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

BACKGROUND: Yellow 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. METHODOLOGY: A 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. RESULT: Of 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. CONCLUSION: Based 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.

Dilution testing using rapid diagnostic tests in a HIV diagnostic algorithm: a novel alternative for confirmation testing in resource limited settings.

BACKGROUND: Current WHO testing guidelines for resource limited settings diagnose HIV on the basis of screening tests without a confirmation test due to cost constraints. This leads to a potential risk of false positive HIV diagnosis. In this paper, we evaluate the dilution test, a novel method for confirmation testing, which is simple, rapid, and low cost. The principle of the dilution test is to alter the sensitivity of a rapid diagnostic test (RDT) by dilution of the sample, in order to screen out the cross reacting antibodies responsible for falsely positive RDT results. METHODS: Participants were recruited from two testing centres in Ethiopia where a tiebreaker algorithm using 3 different RDTs in series is used to diagnose HIV. All samples positive on the initial screening RDT and every 10th negative sample underwent testing with the gold standard and dilution test. Dilution testing was performed using Determine™ rapid diagnostic test at 6 different dilutions. Results were compared to the gold standard of Western Blot; where Western Blot was indeterminate, PCR testing determined the final result. RESULTS: 2895 samples were recruited to the study. 247 were positive for a prevalence of 8.5 % (247/2895). A total of 495 samples underwent dilution testing. The RDT diagnostic algorithm misclassified 18 samples as positive. Dilution at the level of 1/160 was able to correctly identify all these 18 false positives, but at a cost of a single false negative result (sensitivity 99.6 %, 95 % CI 97.8-100; specificity 100 %, 95 % CI: 98.5-100). Concordance between the gold standard and the 1/160 dilution strength was 99.8 %. CONCLUSION: This study provides proof of concept for a new, low cost method of confirming HIV diagnosis in resource-limited settings. It has potential for use as a supplementary test in a confirmatory algorithm, whereby double positive RDT results undergo dilution testing, with positive results confirming HIV infection. Negative results require nucleic acid testing to rule out false negative results due to seroconversion or misclassification by the lower sensitivity dilution test. Further research is needed to determine if these results can be replicated in other settings. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01716299 .

Evaluation of HIV testing algorithms in Ethiopia: the role of the tie-breaker algorithm and weakly reacting test lines in contributing to a high rate of false positive HIV diagnoses.

BACKGROUND: In Ethiopia a tiebreaker algorithm using 3 rapid diagnostic tests (RDTs) in series is used to diagnose HIV. Discordant results between the first 2 RDTs are resolved by a third 'tiebreaker' RDT. Médecins Sans Frontières uses an alternate serial algorithm of 2 RDTs followed by a confirmation test for all double positive RDT results. The primary objective was to compare the performance of the tiebreaker algorithm with a serial algorithm, and to evaluate the addition of a confirmation test to both algorithms. A secondary objective looked at the positive predictive value (PPV) of weakly reactive test lines. METHODS: The study was conducted in two HIV testing sites in Ethiopia. Study participants were recruited sequentially until 200 positive samples were reached. Each sample was re-tested in the laboratory on the 3 RDTs and on a simple to use confirmation test, the Orgenics Immunocomb Combfirm® (OIC). The gold standard test was the Western Blot, with indeterminate results resolved by PCR testing. RESULTS: 2620 subjects were included with a HIV prevalence of 7.7%. Each of the 3 RDTs had an individual specificity of at least 99%. The serial algorithm with 2 RDTs had a single false positive result (1 out of 204) to give a PPV of 99.5% (95% CI 97.3%-100%). The tiebreaker algorithm resulted in 16 false positive results (PPV 92.7%, 95% CI: 88.4%-95.8%). Adding the OIC confirmation test to either algorithm eliminated the false positives. All the false positives had at least one weakly reactive test line in the algorithm. The PPV of weakly reacting RDTs was significantly lower than those with strongly positive test lines. CONCLUSION: The risk of false positive HIV diagnosis in a tiebreaker algorithm is significant. We recommend abandoning the tie-breaker algorithm in favour of WHO recommended serial or parallel algorithms, interpreting weakly reactive test lines as indeterminate results requiring further testing except in the setting of blood transfusion, and most importantly, adding a confirmation test to the RDT algorithm. It is now time to focus research efforts on how best to translate this knowledge into practice at the field level. TRIAL REGISTRATION: Clinical Trial registration #: NCT01716299.

