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Use of Dried Blood Spot to Improve the Diagnosis and Management of HIV in Resource-Limited Settings  [PDF]
Chatté Adawaye, Erick Kamangu, Ali Mahamat Moussa, Bertin Tchoumbou, Dolores Vaira, Michel Moutschen
World Journal of AIDS (WJA) , 2013, DOI: 10.4236/wja.2013.33033

Over 75% of people infected with HIV live in countries where health resources are very limited for the diagnosis and biological monitoring of people infected by the virus. In resource-limited settings, the use of DBS is a valuable alternative. It has provided technical and economical alternative to the collection of blood in the tubes for testing HIV infection. The DBS can be kept for over a year, it is economical in storage space and facilitates storage conditions because it can be stored at room temperature. It is more discreet and easier to carry over liquid samples that require tubes and other appropriate materials. The amount is sufficient for certain analyses of DNA generally, but may be insufficient for the analysis of viral RNA if the viral load is low. Its disadvantage is often associated with small amounts of blood collected available for testing, and the difficulties encountered in laboratories to extract the maximum possibilities without material contamination. DBS can be stored at room temperature (25 - 35), at 4, -20 or even -70. With PCR, the DBS is a suitable medium for the diagnosis of patients infected with HIV, virological monitoring by the VL and even analyzing viral genotype. It is a handy stand for the collection, transport and analyses of biological monitoring of HIV infection. It is indeed very suitable for environments with limited accessibility where it is difficult for specialized laboratories to monitor these patients. The DBS is suitable for resource-limited settings.

Immunovirologic Evaluation of Triomune (Lamivudine, Stavudine and Nevirapine) Antiretroviral Therapy in First Line HIV-1 Adult Patients in N’Djamena, Chad  [PDF]
Chatté Adawaye, Kamangu Erick, Soudy I. Djibrine, Aoudalkarim Moussa Chahad, Ali Mahamat Moussa, Tchombou HZ Bertin, Vaira Dolores, Moutschen Michel
World Journal of AIDS (WJA) , 2014, DOI: 10.4236/wja.2014.43035

Contexte: The fight against HIV/AIDS epidemics is one of the greatest challenges of this century. The epidemic affects generally under-developed countries, and Sub-Saharan Africa are the most concerned. The combined marketed form known as Triomune was used as first-line treatment in several sub-Saharan African Countries (60% of VIH infected people), including Chad. However, no evaluation has been done for that treatment in the country. Objective: To evaluate the efficacy and safety immuno-virological of Triomune at the General Hospital in N’Djamena/Chad. Methods: 48 HIV-1 positive patients eligible for ARV treatment were enrolled in our study, and they have been then followed for 8 months. We have measured in these patients the CD4 cell count before treatment and at the 8th month of treatment. After 8 months of treatment, we have also evaluated the Lymphocyte T CD4 and the plasma viral load (VL). Comparisons of means of CD4 lymphocytes and plasma CV (≥1000 copies/ml) were used to define treatment failure. Results: 48 patients were under Triomune regime. The average CD4 count was decreased from 462 ± 179.22 [56 - 981] cells/mm3 before treatment to 327.23 ± 153.77 [10 - 1008] cells/mm3 at the 8th month of treatment. The mean plasma viral load for patients was 66008.62 copies/ml. The failure rate to Triomune was 43.75% (21/48). Conclusion: Aside from the side effects already described for Triomune, our study reveals a high treatment failure rate. Hence, there is the need of regular revisions of therapeutic regime administer in the first intention.

