Yohannes Mengistu

Extended-dose nevirapine to 6 weeks of age for infants to prevent HIV transmission via breastfeeding in Ethiopia, India, and Uganda: an analysis of three randomised controlled trials.

BACKGROUND UNICEF/WHO recommends that infants born to HIV-infected mothers who do not have access to acceptable, feasible, affordable, sustainable, and safe replacement feeding should be exclusively breastfed for at least 6 months. The aim of three trials in Ethiopia, India, and Uganda was to assess whether daily nevirapine given to breastfed infants through 6 weeks of age can decrease HIV transmission via breastfeeding. METHODS HIV-infected women breastfeeding their infants were eligible for participation. Participants were randomly assigned to receive either single-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborns after birth) or 6 week extended-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborn babies after birth plus nevirapine 5 mg daily from days 8-42 for the infant). The randomisation sequences were generated by computer at a central data coordinating centre. The primary endpoint was HIV infection at 6 months of age in infants who were HIV PCR negative at birth. Analyses were by modified intention to treat, excluding infants with missing specimens and those with indeterminate or confirmed HIV infection at birth. These studies are registered with ClinicalTrials.gov, numbers NCT00074399, NCT00061321, and NCT00639938. FINDINGS 2024 liveborn infants randomised in the study had at least one specimen tested before 6 months of age (1047 infants in the single-dose group and 977 infants in the extended-dose group). The modified intention-to-treat population included 986 infants in the single-dose group and 901 in the extended-dose group. At 6 months, 87 children in the single-dose group and 62 in the extended-dose group were infected with HIV (relative risk 0.80, 95% CI 0.58-1.10; p=0.16). At 6 weeks of age, 54 children in the single-dose group and 25 in the extended-dose group were HIV positive (0.54, 0.34-0.85; p=0.009). 393 infants in the single-dose group and 346 in the extended-dose group experienced grade 3 or 4 serious adverse events during the study (p=0.54). INTERPRETATION Although a 6-week regimen of daily nevirapine might be associated with a reduction in the risk of HIV transmission at 6 weeks of age, the lack of a significant reduction in the primary endpoint-risk of HIV transmission at 6 months-suggests that a longer course of daily infant nevirapine to prevent HIV transmission via breast milk might be more effective where access to affordable and safe replacement feeding is not yet available and where the risks of replacement feeding are high. FUNDING US National Institutes of Health; US National Institute of Allergy and Infectious Diseases; Fogarty International Center.

Intensified tuberculosis case finding among HIV-Infected persons from a voluntary counseling and testing center in Addis Ababa, Ethiopia.

Objective:To evaluate commonly available screening tests for pulmonary tuberculosis (TB), using sputum bacteriology as a gold standard, in HIV-infected persons attending an urban voluntary counseling and testing clinic in Addis Ababa, Ethiopia. Design:Prospective enrollment of HIV-infected persons, all of whom underwent TB screening, regardless of symptoms, with: (1) symptom screening and physical examination, (2) 3 sputum specimens for smear microscopy, and (3) chest radiograph. One sputum was also sent for concentrated smear microscopy and mycobacterial culture. Chest radiographs were reviewed by 2 independent radiologists. A confirmed TB diagnosis was defined as 1 positive sputum smear and/or 1 positive sputum culture. Results:We enrolled 438 HIV-infected persons: 265 (61%) females, median age 34 years (range: 18-65), median CD4 cell count 181 cells per cubic millimeter (range: 2-1185). Overall, 32 (7%) persons were diagnosed with TB, of whom 5 (16%) were asymptomatic but culture-confirmed TB cases. Screening for cough >2 weeks would have detected only 12 (38%) confirmed TB cases; screening for cough or fever, of any duration, would have detected 24 (75%) cases, with specificity of 64%. Negative predictive value of screening for these 2 symptoms was 97%. Simulation of the current Ethiopian national guidelines had a sensitivity of 63% and specificity of 83% for diagnosing TB disease among study patients. Conclusions:Traditional symptom screening is insufficient for detecting TB disease among HIV-infected persons but may serve to exclude TB disease. More sensitive, rapid, and low-cost diagnostic tests are needed to meet the demand of resource-limited settings.

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

ABSTRACT Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1∧E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1∧E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1∧E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1∧E4 (in vivo and in vitro) and could be immunoprecipitated as an E1∧E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1∧E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1∧E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with threeEscherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1∧E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1∧E4-associated protein indicates a possible role for E1∧E4 in virus synthesis.

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.

