Ochratoxin a in breast milk in Morocco: the affecting dietary habits of the lactating mothers and the degree of exposure of newborns "CONTAMILK study".

The aim of the present study is to evaluate the level of contamination of breast milk (BM) by ochratoxin A, among Moroccan lactating mothers in the city of Rabat, and to identify the associated factors of exposure, also to estimate the degree of exposure of the breastfeed infant. The analysis of ochratoxin A (OTA) was accomplished by ELISA method on 82 colostrum samples. OTA was detectable (>0.08 ng/mL) in 55% of samples with a maximum concentration of 10.04 ng/mL, and the levels exceeded 0.5 ng /mL in 50 % of the samples. In addition, several factors and dietary habits affect significantly the level of OTA in the analyzed samples of breast milk including, the consumption of industrial dairy products, the frequency of consumption of canned foods, dried fruits and legumes, also the period of breast milk collection. Besides, OTA was higher than the tolerable daily intake for 49% newborns. However, these results need to be confirmed by multicenter studies to more broadly estimate the levels of exposition of Moroccan population to OTA. Furthermore, awareness campaigns are recommended to inform the public, especially pregnant women and lactating women about appropriate preventive measures to limit exposure to this mycotoxin.

Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection.

Lateral flow assays (lateral flow immunoassays and nucleic acid lateral flow assays) have experienced a great boom in a wide variety of early diagnostic and screening applications. As opposed to conventional examinations (High Performance Liquid Chromatography, Polymerase Chain Reaction, Gas chromatography-Mass Spectrometry, etc.), they obtain the results of a sample's analysis within a short period. In resource-limited areas, these tests must be simple, reliable, and inexpensive. In this review, we outline the production process of antibodies against drugs of abuse (such as heroin, amphetamine, benzodiazepines, cannabis, etc.), used in lateral flow immunoassays as revelation or detection molecules, with a focus on the components, the principles, the formats, and the mechanisms of reaction of these assays. Further, we report the monoclonal antibody advantages over the polyclonal ones used against drugs of abuse. The perspective on aptamer use for lateral flow assay development was also discussed as a possible alternative to antibodies in view of improving the limit of detection, sensitivity, and specificity of lateral flow assays.

Development and validation of an RT-qPCR assay for rapid detection and quantification of hepatitis C virus RNA for routine testing in Moroccan clinical specimens.

A one-step reverse transcription quantitative PCR (RT-qPCR) assay in combination with rapid RNA extraction was evaluated for routine testing of hepatitis C virus (HCV) RNA. Specific primers and probes were designed for the detection of a 150 bp sequence located in the 5'untranslated region (5'UTR) of HCV RNA. The target sequence was selected as the most conserved region between the six known HCV subtype sequences following an alignment. The assay was able to quantify a dynamic linear range of 108 to 101 plasmid copies/reaction (r2 = 0.98) containing the target sequence. Two copies of this HCV plasmid corresponds to one international unit (IU) measured using a standard obtained by serial dilutions of the World Health Organization (WHO) standard. The detection limit of the assay was about 10 IU/mL of HCV RNA (20 copies/mL) in plasma samples. The assay was comparable to Cobas AmpliPrep/Cobas TaqMan® HCV Test, v2.0 Quantitative assay (Roche Molecular Systems, Inc., Branchburg, NJ) with correlation coefficient r2 = 0.98. The present assay could be completed within 3 hours from RNA extraction to data analysis of at least 30 plasma samples. Our test provides sufficient sensitivity, specificity, and reproducibility and proved to be fast, labor-saving, and cost-effective. Indeed, our system will definitely allow low-income countries to monitor accurately this viral infection and to efficiently treat their infected patients.

Human papillomavirus genotypes distribution by cervical cytologic status among women attending the General Hospital of Loandjili, Pointe-Noire, Southwest Congo (Brazzaville).