Molecular typing and drug sensitivity testing of Mycobacterium tuberculosis isolated by a community-based survey in Ethiopia.

BACKGROUND: The identification of circulating TB strains in the community and drug sensitivity patterns is essential for the tuberculosis control program. This study was undertaken to identify M. tuberculosis strains circulating in selected communities in Ethiopia as well as to evaluate the drug sensitivity pattern of these strains. METHOD: This study was a continuation of the Ethiopian National TB Prevalence Survey that was conducted between 2010 and 2011. Culture-positive isolates of M. tuberculosis from previous study were typed using region of difference (RD) 9-based polymerase chain reaction (PCR) and spoligotyping. Drug sensitivity testing was conducted using the indirect proportion method on Lowenstein-Jensen media. RESULT: All 92 isolates were confirmed as M. tuberculosis by RD9-based PCR and spoligotyping of 91 of these isolates leds to the identification of 41 spoligotype patterns. Spoligotype revealed higher diversity (45 %) and among this 65.8 % (27/41) were not previously reported. The strains were grouped into 14 clusters consisting of 2-15 isolates. The dominant strains were SIT53, SIT149 and SIT37 consisting of 15, 11, and 9 isolates, respectively. Our study reveals 70 % (64/91) clustered strains and only 39.1 % (25/64) occurred within the same Kebele. Further assignment of the strains to the lineages showed that 74.7 % (68/91) belonged to Euro-American lineage, 18.6 % (17/91) to East Africa Indian lineage and the remaining 6.5 % (6/91) belonged to Indo-oceanic lineage. Valid drug susceptibility test results were available for 90 of the 92 isolates. Mono-resistance was observed in 27.7 % (25/90) and poly-resistance in 5.5 % (5/90) of the isolates. Moreover, multi-drug resistance (MDR-TB) was detected in 4.4 % of the isolates whilst the rest (60/90) were susceptible to all drugs. The highest level of mono-resistance, 26.6 % (24/90), was observed for streptomycin with majority (91.1 %) of streptomycin mono-resistant strains belonging to the Euro-American lineage. CONCLUSION: In this study, the strains of M. tuberculosis circulating in selected sites of Ethiopia were identified along with the drug sensitivity patterns. Thus, these findings are useful for the TB Control Program of the country.

Toxicity study and evaluation of biochemical markers towards the identification of the causative agent for an outbreak of liver disease in Tahtay Koraro Woreda, Tigray.

BACKGROUND: A team of experts of the Faculty of Medicine, Addis Ababa University reported the emergence of unidentified fatal liver disease in Tahtay Koraro Woreda, Tigray in the mid of December 2005. The EHNRI has been then instructed to investigate the possible etiological agent that are likely to be responsible in triggering the health problem and a field survey team consisting of experts were went to the affected area to investigate the situations surrounding the disease. OBJECTIVES: This investigation was conducted to determine the possible etiological agent(s) for the stated health problem in the affected village. METHOD: Acute toxicity study was performed on animal model for the various samples used in human consumption, which was followed by histopathological examination of the liver of the sacrificed laboratory animals. In order to facilitate the elucidation of the causative agent for the alleged health problem further tests for clinical markers and antigens were also performed on the serum collected from affected persons. RESULT: Neither death nor toxic symptoms manifestations were observed on laboratory animals when feeding the consumable samples for a period of two weeks, however histopathological examination of the liver of the sacrificed animals that were given the unprotected pond water and Tela samples from the affected village as a drink revealed severe hepatoic necrosis. Biochemical test results of the serum samples revealed raised level of some clinical markers that are highly significant for detecting liver abnormality of toxic origin. Serological test for surface antigen ruled out the possible causes of infectious origin such as viral hepatitis. CONCLUSION: The overall results confirmed that the causative agent for the outbreak of the liver disease was of toxic origin rather than due to infectious agent and this was found to be associated with consumption of contaminated water as well as Tela.

