in Germany receive antenatal anti-D prophylaxis without knowledge of fetal RhD status. Non-invasive prenatal testing (NIPT) of cell-free fetal DNA in maternal plasma could avoid unnecessary anti-D administration. In this paper, we systematically reviewed the evidence on the benefit of NIPT for fetal RhD status in RhD-negative pregnant women. METHODS: We systematically searched several bibliographic databases, trial registries, and other sources (up to October 2019) for controlled intervention studies investigating NIPT for fetal RhD versus conventional anti-D prophylaxis. The focus was on the impact on fetal and maternal morbidity. We primarily considered direct evidence (from randomized controlled trials) or if unavailable, linked evidence (from diagnostic accuracy studies and from controlled intervention studies investigating the administration or withholding of anti-D prophylaxis). The results of diagnostic accuracy studies were pooled in bivariate meta-analyses. RESULTS: Neither direct evidence nor sufficient data for linked evidence were identified. Meta-analysis of data from about 60,000 participants showed high sensitivity (99.9%; 95% CI [99.5%; 100%] and specificity (99.2%; 95% CI [98.5%; 99.5%]). CONCLUSIONS: NIPT for fetal RhD status is equivalent to conventional serologic testing using the newborn's blood. Studies investigating patient-relevant outcomes are still lacking.