Quality and Quantity of Nucleic Acids Extracted from Formalin-Fixed Paraffin-Embedded Lymphoma Biopsies from Nigerian Archived Biopsy.

BACKGROUND: Integrity of nucleic acids derived from archived formalin-fixed paraffin-embedded (FFPE) cancer specimens affects diagnosis, prognosis, and therapy. Several factors affect the quality and quantity of extracted nucleic acids and one of such factors is storage period. AIM: We investigated the impact of storage duration on the quality and quantity of nucleic acids extracted from archived FFPE lymphoma biopsies in Nigeria. MATERIALS AND METHODS: A total of 53 FFPE biopsies diagnosed as lymphoma stored over several years (2008-2019) were analyzed. They were 22 chronic lymphocytic leukemia (CLL) cases, 17 Hodgkin lymphoma (HL) cases, and 14 diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). DNA was extracted from all the lymphoma samples which were analyzed for integrity and amplifiability using the four pairs of control genes polymerase chain reaction (PCR) primers of BIOMED-2 protocol, whereas RNA extraction was from 6 CLL cases used for qPCR analysis of RNU43. RESULTS: For CLL, the mean DNA yield was 193.6 ng/µl (range: 3.0-533.0 ng/µl), whereas the mean A260/A280 ratio was 1.7 (1.2-1.9). For DLBCL, NOS, and HL, 255.5 ng/µl (range: 32.9-605.4 ng/µl), 1.8 (1.5-2.0) and 242.7 ng/µl (range: 1.3-886.0 ng/µl), and 1.7 (0.9-1.8), respectively. The extracted DNA gave amplifiable products of at least 200bp, whereas the RNA analysis showed CT values of <38 in all the samples. The mean RNA yield was 462.2 ng/µl (range: 74.7-1082.1), whereas the mean A260/A280 was 1.7 (1.5-1.8). CONCLUSION: Quantity and quality of nucleic acids from FFPE tissues stored for different time periods showed no significant difference in yield and quality.

Sequence analysis for SNP detection and phylogenetic reconstruction of SARS-cov-2 isolated from Nigerian COVID-19 cases.

BACKGROUND: Coronaviruses are a group of viruses that belong to the Family Coronaviridae, genus Betacoronavirus. In December 2019, a new coronavirus disease (COVID-19) characterized by severe respiratory symptoms was discovered. The causative pathogen was a novel coronavirus known as 2019-nCoV and later as SARS-CoV-2. Within two months of its discovery, COVID-19 became a pandemic causing widespread morbidity and mortality. METHODOLOGY: Whole genome sequence data of SARS-CoV-2 isolated from Nigerian COVID-19 cases were retrieved by downloading from GISAID database. A total of 18 sequences that satisfied quality assurance (length ≥29,700 nts and number of unknown bases denoted as "N" ≤ 5%) were used for the study. In addition, genome sequence of SARS-CoV-2 obtained from Nigeria's COVID-19 index case (Accession ID: EPI_ISL_413550) and the reference genome (Accession NC_ 045512.2) were obtained from GISAID and the GenBank databases, respectively. Multiple sequence alignment (MSA) was done in MAFFT (Version 7.471) while SNP calling was implemented in DnaSP (Version 6.12.03), respectively and then visualized in Jalview (Version 2.11.1.0). Phylogenetic analysis was with MEGA X software. RESULTS: Nigerian SARS-CoV-2 had 99.9% genomic similarity with four large conserved genomic regions. A total of 66 SNPs were identified out of which 31 were informative. Nucleotide diversity assessment gave Pi = 0.00048 and average SNP frequency of 2.22 SNPs per 1000 nts. Non-coding genomic regions particularly 5'UTR and 3'UTR had a SNP density of 3.77 and 35.4, respectively. The region with the highest SNP density was ORF10 with a frequency of 8.55 SNPs/1000 nts). This value was significantly higher (P < 0.01) than that of the spike gene, the region of greatest interest in SARS-CoV-2 genomics. Majority (72.2%) of viruses in Nigeria are of L lineage with preponderance of D614G mutation which accounted for 11 (61.1%) out of the 18 viral sequences. Nigeria SARS-CoV-2 revealed 3 major clades namely Oyo, Ekiti and Osun on a maximum likelihood phylogenetic tree. CONCLUSION AND RECOMMENDATION: There was a preponderance of L lineage (to include the new lineage scheme) and D614G mutants. Nigerian SARS-CoV-2 genome revealed ORF1ab as the region containing the highest SNP density as compared to the spike gene. The implication of this distribution of SNPs for the empirical lower infectivity of SARS-CoV-2 in Nigeria is discussed. This also underscores the need for more aggressive testing and treatment of COVID-19 in Nigeria. Additionally, attempt to produce testing kits for COVID-19 in Nigeria should consider the conserved regions identified in this study. Strict adherence to COVID-19 preventive measure is recommended in view of Nigerian SARS-CoV-2 phylogenetic clustering pattern, which suggests intensive community transmission possibly rooted in communal culture characteristic of many ethnicities in Nigeria.