Genotypes and viral load of hepatitis C virus among persons attending a voluntary counseling and testing center in Ethiopia.

The prevalence of different genotypes of hepatitis C virus (HCV) in Ethiopia is not known. HCV genotypes influence the response to therapy with alpha-interferon alone or in combination with ribavirin. A cross sectional study was conducted on attendees of voluntary counseling and testing center. Serum samples from 1,954 (734 HIV positive and 1,220 HIV negative) individuals were screened for HCV antibody. Active HCV infection was confirmed by quantitative PCR in 18 of the 71 samples with anti-HCV antibodies. The HCV viral load ranged from 39,650 to 9,878,341 IU/ml (median 1,589,631 IU/ml) with no significant difference [χ(2)(17) = 18.00, P = 0.389] between persons positive or negative for HIV. The viral load of HCV was, however, higher in older study subjects (r = 0.80, P = 0.000). HCV genotypes were determined using the VERSANT HCV Genotype Assay (LiPA) and sequence analysis of the NS5b region of the HCV genome. Diverse HCV genotypes were found including genotypes 1, 2, 4, and 5. There was no difference in the distribution regarding the HIV status. As in other parts of the world, genotyping of HCV must be considered whenever HCV is incriminated as a cause of hepatitis.

Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network.

Severe rotavirus diarrhea in children <5 years of age is a major public health problem; however, limited regional and country specific data on rotavirus disease burden are available from sub-Saharan Africa. In June 2006, the World Health Organization Regional Office for Africa initiated rotavirus surveillance in selected African countries. With use of standardized methodology developed by the World Health Organization, children <5 years of age who were hospitalized with severe diarrhea were enrolled, and stool specimens were collected for detection of rotavirus strains with use of a commercial enzyme immunoassay. Rotavirus strains were further characterized for G and P types with use of a reverse-transcriptase polymerase chain reaction. From June 2006 through December 2008, rotavirus surveillance was established at 14 sites in 11 African countries. Of 5461 stool samples collected from children enrolled in 8 countries with 1 or 2 complete years of data, 2200 (40%) were positive for rotavirus. Ninety percent of all rotavirus hospitalizations occurred among children aged 3-12 months. Predominant types included G1P[8] (21%), G2P[4] (7%), and P [8] (29%); however, unusual types were also detected, including G8P[6] (5%), G8P[8] (1%), G12P[6] (1%), and G12P[6] (1%). A high percentage of mixed rotavirus infections was also detected. These preliminary results indicate that rotavirus is a major cause of severe diarrheal disease in African children.

Whole Genome Analysis of African G12P[6] and G12P[8] Rotaviruses Provides Evidence of Porcine-Human Reassortment at NSP2, NSP3, and NSP4.

Group A rotaviruses (RVA) represent the most common cause of pediatric gastroenteritis in children <5 years, worldwide. There has been an increase in global detection and reported cases of acute gastroenteritis caused by RVA genotype G12 strains, particularly in Africa. This study sought to characterize the genomic relationship between African G12 strains and determine the possible origin of these strains. Whole genome sequencing of 34 RVA G12P[6] and G12P[8] strains detected from the continent including southern (South Africa, Zambia, Zimbabwe), eastern (Ethiopia, Uganda), central (Cameroon), and western (Togo) African regions, were sequenced using the Ion Torrent PGM method. The majority of the strains possessed a Wa-like backbone with consensus genotype constellation of G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while a single strain from Ethiopia displayed a DS-1-like genetic constellation of G12-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. In addition, three Ethiopian and one South African strains exhibited a genotype 2 reassortment of the NSP3 gene, with genetic constellation of G12-P[8]-I1-R1-C1-M1-A1-N1-T2-E1-H1. Overall, 10 gene segments (VP1-VP4, VP6, and NSP1-NSP5) of African G12 strains were determined to be genetically related to cognate gene sequences from globally circulating human Wa-like G12, G9, and G1 strains with nucleotide (amino acid) identities in the range of 94.1-99.9% (96.5-100%), 88.5-98.5% (93-99.1%), and 89.8-99.0% (88.7-100%), respectively. Phylogenetic analysis showed that the Ethiopian G12P[6] possessing a DS-1-like backbone consistently clustered with G2P[4] strains from Senegal and G3P[6] from Ethiopia with the VP1, VP2, VP6, and NSP1-NSP4 genes. Notably, the NSP2, NSP3, and NSP4 of most of the study strains exhibited the closest relationship with porcine strains suggesting the occurrence of reassortment between human and porcine strains. Our results add to the understanding of potential roles that interspecies transmission play in generating human rotavirus diversity through reassortment events and provide insights into the evolutionary dynamics of G12 strains spreading across selected sub-Saharan Africa regions.

