Rapid screening and diagnosis of tuberculosis: a real challenge for the mycobacteriologist.

Tuberculosis (TB) is an infectious, devastating and contagious disease, which infects third of the global population worldwide with high rates of incidence in the developing countries, where the health care providers face a serious problem and a real challenge during their clinical practice for controlling and preventing the transmission of this illness. Indeed the first step of control is the correct diagnosis and the initiation of the drug treatment regimen at the early stage of infection, which mandate the rapidity of screening and the accuracy of laboratory testing. In this paper we aim to highlight the different actual techniques, regarding the rapid screening and diagnosis of tuberculosis.

[Hepatitis B prevalence and risk factors in Morocco]. / Prévalence de l'infection par le virus de l'hépatite B et l'évaluation des facteurs de risque au Maroc.

AIM: The aim of this study was to determine the prevalence of hepatitis B and the risk factors in Morocco. STUDY DESIGN: A total number of 16,634 individuals were screened for HBsAg using the Murex HBsAg Version 3 assay and were interviewed using a structured standard questionnaire to collect information about risk factor. RESULTS: Two hundred seventy-six subjects were positive for HBsAg, the prevalence of HBV infection was 1.66%. Using a structured standard questionnaire we reported that sexual behaviours (43.84%) are among the main risk factors for HBV transmission. CONCLUSION: This study indicates that the prevalence of HBsAg in Morocco is currently estimated at 1.66% in the active population. The risk factors for HBV infection identified here indicate that prevention is the most cost-effective method for successfully controlling HBV infection, so vaccination remains the best way to control this infection and its related complications.

In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells.

The antiproliferative effect of different extracts obtained from Retama monosperma L. was investigated on human SiHa and HeLa cervical cancer cell lines using a MTT colorimetric assay. The Retama monosperma L. dichloromethane fraction (Rm-DF) was the most active extract, exhibiting a significant cytotoxic activity on both cell lines in a dose-dependent manner, after 72 h of treatment. IC50 values obtained were 14.57 ± 4.15 µg/ml and 21.33 ± 7.88 µg/ml, for SiHa and HeLa cell lines respectively. The morphological features assessment of apoptosis in Rm-DF-treated cells showed a condensation of chromatin and apoptotic bodies, accompanied by a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species in both cell lines. The induction of apoptosis was further confirmed by Western blotting pro-caspase 3, Bcl2 and PARP; caspase 3 activity assay; and Annexin V labelling. Analysis of Rm-DF by CG/MS revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10,97%), L-methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). This study shows that Retama monosperma L. extract exhibits a potential anticancer activity against cervical cancer cell lines in vitro through the inhibition of proliferation and induction of apoptosis, which may involve a mitochondria-mediated signaling pathway.

Polyfunctional Mycobacterium tuberculosis-specific effector memory CD4+ T cells at sites of pleural TB.

Pleural tuberculosis (TB) is a common presentation of Mycobacterium tuberculosis (MTB) infection, and despite spontaneous resolution remains a strong risk factor for reactivation pulmonary TB in a majority of individuals. This study was undertaken to further understand the characteristics of immune cells at sites of pleural TB. A significant shift toward memory CD4+ T cells with an effector phenotype and away from naïve CD4+ T cells in pleural fluid as compared to blood mononuclear cells was found. These data suggest that effector T cells are capable of migrating to sites of active TB infection and/or the differentiation to effector phenotype T cells in situ is highly amplified. Using multi-parameter flow cytometry analysis, a significant portion of MTB-specific CD4+ T cells in the pleural space were polyfunctional demonstrating two, three or four simultaneous functions including IFN-gamma, IL-2, TNF-alpha, and or MIP-1 alpha production. A greater proportion of these polyfunctional cells were of effector memory rather than central memory phenotype. The role of these polyfunctional MTB-specific CD4+ T cells at sites of pleural TB requires further study.

Use of pathophysiological indicators for individual decision of anthelmintic treatment of ewes against gastro-intestinal nematodes in Morocco.