Involvement of the Genetic Diversity of HIV-1 in the Virological Treatment Failure of First Line Antiretroviral in Kinshasa  [PDF]
Erick Ntambwe Kamangu, Richard Lunganza Kalala, Georges Lelo Mvumbi, Dolores Vaira, Marie-Pierre Hayette
World Journal of AIDS (WJA) , 2017, DOI: 10.4236/wja.2017.71003
Abstract: Background: Genetic diversity of human immunodeficiency virus affects the treatment and the emergence of resistance. Some subtypes would develop resistance more frequently than others. The aim of this study is to determine the rate of virological treatment failure and the involvement of genetic diversity and different mutations in this failure in Kinshasa. Methods: Of the 153 Antiretroviral-naive patients who were included in the cohort, 138 patients have been received for the appointment of the 6th month. Clinical parameters were recorded on individual patient charts. The determination of Viral Load (VL) was done at the Laboratory of Molecular Biology. Clinical and biological parameters of the 6th month were compared with those taken at baseline of the cohort to determine the evolution of patients under treatment. Results: At the consultation of the 6th month, 138 patients (90.2%) had returned out of the 153 included. Eighty-one (58.7%) patients were women and 57 (41.3%) men. The age of patients is between 18 and 65 with an average of 37 years. Ten deaths (6.5%) and 5 (3.3%) lost have been reported. One hundred twenty-five patients (90.5%) were in clinical stage 3 and 13 (9.5%) in clinical stage 4. The median CD4 T cells is 560 cells mm3. The median VLs of patients was 0.90 log10 RNA copies/ml. Of the 34 patients in virological failure, 8 (23.5%) are minimal failure, 23 (67.7%) in moderate failure and 3 (8.8%) in severe failure. According to the Pearson’s test, VLs at 6th months were highly correlated with that of inclusion, with V75 and K70 mutations for NRTIs, with V108 mutation for NNRTI well as the virological failure of treatment. Conclusion: Our results confirmed the hypothesis that high Viral Load at the start of the treatment is a poor prognosis for the development of therapy. Transmitted mutations are involved in treatment failure.
Implementation of an In-House Quantitative Real-Time PCR for Determination of HIV Viral Load in Kinshasa  [PDF]
Erick Ntambwe Kamangu, Adawaye Chatte, Raphael Boreux, Richard Lunganza Kalala, Georges Lelo Mvumbi, Patrick Demol, Dolores Vaira, Pierre Marie Hayette
Open Access Library Journal (OALib Journal) , 2014, DOI: 10.4236/oalib.1100855
Abstract: Background: Measurement of Viral Load (VL) is the most reliable mean for evaluating virological monitoring of the Human Immunodeficiency Virus (HIV) infection. It allows determination of the amount of virus present in a given volume. Due to the constraints of costs, the VL is not often requested for patient’s follow-up in countries with limited resources. Hence the objective of this study is to implement an in-house Quantitative Real-Time PCR to assess the VL of HIV infected patients in Kinshasa. Methods: One hundred and fifty five patients positive for HIV type 1, naive of Antiretroviral Therapy (ART) and eligible for treatment were included in the study. Five milliliter of blood was collected in a tube with anticoagulant. One milliliter of plasma was sent to the laboratory for analysis. After RNA extraction, a Quantitative Real time PCR was performed on a portion of the region of the Long Terminal Repeat (LTR) of the virus. Results: Of 155 samples received for determination of VL by Quantitative Real-Time PCR, 153 were successfully amplified according to the protocol. The median VL was 301052.97 copies/ml or 5.48 log10. Conclusions: The results of VL were used to assess the feasibility of the Real-Time Quantitative PCR. It turns a simple, reliable and less expensive alternative for the diagnosis and virological monitoring of HIV patients under ART.
Correlation between Sequencing Results from Liquid Plasma and Dried Plasma Spot (DPS) for Determination of HIV Type 1 Non-B Subtypes  [PDF]
Erick Ntambwe Kamangu, Adawaye Chatté, Dolores Vaira, Patrick de Mol, Georges Lelo Mvumbi, Richard Lunganza Kalala, Marie-Pierre Hayette
Open Access Library Journal (OALib Journal) , 2017, DOI: 10.4236/oalib.1102922
Background: The blotting paper is an alternative to the collection of blood in the tubes for analysis, especially in the field of Human Immunodeficiency Virus infection. This technique allows to easily send the collected samples to specialized laboratories while limiting the stresses of storage and transport. Objective: The objective of this study was to compare the results of sequencing performed on liquid plasma and Dried Plasma Spot (DPS) for the variants of HIV-1 non-B. Methodology: Fifty subjects diagnosed positive for HIV Type 1 using the Rapid Screening Tests voluntarily participated in this study. Two hundred microliters of plasma are deposited on blotting paper Whatman 903 and 500 μl in a micro tube. RNA was extracted from 140 μl of plasma fluid and from a piece of DPS of 5 mm of diameter using the QIAamp RNA Mini Kit QIAGEN. After extraction, the Viral Load (VL) was performed on each sample of liquid plasma. A Reverse Transcription PCR and Nested PCR were used to amplify the regions of interest on the Protease and Reverse Transcriptase for subsequent sequencing. Results: Protease and Reverse Transcriptase were amplified and sequenced respectively for 44 (88%) and 48 (96%) with the liquid plasma samples and 40 (80%) and 45 (90%) with the DPS. The results of Viral Loads were in the range of 2.5 log10 and 6.5 log10. The results of sequencing are comparable for plasma samples and DPS. The correlation coefficient (R2) between the two methods is good (R2 = 0.903, p < 0.001). Conclusion: Liquid Plasma and Dried Plasma Spot give highly correlated results for sequencing strains of HIV type 1 non-B.