Human herpesvirus 8 infection in HIV-1-infected and uninfected pregnant women in Ethiopia.

BACKGROUND: Human Herpes Virus (HHV-8) is related to Kaposi Saracoma, an opportunistic infection occurring with HIV infection. Little is known about the seroepidemiology of Human Herpesvirus 8 (HHV-8) infection among Ethiopian women, even though women are a major HIV risk group in Ethiopia. OBJECTIVES: This study aimed at determining the seroprevalence of HHV-8 infection in HIV-1-infected and uninfected pregnant women in five selected regions of Ethiopia. METHODS: A cross-sectional study was conducted from December 2006 to June 2007 where pregnant women were recruited after age-matching in groups. A total of 400 pregnant women were enrolled, with 200 being HIV-infected and 200 being HIV-uninfected Sera were screened for IgG lytic antibody to HHV-8 using an Indirect Fluorescence Assay (IFA) in Virology Unit of Ethiopian Health and Nutrition Research Institute (EHNR1). RESULTS: Of 400 pregnant women attending antenatal clinic (ANC) testing sites of five regions in Ethiopia, 212 (53.0%) were positive for HHV-8 IgG lytic antibody. There was a high prevalence of HHV-8 infection among HIV-1-infected pregnant women (138, 69.0%) as compared with HIV-1-uninfected pregnant women (74, 37.0%). CONCLUSION: The study shows a high prevalence of HHV-8 infection among HIV-1-infected pregnant women as compared with HIV-1-uninfected pregnant women. Therefore, creating awareness and educating women on safe sexual practice and avoiding deep kissing may be a fundamental ways to limit the roots of transmission. Moreover, initiating strong antiretroviral therapy (ART) for HIV infected women would be best treatment prior to the development of Kaposi's sarcoma (KS).

Rotavirus-associated acute diarrhea outbreak in West Shewa Zone of Oromia Regional State, Ethiopia, 2017.

INTRODUCTION: Rotavirus causes severe-diarrheal diseases in infants. An estimation of 138 million rotavirus-associated diarrheal cases and 215,000 deaths occur every year globally. In December 2016, West-Shewa zone in Ethiopia reported unidentified gastrointestinal diarrhea outbreak. We investigated to identify the causative agent of the outbreak to support response operations. METHODS: Medical records were reviewed, and the daily line list was collected from health facilities. Descriptive data analysis was done by time, person and place. Stool specimens were first tested by antigen capture enzyme immunoassay (EIA) technique and further confirmed by reverse-transcription polymerase chain reaction (RT-PCR) as a gold standard. The product of RT-PCR was genotyped for each gene using G1-G4, G8-G9 and G12 primers for VP7 gene and P(4), P(6), P(8) and P(14) primers for VP4 gene. RESULTS: A total of 1,987 diarrheal cases (5.7 per 1000) and five deaths (case-fatality rate 0.25%) were identified and epidemiologically-linked to confirmed rotavirus from December 2016 to February 2017. Among the cases, 1,946 (98%) were < 5 children. Fourteen (74%) of the 19 tested stool specimens were positive for rotavirus by EIA and RT-PCR. Majority of strains detected were G12P(6) (25%) and G-negative P(8) (25%) followed by G9P(8) (19%), G1P(8) (13%) and G3/G2 P(8), G12P(8), and G-negative P(6) (6% each). CONCLUSION: Diarrheal outbreak which occurred in West-Shewa zone of Ethiopia was associated with rotavirus and relatively more affected districts with low vaccination coverage. Routine rotavirus vaccination quality and coverage should be evaluated and the surveillance system needs to be strengthened to detect, prevent and control a similar outbreak.