The targeted selective treatments (TST) aim at reducing the number of anthelmintic treatments but also to maintain productivity of animals. The aim of this work was to assess the validity of pathophysiological indicators for detecting individually ewes in need for treatments in two regions of Morocco with different management and climatic environment (Chaouia plain-seven farms, and Middle-Atlas-three farms). Although resistance to benzimidazoles was already present the same drug was used for TST. The indicators tested were: FAMACHA(©) (associated with anaemia), DISCO (diarrhoea score), and BODCON (body condition score). Only FAMACHA(©) and DISCO indicators were well correlated to the EPGs. DISCO only did permit a substantial reduction (up to 85%) of the number of treatment and EPG (nematode eggs per gramme) remained low on average (less than 160).

Mycobacterial species as case-study of comparative genome analysis.

The genus Mycobacterium represents more than 120 species including important pathogens of human and cause major public health problems and illnesses. Further, with more than 100 genome sequences from this genus, comparative genome analysis can provide new insights for better understanding the evolutionary events of these species and improving drugs, vaccines, and diagnostics tools for controlling Mycobacterial diseases. In this present study we aim to outline a comparative genome analysis of fourteen Mycobacterial genomes: M. avium subsp. paratuberculosis K­10, M. bovis AF2122/97, M. bovis BCG str. Pasteur 1173P2, M. leprae Br4923, M. marinum M, M. sp. KMS, M. sp. MCS, M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis H37Ra, M. tuberculosis H37Rv, M. tuberculosis KZN 1435 , M. ulcerans Agy99,and M. vanbaalenii PYR­1, For this purpose a comparison has been done based on their length of genomes, GC content, number of genes in different data bases (Genbank, Refseq, and Prodigal). The BLAST matrix of these genomes has been figured to give a lot of information about the similarity between species in a simple scheme. As a result of multiple genome analysis, the pan and core genome have been defined for twelve Mycobacterial species. We have also introduced the genome atlas of the reference strain M. tuberculosis H37Rv which can give a good overview of this genome. And for examining the phylogenetic relationships among these bacteria, a phylogenic tree has been constructed from 16S rRNA gene for tuberculosis and non tuberculosis Mycobacteria to understand the evolutionary events of these species.

Rubella seroprevalence among women aged 15-39 years in Morocco.

This study was designed to determine the age-specific rubella seroprevalence in women of childbearing age in Morocco and to contribute to the development of a rubella vaccination strategy in the country. Of 967 women aged 15-39 years tested in 2000, 161 (16.6%) were susceptible to rubella based on absence of IgG antibodies. A significantly higher rate of susceptibility among women aged 15-19 years was observed (29.3%) compared with age 35-39 years (8.3%). An estimated 77,562 live births occur annually to rubella-susceptible women. No statistical difference in seroprevalence was seen between women in rural and urban areas (81.5% and 85.0% respectively). A substantial risk of rubella infection exists for Moroccan women of childbearing age.

Evaluation of a multi-antigen test based on B-cell epitope peptides for the serodiagnosis of pulmonary tuberculosis.

SETTING: Two sample panels: 1) 20 pulmonary tuberculosis (PTB) patients and 10 healthy subjects from a country with a low incidence of TB (Italy); and 2) 47 PTB patients and 26 healthy subjects from a country with a high incidence of TB (Morocco). OBJECTIVE: To identify a combination of Mycobacterium tuberculosis peptides useful for the serodiagnosis of active PTB. METHODS: Fifty-seven B-cell epitope peptides of M. tuberculosis were evaluated by immunoenzymatic assay and the data were analysed using logistic regression analysis and the random forest method. RESULTS: The best discriminating peptide between PTB patients and healthy subjects from the sample of the low TB incidence country was the 23 amino acid peptide of the Rv3878 protein. The sensitivity and specificity were respectively 65% and 100%. The same peptide had a sensitivity and specificity of respectively 47% and 100% for the sample from the high TB incidence country. The best combination of peptides was a pool of nine peptides which had a sensitivity of 70.2% and a specificity of 100% in the high TB incidence country. CONCLUSIONS: The 9-peptide pool can be useful in identifying patients with active PTB.