Variación de la coloración en poblaciones argentinas de Melanophryniscus rubriventris (Vellard, 1947)
Vaira, Marcos
Cuadernos de Herpetología , 2002,
Abstract: Tanto los patrones como el tono de la coloración dorsal y ventral han sido caracteres comúnmente utilizados para el reconocimiento y diferenciación de numerosas especies y subespecies del género de Bufonidae Melanophryniscus. Sin embargo, raramente se han presentado análisis detallados y sistematizados que indiquen la ausencia o la baja frecuencia de polimorfismos en estos caracteres para asegurar que son realmente diagnósticos. Mediante una metodología sistematizada se estableció la validez diagnóstica de estos caracteres para las poblaciones argentinas de Melanophryniscus rubriventris. La información obtenida en este trabajo, sugiere la existencia de poblaciones con variaciones morfológicas distintivas aunque no exclusivas y se propone que las actuales subespecies sean sinonimizadas. Dada la importante variabilidad registrada en los patrones de coloración de ejemplares conservados y de la intensidad de la coloración in vivo en todo el rango de distribución de Melanophryniscus rubriventris, así como la multiplicidad de fuentes de variabilidad que estos caracteres pueden expresar, se sugiere que el uso de la coloración como carácter diagnóstico único para la delimitación de especies sea utilizado con cierta precaución o empleando una metodología de muestreo rigurosa que contemple la edad, el sexo y la mayor cantidad posible de poblaciones en todo el rango de distribución del grupo a investigar. Both, patterns and hue of dorsal and ventral coloration were commonly utilized in species and subspecies recognition in the genus of Bufonidae Melanophryniscus. However, no detailed and systematized analysis were presented to determine if such characters are truly fixed. I determine the validity of these attributes as truly diagnostic characters in different populations of Melanophryniscus rubriventris of Argentina. The evidence obtained suggest the existence of sets of populations which differ only in trait frequencies and I propose the synonymy of the actual subspecies. I suggest that, because of relatively large intrapopulational variability of patterns and hues, coloration may be a character inadequate for making clear delimitations between putative species. Also, I suggests that age and sex of the individuals and a geographically comprehensive sample of populations should be considered when coloration were applied to species delimitation in the genus Melanophryniscus.
Distribución espacial de una comunidad de anuros de las Yungas Andinas de Argentina
Vaira, Marcos
Cuadernos de Herpetología , 2001,
Abstract: Se determina la distribución espacial de una comunidad de anuros en las selvas subtropicales de monta a del NW de Argentina (Yungas Andinas) y su relación con el gradiente altitudinal y la disponibilidad de ambientes acuáticos para la reproducción. Un total de 19 especies fueron detectadas en los muestreos efectuados en trece sitios diferentes. La riqueza de especies disminuyó con el incremento de la altitud, registrándose cambios en la composición entre los sectores bajos y altos. Ninguna de las especies se distribuyó en forma regular en todos los sitios analizados ni utilizó todas las clases de ambientes acuáticos disponibles para la reproducción. Una gran proporción de la especies utilizó principalmente ambientes lénticos. Las distribuciones restringidas de la mayoría de las especies no parece explicarse exclusivamente por la ausencia de ambientes potenciales para su reproducción o por efectos de la competencia interespecífica. La composición y distribución de las especies en este sector de selvas podría entenderse mejor si se consideran además de las restricciones ecológicas y ambientales los patrones globales de distribución de las especies. La determinación de la distribución de las especies permitiría aplicar medidas de conservación más eficaces para las poblaciones locales del sector de Yungas Andinas estudiado. The spatial distribution and its relationship with altitudinal gradient and aquatic breeding sites availability were determined for an anuran assemblage of an area of Subtropical Montane Forest in northwest Argentina (Andean Yungas). Nineteen species were recorded in 13 sites sampled. Species richness decreased at upper sites and species composition differed from lowland to upland. None of any of the species was distributed regularly within the sites and no species was found at each of the aquatic breeding sites studied. Most species breed in temporary or permanent lentic waters, and a few breeds primarily in streams. Availability of aquatic breeding sites or interspecific competition do not seem to be the main restricting factors to species distributions. Global patterns of distribution added to studies on habitat availability and competition could provide a better explanation to local distributions. To determine species distribution yielded valuable information to conservation efforts of local populations.