Enteric pathogens and associated risk factors among under-five children with and without diarrhea in Wegera District, Northwestern Ethiopia.

INTRODUCTION: Childhood diarrhea is highly prevalent in slums in developing countries, but it remains understudied. The objectives of this study were to explore the prevalence of Giardia, rotavirus and bacterial enteropathogens among diarrheic and non-diarrheic children and investigate socio-environmental determinants of diarrhea in two Ethiopian towns. METHODS: A cross-sectional study was conducted from June to October 2016. Prevalence of childhood diarrhea was established using information gathered during interviews with mothers/guardians. Saline wet mounts of fresh stool samples were used to test for the presence of Giardia. Stool samples were cultured on MacConkey agar and suspected colonies were characterized using biochemical tests. Susceptibility testing was done by the disk diffusion method. ELISA was used to screen for rotavirus. RESULTS: A total of 225 children were included in this study. Four enteropathogens (Giardia, rotavirus, Shigella and Salmonella) were identified from 31% (35/112) diarrheic and 12% (14/113) from non-diarrheic children (p < 0.001). The prevalence of rotavirus infection was 18.0% among diarrheic children and 3.3% among non-diarrheic children unvaccinated against rotavirus (p < 0.01). The prevalence of Giardia was 21.0% among diarrheic and 8.0% among non-diarrheic children (p < 0.01). Diarrheic children had significantly higher rates of bloody stool (p < 0.02), vomiting, fever and breastfeeding for children beyond 23 months of age (p < 0.001). Giardia and rotavirus were identified in more diarrheic than non-diarrheic children. CONCLUSION: The high prevalence of Giardia and rotavirus in the study area indicates the need for coordinated healthcare activities in the two communities. Vaccination against rotavirus infections and educational interventions are recommended.

HIV-genetic diversity and drug resistance transmission clusters in Gondar, Northern Ethiopia, 2003-2013.

BACKGROUND: The HIV-1 epidemic in Ethiopia has been shown to be dominated by two phylogenetically distinct subtype C clades, the Ethiopian (C'-ET) and East African (C-EA) clades, however, little is known about the temporal dynamics of the HIV epidemic with respect to subtypes and distinct clades. Moreover, there is only limited information concerning transmission of HIV-1 drug resistance (TDR) in the country. METHODS: A cross-sectional survey was conducted among young antiretroviral therapy (ART)-naïve individuals recently diagnosed with HIV infection, in Gondar, Ethiopia, 2011-2013 using the WHO recommended threshold survey. A total of 84 study participants with a median age of 22 years were enrolled. HIV-1 genotyping was performed and investigated for drug resistance in 67 individuals. Phylogenetic analyses were performed on all available HIV sequences obtained from Gondar (n = 301) which were used to define subtype C clades, temporal trends and local transmission clusters. Dating of transmission clusters was performed using BEAST. RESULT: Four of 67 individuals (6.0%) carried a HIV drug resistance mutation strain, all associated with non-nucleoside reverse transcriptase inhibitors (NNRTI). Strains of the C-EA clade were most prevalent as we found no evidence of temporal changes during this time period. However, strains of the C-SA clade, prevalent in Southern Africa, have been introduced in Ethiopia, and became more abundant during the study period. The oldest Gondar transmission clusters dated back to 1980 (C-EA), 1983 (C-SA) and 1990 (C'-ET) indicating the presence of strains of different subtype C clades at about the same time point in Gondar. Moreover, some of the larger clusters dated back to the 1980s but transmissions within clusters have been ongoing up till end of the study period. Besides being associated with more sequences and larger clusters, the C-EA clade sequences were also associated with clustering of HIVDR sequences. One cluster was associated with the G190A mutation and showed onward transmissions at high rate. CONCLUSION: TDR was detected in 6.0% of the sequenced samples and confirmed pervious reports that the two subtype C clades, C-EA and C'-ET, are common in Ethiopia. Moreover, the findings indicated an increased diversity in the epidemic as well as differences in transmission clusters sizes of the different clades and association with resistance mutations. These findings provide epidemiological insights not directly available using standard surveillance and may inform the adjustment of public health strategies in HIV prevention in Ethiopia.