Rubella seroprevalence among women aged 15-39 years in Morocco

This study was designed to determine the age-specific rubella seroprevalence in women of childbearing age in Morocco and to contribute to the development of a rubella vaccination strategy in the country. Of 967 women aged 15-39 years tested in 2000, 161 [16.6%] were susceptible to rubella based on absence of IgG antibodies. A significantly higher rate of susceptibility among women aged 15-19 years was observed [29.3%] compared with age 35-39 years [8.3%]. An estimated 77 562 live births occur annually to rubella-susceptible women. No statistical difference in seroprevalence was seen between women in rural and urban areas [81.5% and 85.0% respectively]. A substantial risk of rubella infection exists for Moroccan women of childbearing age

Frequency of the severe combined immunodeficiency disease gene among horses in Morocco.

Severe combined immunodeficiency disease (SCID) of horses is an autosomal, recessive hereditary disease occurring among Arabian or crossbred Arabian horses. The genetic defect responsible was previously identified as a 5-base pair deletion in the gene encoding the catalytic subunit of the DNA dependant protein kinase (DNA-PKcs). This study was carried out to determine the frequency of SCID and identify horses carrying the gene for SCID among Arabian and Arabian crossbred stallions and mares in Morocco using a DNA-based test. Twenty-one horses were SCID carriers: 14 (7%) Arabians, 6 (4%) Arab-Barbs and one (33%) Anglo-Arab. After analysing their genealogy, 3 imported stallions were identified that disseminated the mutant gene of DNA-PKcs in Morocco.

Protective effect of saponins from Argania spinosa against free radical-induced oxidative haemolysis.

Saponins from Argania spinosa at a non-haemolytic concentration diminish by 53.2% erythrocyte haemolysis induced by free radicals. 2 mM aspirin and acetaminophen diminish by 75% and 68% , respectively, erythrocyte haemolysis induced by free radicals, while 0.3 microM vitamin E shows no significant antioxidant activity. Interestingly, a combination of 1 mg/l of A. spinosa saponins and vitamin E at 0.3 microM resulted in a 68% level of protection against free radical-induced erythrocyte haemolysis, which may suggest that A. spinosa saponins enhance the antioxidant effect of vitamin E. In contrast, no synergic effect was observed for acetaminophen (2 mM) when in combination with vitamin E (0.3 microM). These results demonstrate the antioxidant properties of saponins from A. spinosa and their ability to potentate the antioxidant effect of vitamin E.

[Diagnosis of pulmonary tuberculosis: evaluation of the TB IgA EIA assay in Morocco]. / Diagnostic de la tuberculose pulmonaire: evaluation du test "TB IgA EIA" au Maroc.

We analysed 2 evaluation lots of the TB IgA EIA test in pulmonary tuberculosis patients (TBp). Sera were obtained from 345 TBp, 18 healthy subjects (HS), 28 subjects in contact with tuberculous patients (CS) and 16 non-tuberculous lung disease patients (N-TB) for the first evaluation lots and 302 TBp, 60 HS, 21 CS and 18 N-TB for the second. IgA titres against p-90 antigen with the second evaluation lot were significantly higher than the first evaluation lot. With the second evaluation lots, the sensitivity was 78.8% whereas with the first evaluation lot, the sensitivity was 75.9%. Specificity for the first and second evaluation lots was 50% and 70.7% respectively. The sensitivity of this test is still not satisfactory to establish pulmonary tuberculosis diagnosis.