An Improved Protocol for Efficient Engraftment in NOD/LTSZ-SCIDIL-2RγNULL Mice Allows HIV Replication and Development of Anti-HIV Immune Responses
Maneesh Singh, Pratibha Singh, Gilles Gaudray, Lucia Musumeci, Caroline Thielen, Dolores Vaira, Claire Vandergeeten, Laurence Delacroix, Ellen Van Gulck, Guido Vanham, Laurence de Leval, Souad Rahmouni, Michel Moutschen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038491
Abstract: Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγnull (NSG) and NOD/SCID/IL2Rγnull (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3–4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.
Downregulation of CD94/NKG2A inhibitory receptors on CD8+ T cells in HIV infection is more pronounced in subjects with detected viral load than in their aviraemic counterparts
Mustapha Zeddou, Souad Rahmouni, Arnaud Vandamme, Nathalie Jacobs, Frédéric Frippiat, Philippe Leonard, Nicole Schaaf-Lafontaine, Dolores Vaira, Jacques Boniver, Michel Moutschen
Retrovirology , 2007, DOI: 10.1186/1742-4690-4-72
Abstract: The CD94/NKG2 heterodimer is a C-type lectin receptor, formed by the covalent association of CD94, a protein with a short non-signaling intracytoplasmic tail [1], and one of the NKG2 molecules. To generate a functional receptor, CD94 is disulfide linked with a member of the NKG2 family, namely NKG2A, -B, -C or -E [2,3]. In humans, CD94/NKG2A interacts with complexes of non-classical HLA-E molecules [4,5]. The intracellular domain of NKG2A contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), responsible for transducing inhibitory signals [6]. The other NKG2 members lack ITIMs and are linked to transmembrane proteins, such as DAP10 and DAP12 which contain immunoreceptor tyrosine-based activating motifs and transduce activating signals [7]. CD94/NKG2A is normally expressed on most NK cells and on a small fraction of CD8+ T lymphocytes. The proportion of NK cells bearing the CD94/NKG2A inhibitory receptor decreases in advanced HIV infection [8], in contrast with other inhibitory receptors of the KIR family which are upregulated. It is presently unknown if HIV infection has similar effects on the expression of the CD94/NKG2A inhibitory receptor by CD8+ T cells. A few studies have shown that CD94 expression by CD8+ T cells is increased during HIV infection [9-11] and have led to postulate that increased expression of the CD94/NKG2A inhibitory receptors is one of the mechanisms rendering HIV-specific CD8+ T lymphocytes unable to control HIV-1 infection [12]. Nevertheless, the simultaneous expression of both subunits of the inhibitory receptor on CD8+ T cells has hardly been studied in HIV infection. Costa et al. using two-color FACS analysis to study CD3+ NKG2A+ T cells, showed no difference between uninfected controls, long term non progressors or aviraemic subjects under HAART. A slight increase was noted in subjects with active viral replication [13], in contradiction with the downregulation previously observed on NK cells from infected subjects.In the pres
Comparison of an In-House Quantitative Real-Time PCR and COBAS AmpliPrep/TaqMan Roche for Determination of Viral Load for HIV Type 1 Non-B  [PDF]
Erick Ntambwe Kamangu, Adawaye Chatte, Raphael Boreux, Fabrice Susin, Richard Lunganza Kalala, Georges Lelo Mvumbi, Patrick De Mol, Marie-Pierre Hayette, Dolores Vaira
Open Access Library Journal (OALib Journal) , 2015, DOI: 10.4236/oalib.1101402
Abstract: Context: The in-house techniques or experimental methods are increasingly recommended for their low-cost reagents for the determination of the Viral Load (VL) in resource-limited settings. The objective of this study was to compare the determination of VL from HIV-1 non-B samples by an in-house technique with the COBAS AmpliPrep/TaqMan version 2.0. Method: In this cross-sectional study, 39 plasma samples from patients infected with HIV type 1 non-B from N’Djamena and Kinshasa were used to determine the VL using the two techniques. Results: The mean values of VL are respectively 4.68 ± 1.26 and 4.58 ± 1.33 log10 RNA copies/ml for the COBAS AmpliPrep/TaqMan assays and the in-house assays. A good correlation (Spearman Correlation) was obtained, with a coefficient (R2) of 0.9452. Conclusion: This study demonstrates that there is no significant difference between the results of VL determined by the COBAS AmpliPrep/TaqMan assays and the in-house assays used.
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