HPV infection is associated with cervical cancer, one of the major public health problems in developing countries. In the Republic of Congo, despite of the high age-standardized incidence rate estimated at 25.2 per 100,000 women, molecular epidemiology data on HPV infections are very limited. We investigated HPV genotypes distribution in cervical smears among patients attending the General Hospital of Loandjili, Southwest Congo. A cross-sectional hospital-based study was conducted on 321 women. Liquid-based cytology samples were collected for cytological diagnosis and HPV detection. Nested-PCR was performed using MY09/MY11 and GP5+/GP6+ primers with genotyping by direct sequencing. Type-specific PCR for HPV-6, -11, -16, -18, -31 and -33 was also used to assess multiple infections. Out of 321 women examined, 189 (58.8%) had normal cytology, 16 (5.0%) had ASCUS and 116 (36.1%) had cytological abnormalities. HPV-DNA was detected in 22 (11.6%), 6 (37.5%), and 104 (89.6%) normal cytology, ASCUS and cytological abnormalities respectively. HPV16 was the most prevalent genotype regardless of cytological status followed by HPV70 in women without lesions and HPV33 among those with lesions. HR-HPV prevalence varied significantly according to the cervical cytology (P = 0.000). Among women without lesions, two peaks of HPV infections were observed in age group less than 30 years (60.0%) and in age group 50-59 years (7.1%). Age, age of first sex, multiple sexual partners and pregnancies were the risk factors for HPV infection in women without lesions. Our findings could be used as evidence data base for future epidemiological monitoring in this region.

Alteration of glucose metabolism and expression of glucose transporters in ovarian cancer.

Aerobic glycolysis also known as the Warburg effect, remains a hallmark of various cancers, including ovarian cancer. Cancer cells undergo metabolic changes to sustain their tumorigenic properties and adapt to environmental conditions, such as hypoxia and nutrient starvation. Altered metabolic pathways not only facilitate ovarian cancer cells' survival and proliferation but also endow them to metastasize, develop resistance to chemotherapy, maintain cancer stem cell phenotype, and escape anti-tumor immune responses. Glucose transporters (GLUTs), which play a pivotal role as the rate-limiting step in glycolysis, are frequently overexpressed in a variety of tumors, including ovarian cancer. Multiple oncoproteins can regulate GLUT proteins, promoting tumor proliferation, migration, and metastasis, either dependent or independent of glycolysis. This review examines the alteration of GLUT proteins, particularly GLUT1, in ovarian cancer and its impact on cancer initiation, progression, and resistance to treatment. Additionally, it highlights the role of these proteins as biomarkers for diagnosis and prognosis in ovarian cancer, and delves into novel therapeutic strategies currently under development that target GLUT isoforms.

Analysis of mutations in the E6 oncogene of human papillomavirus 16 in cervical cancer isolates from Moroccan women.

BACKGROUND: Worldwide, cervical cancer is the second most common cancer in women. High-risk human papillomavirus (HPV) play a crucial role in the etiology of cervical cancer and the most prevalent genotype is HPV16. HPV 16 intratypic variants have been reported to differ in their prevalence, biological and biochemical properties. The present study was designed to analyze and identify HPV type 16 E6 variants among patients with cervical cancer in Morocco. METHODS: A total of 103 HPV16 positive samples were isolated from 129 cervical cancer cases, and variant status was subsequently determined by DNA sequencing of the E6 gene. RESULTS: Isolates from patients were grouped into the European (E), African (Af) and North-American (NA1) phylogenetic clusters with a high prevalence of E lineage (58.3%). The Af and NA1 variants were detected in 31.1% and 11.6% of the HPV16 positive specimens, respectively, whereas, only 3% of cases were prototype E350T. No European-Asian (EA), Asian (As) or Asian-American (AA) variants were observed in our HPV16-positive specimens. At the amino acid level, the most prevalent non-synonymous variants were L83V (T350G), H78Y (C335T), E113D (A442C), Q14D (C143G/G145T) and R10I (G132T), and were observed respectively in 65%, 41.8%, 38.8%, 30.1% and 23.3% of total samples.Moreover, HPV16 European variants were mostly identified in younger women at early clinical diagnosis stages. Whereas, HPV16 Af variants were most likely associated with cervical cancer development in older women with pronounced aggressiveness. CONCLUSION: This study suggests a predominance of E lineage strains among Moroccan HPV 16 isolates and raises the possibility that HPV16 variants have a preferential role in progression to malignancy and could be associated with the more aggressive nature of cervical cancer.

The Tumor Suppressor BRCA1/2, Cancer Susceptibility and Genome Instability in Gynecological and Mammary Cancers.