Evaluation of Foetal Haemoglobin Status among Nigerian Patients with Sickle Cell Anaemia Using High Performance Liquid Chromatography.

BACKGROUND: Sickle cell anaemia (SCA, HbSS) is a genetic disorder of haemoglobin with marked variation in clinical manifestation. The aim of this study was to determine the foetal haemoglobin (HbF) status of patients with HbSS, compared with that of individuals with HbAS and HbAA control as well as to establish the relationship between HbF level and age and gender of the participants. METHODS: This was a cross-sectional study in which HbF values of known HbSS patients along with HbAS and HbAA controls were analysed using High Performance Liquid Chromatography. Socio-demographic and other information were obtained with the use of questionnaire. Data was analyzed using SPSS software, version 20.0. Ethical approval was obtained for the study. RESULTS: One hundred and two (102) participants were recruited for the study, comprising 60 patients with HbSS, 22 HbAS and 20 HbAA controls, with mean age of 11.0 years±9.6, 11.7 years ±8.8 and 12.3 years±8.1 respectively. There were 30 (61.2%) males and 30 (56.6%) females for HbSS group, 9 (18.4%) males and 13(24.5%) females for HbAS group and 10(20.4%) males and 10(18.9%) for HbAA group. Mean HbF level among HbSS participants was 8.0 ±6.1% and was significantly higher than that of HbAS (3.0 ±3.4%) and HbAA (2.2 ±4.1%) control (P<0.05). Mean HbF level was higher in children (<18 years) than adults (e"18 years) among HbSS, HbAS and HbAA participants, though not statistically significant (p >0.05). Mean HbF level was also higher among female HbSS, HbAS and HbAA groups compared to corresponding male groups, though only HbSS female group was significant (p = 0.031). CONCLUSION: Patients with HbSS have significantly higher HbF level than individuals with HbAS and HbAA. Foetal haemoglobin level tend to decrease with advancing age and higher in females. Increased HbF level may play a compensatory mechanism in sickling in HbSS, thus the use of agent that increase HbF level may improve clinical outcome.

