Workenesh Ayele

Use of Dried Spots of Whole Blood, Plasma, and Mother's Milk Collected on Filter Paper for Measurement of Human Immunodeficiency Virus Type 1 Burden

ABSTRACT We studied the use of dried spots of bodily fluids (plasma, whole blood, and mothers milk) on filter paper as a means of sample collection and storage for human immunodeficiency virus type 1 (HIV-1) viral load testing under stringent field conditions. Plasma placed directly in lysis buffer, which is customarily used for viral load assays, was used for comparison in all our experiments. Utilizing reconstruction experiments, we demonstrate no statistical differences between viral loads determined for plasma and mothers milk spotted on filter paper and those for the same fluids placed directly in lysis buffer. We found that the addition of whole blood directly to lysis buffer was unreliable and could not be considered a feasible option. However, viral load measurements for whole blood spotted onto filter paper correlated with plasma viral load values for both filter spots and lysis buffer (Pearson correlation coefficients, 0.7706 and 0.8155, respectively). In conclusion, dried spots of plasma, whole blood, or mothers milk provide a feasible means for the collection, storage, and shipment of samples for subsequent viral load measurement and monitoring. Virus material spotted and dried on filter paper is a good inexpensive alternative for collecting patient material to monitor the HIV-1 viral load. Measuring the HIV-1 burden from whole blood dried on filter paper provides a suitable alternative for low-technology settings with limited access to refrigeration, as can be found in sub-Saharan Africa.

Development of a Nucleic Acid Sequence-Based Amplification Assay That Uses gag-Based Molecular Beacons To Distinguish between Human Immunodeficiency Virus Type 1 Subtype C and C′ Infections in Ethiopia

ABSTRACT A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C′) circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C′ by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C′ subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C′ sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C′ beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C′ HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.

Sero-prevalence of yellow fever and related Flavi viruses in Ethiopia: a public health perspective

BackgroundYellow fever (YF) is a viral hemorrhagic fever, endemic in the tropical forests of Africa and Central and South America. The disease is transmitted by mosquitoes infected with the yellow fever virus (YFV). Ethiopia was affected by the largest YF outbreak since the vaccination era during 1960–1962. The recent YF outbreak occurred in 2013 in Southern part of the country. The current survey of was carried out to determine the YF seroprevalence so as to make recommendations from YF prevention and control in Ethiopia.MethodologyA multistage cluster design was utilized. Consequently, the country was divided into 5 ecological zones and two sampling towns were picked per zone randomly. A total of 1643 serum samples were collected from human participants. The serum samples were tested for IgG antibody against YFV using ELISA. Any serum sample testing positive by ELISA was confirmed by plaque reduction neutralization test (PRNT). In addition, differential testing was performed for other flaviviruses, namely dengue, Zika and West Nile viruses.ResultOf the total samples tested, 10 (0.61%) were confirmed to be IgG positive against YFV and confirmed with PRNT. Nine (0.5%) samples were antibody positive for dengue virus, 15(0.9%) forWest Nile virus and 7 (0.4%) for Zika virus by PRNT. Three out of the five ecological zones namely zones 1, 3 and 5 showed low levels (< 2%) of IgG positivity against YFV. A total of 41(2.5%) cases were confirmed to be positive for one of flaviviruses tested.ConclusionBased on the seroprevalence data, the level of YFV activity and the risk of a YF epidemic in Ethiopia are low. However additional factors that could impact the likelihood of such an epidemic occurring should be considered before making final recommendations for YF prevention and control in Ethiopia. Based on the results of the serosurvey and other YF epidemic risk factors considered, a preventive mass vaccination campaign is not recommended, however the introduction of YF vaccine in routine EPI is proposed nationwide, along with strong laboratory based YF surveillance.

RNA Detection and Subtype C Assessment of HIV-1 in Infants with Diarrhea in Ethiopia

In the absence of chemoprophylaxis, HIV-1 transmission occurs in 13-42% of infants born to HIV-1 positive mothers. All exposed infants acquire maternal HIV-1 antibodies that persist for up to 15 months, thereby hampering diagnosis. In resource limited settings, clinical symptoms are the indices of established infection against validated laboratorybased markers. Here we enrolled 1200 children hospitalized for diarrheal and other illnesses. 20-25% of those tested, aged 15 months or younger, were found to be HIV-1-seropositive. Where sufficient plasma was available, HIV-1 RNA detection was performed using a subtype-insensitive assay, with 71.1% of seropositive infants presenting with diarrhea showing positive. From sub-typing analysis, we identified that viruses of the C’ sub-cluster were predominated amongst infants. Although this study may overestimate the HIV-1 frequency through testing symptomatic infants, diarrhea can be seen as a useful marker indicating HIV-1 infection in infants less than 15 months old.