Impact of rotavirus vaccine introduction and genotypic characteristics of rotavirus strains in children less than 5 years of age with gastroenteritis in Ethiopia: 2011-2016.

INTRODUCTION: A monovalent rotavirus vaccine was introduced in the Ethiopian Expanded Program on Immunization from November 2013. We compared impact of rotavirus vaccine introduction on rotavirus associated acute diarrhea hospitalizations and genotypic characteristics of rotavirus strains pre-and post-vaccine introduction. METHODS: Sentinel surveillance for diarrhea among children <5 years of age was conducted at 3 hospitals in Addis Ababa, Ethiopia from 2011 to 2017. Stool specimens were collected from enrolled children and tested using an antigen capture enzyme immunoassay. Rotavirus positive samples (156 from pre- and 141 from post-vaccination periods) were further characterized by rotavirus genotyping methods to identify the predominant G and P types circulating during the surveillance era. RESULTS: A total of 788 children were enrolled during the pre- (July 2011-June 2013) and 815 children during the post-vaccination (July 2014-June 2017) periods. The proportion of diarrhea hospitalizations due to rotavirus among children <5 years of age declined by 17% from 24% (188/788) in the pre-vaccine period and to 20% (161/185) in post-vaccine introduction era. Similarly, a reduction of 18% in proportion of diarrhea hospitalizations due to rotavirus in children <12 months of age in the post (27%) vs pre-vaccine (33%) periods was observed. Seasonal peaks of rotavirus declined following rotavirus vaccine introduction. The most prevalent circulating strains were G12P[8] in 2011 (36%) and in 2012 (27%), G2P[4] (35%) in 2013, G9P[8] (19%) in 2014, G3P[6] and G2P[4] (19% each) in 2015, and G3P[8] (29%) in 2016. DISCUSSION: Following rotavirus vaccine introduction in Ethiopia, a reduction in rotavirus associated hospitalizations was seen in all age groups with the greatest burden in children <12 months of age. A wide variety of rotavirus strains circulated in the pre- and post-vaccine introduction periods.

Surveillance technology for HIV-1 subtype C in Ethiopia: an env-based NASBA molecular beacon assay to discriminate between subcluster C and C'.

Forty-nine samples with known C2V3 sequences were used for the evaluation of an env-based molecular beacon assay to distinguish between the two genetic subclusters C and C' which characterize the HIV-1 epidemic in Ethiopia. Two subcluster C and two subcluster C' beacons targeting two different loci in the C2V3 region were developed. Using a three beacon-based (2C and 1C'=C prime), isothermal amplification assay, concordance with DNA sequencing was achieved for 43 (87.8%) samples. Sensitivity was 81.8% and specificity 97.4% for subcluster C beacons. For the subcluster C' beacon, a sensitivity of 97% and a specificity of 87.5% was achieved. Five samples were ambiguous by sequencing of which two samples were subcluster C' by the beacon assay and one subcluster C. Two of the samples remained ambiguous with different beacon-pair combinations as well. From samples with a clear C or C' phylogeny by sequencing, three were undetected by the first-line beacon genotyping assay. Genotype ambiguity was resolved in the three samples using beacon pair combinations restricted to each targeted locus. The beacons were evaluated further in a panel including all HIV-1 subtypes. Four of five subtype C isolates were identified correctly, and no cross-reactivity was observed with other subtypes.

Accounting for False Positive HIV Tests: Is Visceral Leishmaniasis Responsible?