CONTEXTE: L'anémie falciforme (SCA, HbSS) est un mal de l'hémoglobine avec une variation marquée de la manifestation. Le but de cette étude était de déterminer le fœtus le statut d'hémoglobine (HbF) des patients atteints d'HbSS, par rapport à celle des personnes ayant un contrôle de l'HbAS et de l'HbAA ainsi que établir la relation entre le taux d'HbF et l'âge et le sexe des participants. MÉTHODES: Il s'agissait d'une étude transversale dans laquelle l'HbF valeurs des patients HbSS connus ainsi que l'HbAS et l'HbAA les contrôles ont été analysés à l'aide d'un liquide haute performance Chromatographie. Informations sociodémographiques et autres ont été obtenus à l'aide d'un questionnaire. Les données ont été analysées à l'aide du logiciel SPSS, version 20.0. L'approbation éthique a été obtenue pour l'étude. RÉSULTATS: Cent deux (102) participants ont été recrutés pour l'étude, comprenant 60 patients avec HbSS, 22 HbAS et 20 contrôles HbAA, avec un âge moyen de 11,0 ans ± 9,6, 11,7 ans ± 8,8 et 12,3 ans ± 8,1 respectivement. Il y avait 30 (61,2%) hommes et 30 femmes (56,6%) pour le groupe HbSS, 9 hommes (18,4%) et 13 femmes (24,5%) pour le groupe HbAS et 10 hommes (20,4%) et 10 (18,9%) pour le groupe HbAA. Niveau moyen d'HbF parmi l'HbSS participants était de 8,0 ± 6,1% et était significativement plus élevé que celle du contrôle HbAS (3,0 ± 3,4%) et HbAA (2,2 ± 4,1%) (p <0,05). Le taux moyen d'HbF était plus élevé chez les enfants (<18 ans) que chez les adultes (e "18 ans) chez les participants HbSS, HbAS et HbAA, cependant non statistiquement significatif (p> 0,05). Le taux moyen d'HbF était également plus élevé parmi les groupes féminins HbSS, HbAS et HbAA par rapport aux groupes masculins correspondants, mais seulement au groupe féminin HbSS était significative (p = 0,031). CONCLUSION: Les patients atteints d'HbSS ont des Taux d'HbF que les individus avec HbAS et HbAA. Fœtal le taux d'hémoglobine a tendance à diminuer avec l'âge et plus élevé chez les femmes. L'augmentation du niveau d'HbF peut jouer un rôle compensatoire mécanisme de la faucille dans l'HbSS, donc l'utilisation d'un agent qui l'augmentation du taux d'HbF peut améliorer les résultats cliniques. Mots clés: hémoglobine fœtale, liquide haute performance Chromatographie, drépanocytose, traits drépanocytaires, normaux l'hémoglobine adulte.

Detection of BCR-ABL1 fusion gene transcripts in the saliva of Nigerian patients with chronic myeloid leukemia.

BACKGROUND: The presence of BCR-ABL1 fusion gene resulting from a t(9; 22) reciprocal chromosome translocation is the molecular hallmark of chronic myeloid leukemia (CML). In the diagnosis and treatment of CML, peripheral blood or bone marrow samples are usually taken for analysis. However, both methods are invasive sample collection methods, thus a noninvasive saliva sample method for the detection of the fusion gene transcripts (BCR-ABL) was investigated in some Nigerians with CML. MATERIALS AND METHODS: Real-time (RT)-polymerase chain reaction (PCR) analysis was used to detect BCR-ABL1 fusion gene in the saliva and blood of 42 Nigerian CML patients. RNA was extracted using RNeasy kit and reverse transcribed by random hexamer priming using murine Moloney reverse transcriptase. BCR-ABL1 transcript types were first detected by multiplex PCR and then quantified by a duplex RT-PCR-TaqMan chemistry with MGB probe and Black Hole Quencher. RESULTS: Of the 42 subjects, transcript types were detected in 36 (85.7%) samples, e13a2 fusion transcript sub-type was detected in 9 (21.4%), whereas e14a2 subtype was found in 27 (67.3%); six (14.3%) of the samples did not reveal any of the fusion transcript subtypes. The median BCR-ABL1 messenger RNA values were 9.38 × 102 in saliva and 10.29 × 104 in blood (P < 0.05). Similarly, the median ABL1 value in saliva (3.11 × 103) was significantly lower (P < 0.01) than in blood (4.22 × 103). However, the median BCR-ABL1 ratio in saliva (14.5%) was not significantly different (P = 0.8) from that of blood (12.0%). CONCLUSION: Saliva may offer an alternative easy-to-collect, readily available, and noninvasive sample for the diagnosis and treatment of CML.