BRCA1 and BRCA2 germline alterations highly predispose women to breast and ovarian cancers. They are mostly found within the TNBC (Triple-Negative Breast Cancer) and the HGSOC (High-Grade Serous Ovarian Carcinoma) subsets, known by an aggressive phenotype, the lack of therapeutic targets and poor prognosis. Importantly, there is an increased risk for cervical cancer in BRCA1 and BRCA2 mutation carriers that raises questions about the link between the HPV-driven genome instability and BRCA1 and BRCA2 germline mutations. Clinical, preclinical, and in vitro studies explained the increased risk for breast and ovarian cancers by genome instability resulting from the lack or loss of many functions related to BRCA1 or BRCA2 proteins such as DNA damage repair, stalled forks and R-loops resolution, transcription regulation, cell cycle control, and oxidative stress. In this review, we decipher the relationship between BRCA1/2 alterations and genomic instability leading to gynecomammary cancers through results from patients, mice, and cell lines. Understanding the early events of BRCA1/2-driven genomic instability in gynecomammary cancers would help to find new biomarkers for early diagnosis, improve the sensitivity of emerging therapies such as PARP inhibitors, and reveal new potential therapeutic targets.

BRCA1 Promoter Hypermethylation in Malignant Breast Tumors and in the Histologically Normal Adjacent Tissues to the Tumors: Exploring Its Potential as a Biomarker and Its Clinical Significance in a Translational Approach.

The hypermethylation status of the promoter region of the breast cancer 1 (BRCA1), a well-known tumor suppressor gene, has been extensively investigated in the last two decades as a potential biomarker for breast cancer. In this retrospective study, we investigated the prevalence of BRCA1 promoter methylation in 84 human breast tissues, and we correlated this epigenetic silencing with the clinical and histopathological parameters of breast cancer. We used methylation-specific PCR (MSP) to analyze BRCA1 promoter hypermethylation in 48 malignant breast tumors (MBTs), 15 normal adjacent tissues (NATs), and 21 benign breast lesions (BBLs). The results showed that BRCA1 promoter hypermethylation was higher in MBTs (20/48; 41.67%) and NATs (7/15; 46.67%) compared to BBLs (4/21; 19.05%). The high percentage of BRCA1 hypermethylation in the histologically normal adjacent tissues to the tumors (NATs) suggests the involvement of this epigenetic silencing as a potential biomarker of the early genomic instability in NATs surrounding the tumors. The detection of BRCA1 promoter hypermethylation in BBLs reinforces this suggestion, knowing that a non-negligible rate of benign breast lesions was reported to evolve into cancer. Moreover, our results indicated that the BRCA1 promoter hypermethylated group of MBTs exhibited higher rates of aggressive features, as indicated by the SBR III grade (14/19; 73.68%), elevated Ki67 levels (13/16; 81.25%), and Her2 receptor overexpression (5/20; 25%). Finally, we observed a concordance (60%) in BRCA1 promoter hypermethylation status between malignant breast tumors and their paired histologically normal adjacent tissues. This study highlights the role of BRCA1 promoter hypermethylation as a potential useful biomarker of aggressiveness in MBTs and as an early marker of genomic instability in both histological NATs and BBLs.

Worldwide genetic variations in high-risk human papillomaviruses capsid L1 gene and their impact on vaccine efficiency.