BACKGROUND: Co-infection with HIV and visceral leishmaniasis is an important consideration in treatment of either disease in endemic areas. Diagnosis of HIV in resource-limited settings relies on rapid diagnostic tests used together in an algorithm. A limitation of the HIV diagnostic algorithm is that it is vulnerable to falsely positive reactions due to cross reactivity. It has been postulated that visceral leishmaniasis (VL) infection can increase this risk of false positive HIV results. This cross sectional study compared the risk of false positive HIV results in VL patients with non-VL individuals. METHODOLOGY/PRINCIPAL FINDINGS: Participants were recruited from 2 sites in Ethiopia. The Ethiopian algorithm of a tiebreaker using 3 rapid diagnostic tests (RDTs) was used to test for HIV. The gold standard test was the Western Blot, with indeterminate results resolved by PCR testing. Every RDT screen positive individual was included for testing with the gold standard along with 10% of all negatives. The final analysis included 89 VL and 405 non-VL patients. HIV prevalence was found to be 12.8% (47/ 367) in the VL group compared to 7.9% (200/2526) in the non-VL group. The RDT algorithm in the VL group yielded 47 positives, 4 false positives, and 38 negatives. The same algorithm for those without VL had 200 positives, 14 false positives, and 191 negatives. Specificity and positive predictive value for the group with VL was less than the non-VL group; however, the difference was not found to be significant (p = 0.52 and p = 0.76, respectively). CONCLUSION: The test algorithm yielded a high number of HIV false positive results. However, we were unable to demonstrate a significant difference between groups with and without VL disease. This suggests that the presence of endemic visceral leishmaniasis alone cannot account for the high number of false positive HIV results in our study.

Metals exposures of residents living near the Akaki River in Addis Ababa, Ethiopia: a cross-sectional study.

BACKGROUND: The Akaki River in Ethiopia has been found to contain elevated levels of several metals. Our objectives were to characterize metals exposures of residents living near the Akaki River and to assess metal levels in their drinking water. METHODS: In 2011, we conducted a cross-sectional study of 101 households in Akaki-Kality subcity (near the Akaki River) and 50 households in Yeka subcity (distant to the Akaki River). One willing adult in each household provided urine, blood, and drinking water sample. RESULTS: Urinary molybdenum (p < 0.001), tungsten (p < 0.001), lead (p < 0.001), uranium (p < 0.001), and mercury (p = 0.049) were higher in Akaki-Kality participants compared to Yeka participants. Participants in both subcities had low urinary iodine; 45% met the World Health Organization (WHO) classification for being at risk of moderate iodine deficiency. In Yeka, 47% of households exceeded the WHO aesthetic-based reference value for manganese; in Akaki-Kality, only 2% of households exceeded this value (p < 0.001). There was no correlation between metals levels in water samples and clinical specimens. CONCLUSIONS: Most of the exposures found during this investigation seem unlikely to cause acute health effects based on known toxic thresholds. However, toxicity data for many of these metals are very limited.

HIV type 1 C and C' subclusters based on long terminal repeat sequences in the Ethiopian type 1 subtype C epidemic.

While the Ethiopian HIV-1 epidemic is dominated by subtype C, two distinguishable cocirculating C genotypes have been identified based on sequences of the C2V3 envelope region. In this study we sequenced and analyzed the long terminal repeat (LTR) sequence from 22 Ethiopian HIV-1-positive individuals. The two phylogenetically distinguishable genotypes C (n = 13) and C' (n = 4) are separated by significant bootstrap values. Nucleotide differences between the two groups were identified in the NF-AT, TCF-1alpha, and SP1 transcription factor binding sites, whereas the NF-kappaB and NRE-core sequences were identical between the two groups. Five isolates that could not be classified C or C' were found to be recombinants within the LTR sequence upon boots can analysis. Comparison of all the LTR sequences with their corresponding C2V3 envelope sequence revealed four intersubtype C/C' recombinant isolates. Thus, the prevalence of C/C' recombinant viruses is well over 40%. Interestingly, the C2V3 envelope sequences of all recombinant viruses belonged to the genotype C', whereas every LTR sequence belonged to the genotype C. This result indicates that recombination between the two genotypes is unidirectional, possibly as the result of evolutionary pressure on the respective biological functions of the LTR promoter and the envelope protein.

Recombination of HIV type 1C (C'/C") in Ethiopia: possible link of EthHIV-1C' to subtype C sequences from the high-prevalence epidemics in India and Southern Africa.