[Diagnosis of pulmonary tuberculosis: evaluation of the TB IgA EIA assay in Morroco]

We analysed 2 evaluation lots of the TB IgA EIA test in pulmonary tuberculosis patients [TBp]. Sera were obtained from 345 TBp, 18 healthy subjects [HS], 28 subjects in contact with tuberculous patients [CS] and 16 non-tuberculous lung disease patients [N-TB] for the first evaluation lots and 302 TBp, 60 HS, 21 CS and 18 N-TB for the second. IgA titres against p-90 antigen with the second evaluation lot were significantly higher than the first evaluation lot. With the second evaluation lots, the sensitivity was 78.8% whereas with the first evaluation lot, the sensitivity was 75.9%. Specificity for the first and second evaluation lots was 50% and 70.7% respectively. The sensitivity of this test is still not satisfactory to establish pulmonary tuberculosis diagnosis

Antibacterial and antifungal activity of essential oils of Mentha suaveolens.

The essential oils (EO) of Mentha suaveolens, a wild Labiatae, which grows in several regions in Morocco, were characterized and their antimicrobial activity assessed. The main aromatic constituents of this plant, as characterized by IR, NMR and MS studies, were pulegone, piperitenone oxide (PEO) and piperitone oxide (PO) occurring in different amounts depending on the subspecies. These constituents as well as a series of other aromatic products such as carvone, limonene and menthone, were tested for their antimicrobial activity against 19 bacteria including Gram-positive and Gram-negative and against three fungi, using solid phase and microtitration assays. Pulegone-rich essential oil inhibited efficiently all the micro-organisms tested with MICs ranging between 0.69 and 2.77 ppm. Among the components from Mentha suaveolens EO, pulegone was the most effective against the tested microorganisms, followed by PEO and PO. The structure-activity relationship is discussed on the basis of the activity of the other aromatic derivatives tested such as carvone, limonene, menthone and the profile of the essential oils of Mentha suaveolens was compared with other Mentha species.

Some characteristics of the HIV epidemic in Morocco.

The first case of AIDS in Morocco was declared in 1986 and since then the number of AIDS cases has steadily increased. According to the Ministry of Health, the cumulative number of AIDS cases in December 2002 was 1085. HIV in Morocco is acquired mainly through heterosexual intercourse. Individuals aged between 30 and 39 years and in the regions of Marrakech and Agadir have been the most affected. Monitoring of the trend of the epidemic by sentinel surveillance surveys indicates that Morocco is still a low prevalence zone, since prevalence among pregnant women is less than 1%. The estimated number of HIV-infected people in Morocco is around 15 000. It is not clear why the epidemic here has not evolved as it has in the sub-Saharan countries where it is spreading at an alarming rate. Late introduction of HIV-1 subtype B in Morocco, which is relatively less transmissible, circumcision and reduced risk behaviours of Muslims may explain this. Nonetheless, because prevalence has increased in recent years, unless preventive measures are strengthened, the HIV epidemic will worsen in Morocco.

Some characteristics of the HIV epidemic in Morocco

The first case of AIDS in Morocco was declared in 1986 and since then the number of AIDS cases has steadily increased. According to the Ministry of Health, the cumulative number of AIDS cases in December 2002 was 1085. HIV in Morocco is acquired mainly through heterosexual intercourse. Individuals aged between 30 and 39 years and in the regions of Marrakech and Agadir have been the most affected. Monitoring of the trend of the epidemic by sentinel surveillance surveys indicates that Morocco is still a low prevalence zone, since prevalence among pregnant women is less than 1%. The estimated number of HIV-infected people in Morocco is around 15 000. It is not clear why the epidemic here has not evolved as it has in the sub-Saharan countries where it is spreading at an alarming rate. Late introduction of HIV-1 subtype B in Morocco, which is relatively less transmissible, circumcision and reduced risk behaviours of Muslims may explain this. Nonetheless, because prevalence has increased in recent years, unless preventive measures are strengthened, the HIV epidemic will worsen in Morocco

The susceptibility of macrophages to human immunodeficiency virus type 1 X4 isolates depends on their activation state.