BACKGROUND: Human papillomavirus is the most common sexually transmitted infection. It is associated with different cancers, mainly cervical cancer, which remains the fourth most frequent cancer among women worldwide; it is also related to anogenital (anus, vulvar, vagina, and penis) and oropharyngeal cancers. Vaccination against HPV infection is the major way of prevention, and it has demonstrated impressive efficacy in reducing cervical cancer incidence. Nowadays, all the licensed HPV recombinant vaccines were designed based on HPV major capsid L1 protein. However, some variations in the HPV L1 gene sequence may induce structural changes within the L1 protein, which may alter the affinity and interaction of monoclonal antibodies (MAbs) with L1 protein epitopes, and influence host immune response and recognition. Hence, the importance of accuracy in delineating epitopes relevant to vaccine design and defining genetic variations within antigenic regions in the L1 gene to predict its impact on prophylactic vaccine efficiency. The present review reports the sequence variations in HR-HPV L1 gene isolates from different countries around the world, which may help to understand the effect of HPV L1 gene variations on vaccine efficiency. METHODS: Research studies were retrieved from PubMed, Google Scholar, Science direct, and the National Center for Biotechnology Information (NCBI) database. A total of 31 articles describing genetic variations within the major capsid L1 gene and conducted in Africa, Europe, America and Asia were found. Only 26 studies conducted on HPV16, 18, 31, 33, 58, 45 and 52 which are the targets of HPV prophylactic vaccines, and which reported genetic variations within the L1 gene, were selected and evaluated in this review. FINDINGS: We found a total of 87, 49, 11, 7, 22, 3, and 17 non-synonymous single nucleotide polymorphisms (SNPs) within HPV16, HPV18, HPV31, HPV58, HPV45, and HPV52 L1 gene, respectively. Four mutations were frequently observed in HPV16 L1 sequences: T353P in the HI loop, H228D in the EF loop, T266A in the FG loop, and T292A in the FG loop. Two mutations in HPV58 L1 sequences: T375N in the HI loop and L150F in the DE loop. Three mutations in HPV33 L1 sequences: T56N in the BC loop, G133S in the DE loop, T266K in the FG loop. Other mutations were found in HPV18, HPV45, and HPV52 L1 sequences. Some were found in different countries, and others were specific to a given population. Furthermore, some variations were located on peptide binding epitopes and lead to a modification of epitopes, which may influence MAbs interactions. Others need further investigations due to the lack of studies. CONCLUSION: This study investigated the major capsid L1 genetic diversity of HPV16, 18, 31, 33, 58, 45, and 52 circulating in different populations around the world. Further investigations should be conducted to confirm their effect on immunogenicity and prophylactic vaccine efficiency.

Benzodiazepines use in Morocco: A nation wide consumption database study between 2004 and 2017.

Although information exists regarding the rate of benzodiazepines (BZDs) use in different countries, little information is available concerning the BZDs consumption in Morocco. To describe prescription rate in Morocco, a retrospective descriptive analysis of BZDs and their agonists use with the instituteIQIVIA database was performed during the period 2004-2017. The obtained data provide a dynamic approach to total BZDs consumption using an annual collection of sales data in Morocco and were expressed in terms of daily defined doses/ 1000 inhabitants / day. Data analysis showed that the major BZDs sold in Morocco were Alprazolam, Bromazepam, Nordazepam, Lorazepam, Parazepam, Diazepam and two benzodiazepine agonists, Zolpidem and Zopiclone. The Bromazepam was the molecule the most consumed during 2004-2016. In 2017, Alprazolam was the most consumed followed by Bromazepam, Nordazepam, Zolpidem, Lorazepam, Parazepam, Diazepam, Dipotassium clorazepate, Dipotassium Clorazypate and Zopiclone with 0.94, 0.91, 0.6, 0.55, 0.45, 0.32, 0.18, 0.18, 0.07 and 0.05 daily defined doses/ 1000 inhabitants / day respectively. The total amount consumed each year for all BZDs and their agonists in Morocco was 2.69, 2.77, 3, 3.17, 3.32, 3.54, 3.61, 3.81, 4.06, 4.30, 4.06, 3.94, 3.78 and 3.66 daily defined doses/ 1000 inhabitants / day, respectively during 2004-2017. To our knowledge, this is the first study that describes the consumption of BZDs and their agonists (Zolpidem, Zopiclone) in Morocco. This data may help the analytical toxicology laboratory and health organizations operating in the field of analytical biochemistry to develop specific BZDs quantification and detection methods needed for the Moroccan population.

Aflatoxin M1 prevalence in breast milk in Morocco: Associated factors and health risk assessment of newborns "CONTAMILK study".

The aim of this study was to assess the contamination of breast milk by aflatoxin M1 among nursing mothers from Rabat, Morocco, and to explore its association with several maternal parameters and dietary habits. In addition, the health risk assessment of the newborns by the estimation of the daily intake. A competitive Enzyme Linked Immunosorbent Assay method was used for the analysis of aflatoxin M1 in breast milk samples. Analytical results indicate that out of 82 total samples, 43 samples (52.4%) of milk were positive. Aflatoxin M1 levels ranged from undetectable to 13.33 ng/L, while the mean level was 5.75 ± 3.44 ng/L. Besides, several factors and foodstuffs seem to increase the level of AFM1 in breast milk. As regards the estimated daily intake of aflatoxin M1, it varies between immeasurable and a maximum of 1.16 ng/kg.bw. The degree of exposure to AFB1 and the levels of its metabolite AFM1 in breast milk were low, compared to some studies from other countries. Further investigations and periodic monitoring programs are recommended in large samples and in many cities of morocco to assess the level of exposure of the Moroccan population.