The magnitude and complexity of the HIV-1 genetic diversity are major challenges for vaccine development. Investigation of the genotypes circulating in areas of high incidence, as well as their interactions, will be a milestone in the development of an efficacious vaccine. Because HIV-1 subtype C (HIV-1C) is responsible for most of the 36 million infections worldwide we investigated the HIV-1C strains circulating in Ethiopia in a retrospective, cross-sectional study. Serum samples from HIV-1-positive individuals were collected in seven Ethiopian cities and towns. Nucleotide sequences of the gag, pol, and env genes were analyzed. We performed phylogenetic analysis by the neighbor-joining and maximum-likelihood methods with sequences from 30 isolates, and we determined recombination by the bootscanning method as implemented in the SIMPLOT program. Sequence analyses of a 2600-nucleotide fragment (including the gag gene, the protease, and the 5' half of reverse transcriptase of the pol gene) and the corresponding V1V2/C2V3 envelope regions confirmed that two distinct HIV-1C genotypes (C' and C") are cocirculating in Ethiopia, as shown previously by the analysis of the C2V3 envelope region. We have identified intrasubtype recombination between the two HIV-1C genotypes, C' and C", with 6 of the 30 (20%) analyzed viruses being recombinants. The C' sequences were phylogenetically linked to the fast spreading viruses in India and southern Africa. Furthermore, all the recombinant viruses shared the C' V1V3 region of the envelope, suggesting that the prevalence of viruses with the C' envelope is increasing compared to the C" envelope. The possibility that viruses with a C' envelope have a biological advantage over the viruses with a C" envelope should be further investigated in biological and epidemiological studies.

Rapid CD4+ T-lymphocyte depletion in rhesus monkeys infected with a simian-human immunodeficiency virus expressing the envelope glycoproteins of a primary dual-tropic Ethiopian Clade C HIV type 1 isolate.

Simian-human immunodeficiency virus (SHIV) chimerae with the envelope glycoproteins of X4 or R5/X4 HIV-1 isolates from clade B can cause rapid and severe CD4(+) T cell depletion and AIDS-like illness in infected monkeys. We created a SHIV (SHIV-MCGP1.3) expressing the envelope glycoproteins of a primary R5/X4, clade C HIV-1 isolate. Infection of a rhesus monkey with SHIV-MCGP1.3 resulted in a low level of viremia and no significant alteration in CD4(+) T-lymphocyte counts. However, serial intravenous passage of the virus resulted in the emergence of SHIV-MCGP1.3 variants that replicated efficiently and caused profound CD4(+) T cell depletion during the acute phase of infection. The CD4(+) T cell counts in the infected monkeys gradually returned to normal, and the animals remained healthy. The ability to cause rapid and profound loss of CD4(+) T lymphocytes in vivo is a property shared by passaged, CXCR4-using SHIVs, irrespective of the clade of origin of the HIV-1 envelope glycoproteins.

Decline in prevalence of HIV-1 infection and syphilis among young women attending antenatal care clinics in Addis Ababa, Ethiopia: results from sentinel surveillance, 1995-2001.

From 1995 to 2001, five rounds of sentinel surveillance were carried out for young women attending antenatal care clinics at four health centers in Addis Ababa, the capital city of Ethiopia, to monitor trends in the prevalence of HIV infection and syphilis. Serum samples were tested for antibodies to HIV (enzyme-linked immunosorbent assay and Western blotting) and antibodies to Treponema pallidum (T. pallidum hemagglutination assay and rapid plasma reagin test). Prevalence ratios for an increase in one calendar year were estimated using log-binomial models. Between 1995 and 2001, the prevalence of HIV infection among young women (age range, 15-24 years) attending antenatal care clinics in inner city health centers declined from 24.2% to 15.1% (prevalence ratio for an increase in one calendar year, 0.91; 95% confidence interval, 0.87-0.95). No change was observed for older age groups or in outer city health centers. The decline in the prevalence of active syphilis (T. pallidum hemagglutination assay and rapid plasma reagin testing positive for antibodies to T. pallidum) was more pronounced among and also restricted to the young age groups (age range, 15-24 years) in the inner city (from 7.6% in 1995 to 1.3% in 2001; prevalence ratio, 0.69; 95% confidence interval, 0.59-0.80). The declining trends in the prevalence of HIV infection and syphilis among young women attending antenatal care clinics in the inner city are encouraging, but these findings require confirmation in future years and for other population groups.