The demonstration that macrophages express CXCR4 has led to a reexamination of their susceptibility to human immunodeficiency (HIV)-1 X4 strains. Here, we examined the susceptibility to X4 HIV-1Lai of two previously characterized macrophage populations, obtained either as 1) adherent cells of five-day cultures of blood mononuclear cells (PBMC), followed by two days without nonadherent PBMC nor added cytokines (MDM-5d); or 2) as adherent cells recovered from one-hour incubation of PBMC, which were cultured for seven days with macrophage colony-stimulating factor (MDM-MCSF). Exposing MDM-5d or MDM-MCSF to HIV-1Lai did not lead to productive infection, as indicated by a lack of (MDM-MCSF) or low (MDM-5d) viral p24 levels in culture supernatants. However, MDM-5d vigorously transmitted HIV-1 Lai to autologous T lymphocytes, which was not the case of HIV-1Lai-exposed MDM-MCSF. PCR analysis of the LTR RU5 region showed that X4 HIV-1Lai entered into both types of macrophages in the same manner as R5 HIV-1 BaL. However, in contrast to MDM-5d, there was a block of HIV-1 Lai retrotransciption in MDM-MCSF. Cytokine profile analysis of the two types of macrophages showed that TNF-alpha, IL-6 and RANTES levels were higher in MDM-5d than in MDM-MSCF, while the IL10 level was higher in MDM-MCSF, both producing similar IL16 levels. Altogether, these data indicate that HIV-1 X4 strains enter into macrophages but that their replication is blocked thereafter in a different manner according to the activation status of the cells.

The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication.

Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DC(CD40LT)), or monocyte-conditioned medium (DC(MCM)) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1(LAI) or R5 HIV-1(Ba-L) were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4(low) (CXCR4(low)) CCR5(+) iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1(LAI) entered more efficiently into DC(CD40LT) or DC(MCM) with up-regulated CXCR4. CCR5(low) DC(CD40LT) still allowed entry of HIV-1(Ba-L), whereas CCR5(-) DC(MCM) displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1(Ba-L) reverse transcription was more efficient than that of HIV-1(LAI), but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3.GFP or pNL4-3.Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60-75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-kappa B proteins and SP1 and SP3 transcription factors.

Soluble glycosaminoglycans Do not potentiate RANTES antiviral activity on the infection of primary macrophages by human immunodeficiency virus type 1.

Macrophages play an important role in human immunodeficiency virus (HIV)-1 infection. They exist in various differentiation and activation states in vivo, a heterogeneity that may affect their interactions with HIV-1 and susceptibility to drugs. Here, we found that RANTES and MIP-1beta, heparin, or soluble chondroitin sulfate B, but not chondroitin sulfate A, inhibited HIV-1(BaL) infection of macrophages obtained as the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without either nonadherent PBMC or added cytokines (MDM-5d), whereas they did not affect infection of macrophages obtained as the adherent cells recovered from 1-h incubation of PBMC and subsequent 7-day culture with macrophage colony-stimulating factor (MDM-MCSF). Such different behavior was not related to differences in HIV-1 binding but rather to postbinding steps, as HIV-1(BaL) attached similarly to MDM-5d and MDM-MCSF, a binding that was affected by soluble glycosaminoglycans but not by RANTES. Of note, CCR5 expression on both types of MDM was comparable, and it was not downregulated by RANTES on either. Mixing RANTES with each of the glycosaminoglycans did not restore inhibition of MDM-MCSF infection by HIV-1; however, heparin at concentrations that had low antiviral activity for MDM-5d counteracted RANTES anti-HIV-1 activity for these cells, whereas chondroitin sulfate B had no additive effect on that of RANTES. Both glycosaminoglycans affected RANTES binding to MDM. Thus, in contrast to cell surface proteoglycans that contribute to the attachment of RANTES to macrophages and enhance its anti-HIV-1 activity, soluble glycosaminoglycans do not facilitate, and may even offset, the anti-HIV-1 activity of RANTES.