Analytical Methods Used for the Detection and Quantification of Benzodiazepines.

The prescription of psychotropic drugs, especially benzodiazepines (BZDs), occupies a preponderant place in the management of mental illnesses. Indeed, the BZDs have been used in different therapeutic areas including insomnia, anxiety, seizure disorders, or general anesthesia. Unfortunately, these drugs are present in the illegal street market, leading to a lot of drug abuse amongst some addicted users, road insecurity, and suicide. Hence, it has become essential to analyze the BZDs drugs in human biological specimens for drug abuse in forensic sciences. The present review provides a summary of sample preparation techniques (solid-phase extraction and Liquid-liquid phase extraction) and the methods for the detection and quantification of BZDs molecules in the commonly used biological specimens over the ten last years which may potentially lead to better and accurate evaluation of the physiological state of a given person. The commonly used methods for the detection and quantification of BZDs include nuclear magnetic resonance (NMR), chromatography (GC-MS, HPLC, and TLC), immunoassay (ELISA, RIA, LFA, CEDEA, FPIA, and KIMS), and electroanalytical methods (voltammetry and potentiometry).

Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the first study using the specific combination of RPL30 and RPL37 as reference genes for an accurate HER2 gene quantification in FFPE biopsy samples. Although further clinical validation regarding evolution and therapeutic response using RT-qPCR for the quantification of HER2 expression are still needed, the present study constitutes definitely a factual element that the RT-qPCR based assay may constitute a valid complementary test to accurately measure HER2 expression for a better treatment orientation.

Evaluation of Combined Quantification of PCA3 and AMACR Gene Expression for Molecular Diagnosis of Prostate Cancer in Moroccan Patients by RT-qPCR

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

Detection of MMTV-Like sequences in Moroccan breast cancer cases.

INTRODUCTION: The mouse mammary tumor virus (MMTV) like sequences have been reported to be present in some human breast cancers, but their association with breast cancer development is still controversial. METHODS: In this retrospective study, we investigated the status of MMTV-like in 42 tumor biopsies and 18 paired normal tissues from Moroccan patients with breast cancer. MMTV-like env sequences were identified by PCR and confirmed by direct DNA sequencing. RESULTS: Specific MMTV-like env sequences were found in 24 (57.14%) cases of breast carcinomas, and 6 (33.3%) cases of matched normal breast tissues. Comparison to sociologic and clinicopathological parameters showed no significant association between the presence of MMTV-like sequences and age, menopausal status, histological subtype, histological grade, tumor size and the expression of hormone receptors (estrogen ER and/or progesterone PgR) and Her 2. However, a significant correlation was found between MMTV-like presence and parity (p = 0.024). CONCLUSIONS: This present study confirms the presence of MMTV-like env sequences in breast cancer in Moroccan women, prompting further evaluation, on large sampling, to elucidate the probable causal roles of MMTV-like in breast cancer development.

Human papillomavirus detection in Moroccan patients with bladder cancer.

INTRODUCTION: Human papillomavirus (HPV) is associated with more human cancers than any other virus. Many studies have investigated the association between bladder cancer and HPV but the results remain controversial. The aim of the present study is to evaluate whether HPV have an etiological role in bladder carcinogenesis among Moroccan patients. METHODOLOGY: Forty-eight fresh biopsies (43 bladder tumors and 5 non-tumor samples) were collected for this purpose. Nested PCR with the consensus MY09/MY11 and GP5+/GP6+ primers was performed to detect the presence of HPV L1 gene DNA. RESULTS: The results showed that 52.4% of bladder cancer patients were positive for HPV. Subsequent DNA sequencing of positive cases of HPV revealed the presence of HPV16 in 95.5% of bladder tumor samples. The occurrence of HPV infection varies according to clinicopathological features, but there is no significant correlation between the viral infection and tumor stage or grade. In addition, statistical analysis demonstrated that there is no association between age or sex and HPV infection. CONCLUSION: Our data indicate for the first time that bladder tumors from Moroccan patients harbor HR-HPV genotypes, especially HPV16, and thereby suggest that this virus may play a causative role in bladder cancer.