Prevalence of Helicobacter pylori infection among adult dyspeptic patients in Ethiopia

Abstract In developing countries such as Ethiopia, where chronic gastritis and peptic-ulcer disease are the most common endoscopic findings, it is important to study the association between Helicobacter pylori infection and gastroduodenal diseases. Both invasive and non-invasive diagnostic methods were therefore used to investigate 300, consecutive, adult patients with dyspepsia, from the gastrointestinal clinic of Tikur Anbassa University Hospital, Addis Ababa. The apparent overall prevalence of H. pylori infection varied according to the detection method employed. Culture revealed H. pylori in only 69% of the patients but this pathogen appeared more common when rapid urease tests (71%), PCR-denaturating gradient gel electrophoresis (91%), histopathology (81%), silver staining (75%) or stool-antigen tests (81%) were employed. Antibodies to H. pylori were detected, both by enzyme immuno-assay (EIA) and immunoblotting, in approximately 80% of the patients, whether the antigens used were of a reference strain or from a local isolate of H. pylori. When some of the EIA-positive and EIA-negative sera were cross-absorbed with antigens of Campylobacter jejuni and re-tested by EIA, the H. pylori-positive sera remained positive and the negative sera remained negative. Dyspeptic patients in Ethiopia, like most of those previously observed elsewhere in Africa, are often infected with H. pylori. It is important that the management of these patients should not be hampered by the misinterpretation of the African epidemiology of this pathogen.

Prevalence of Helicobacter pylori vacA and cagA Genotypes in Ethiopian Dyspeptic Patients

ABSTRACT A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).

Dermatophytosis in Tulugudu Island, Ethiopia

The objective of this investigation was to assess the prevalence of dermatophytoses in children in a geographically restricted area in the Ethiopian countryside, and to determine the aetiological agents of these infections. Demographical and clinical-dermatological data were collected from all children 4-15 years of age on Tulugudu Island, Southern Ethiopia. Mycological specimens were taken and species identification determined through morphological observations and biochemical tests, complemented with sequencing of rDNA ITS2 region when necessary. Of 171 children, 96% shared combs, 85% shared beds and 97% had animal contact. Family size was > 5 persons in 50% of the test subjects and prevalence of tinea capitis was elevated in this group (P < 0.005). Dermatophytoses were clinically diagnosed in 136 cases (79.5%). Tinea capitis (T. capitis) was the most common manifestation with 104 cases (76.5%). T. capitis was combined with dermatophytic infections at other sites in 19 cases. Tinea faciae and Tinea corporis were found in four and two cases, respectively, and pediculosis capitis was diagnosed in 2.9% of the test subjects. Of 135 samples from hair (n = 112), skin (n = 19) and finger-nail (n = 4), 74.1% were microscopy-positive for dermatophytes, 73% were positive in culture, giving an overall prevalence of dermatophytoses in 57.3% of all children examined. Trichophyton violaceum was identified in 80.6% of cultures, Trichophyton verrucosum in 16.3% and Trichophyton tonsurans in 2.0%. One isolate was identified as a white variant of T. violaceum. Tinea capitis was highly prevalent in children on Tulugudu Island, Southern Ethiopia. The anthropophilic species T. violaceum dominated as an aetiological agent. Zoophilic dermatophytes were relatively rarely isolated from clinical specimens, despite the childrens frequent contact with animals.

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.

Use of Dried-Blood-Spot Samples and In-House Assays To Identify Antiretroviral Drug Resistance in HIV-Infected Children in Resource-Constrained Settings

ABSTRACT Monitoring HIV drug resistance is an important component of the World Health Organizations global HIV program. HIV drug resistance testing is optimal with commercially available clinically validated test kits using plasma; however, that type of testing may not be feasible or affordable in resource-constrained settings. HIV genotyping from dried blood spots (DBS) with noncommercial (in-house) assays may facilitate the capture of HIV drug resistance outcomes in resource-constrained settings but has had varying rates of success. With in-house assays for HIV reverse transcriptase, we evaluated the yield of genotyping DBS samples collected from HIV-infected children who were enrolled in two clinical trials conducted in sub-Saharan Africa (median HIV viral load, 5.88 log10 HIV RNA copies/ml; range, 4.04 to 6.99). Overall, HIV genotypes were obtained for 94 (89.5%) of 105 samples tested (95% and 84% from clinical trials #1 and #2, respectively); however, successful analysis of 15 (16.1%) of the 94 samples required repeat testing using a different set of primers on previously synthesized cDNA. The yield of genotyping was lower on the DBS that were stored suboptimally from clinical trial #2 (56% versus 88% for optimally stored). Concordance with plasma genotypes derived using a clinically validated, commercial kit-based assay (ViroSeq HIV-1 genotyping system) was also assessed in a subset of children with paired testing. For 34 samples with paired DBS and plasma genotypes, there was 100% concordance for major drug resistance mutations. DBS genotyping using in-house assays provides an alternative for antiretroviral drug resistance testing in children in resource-constrained regions but may require region-specific optimization before widespread use.

Viral Subtypes, Coreceptor Usage and Phylogenetic Relationships of Hiv-1 Isolates in Discordant Positive and Concordant Couples

There are various cardiac biomarkers available for diagnosis and stratification of patients with acute coronary syndrome but only few are cardiospecific and sensitive. Troponins are protein molecules that are part of cardiac and skeletal muscle. Smooth muscle cells do not contain troponins. There are three subunits of troponin T, I and C subunits. Cardiac troponins are known to increase on myocardial cell damage leading to myocardial necrosis. The kinetic pattern of release of troponin is known. The technological advancements in Immunoassay has led to improvement in the limit of detection, (LoD) Limit of quantitation (LoQ) and cut off detection limits. There are decreases in false positive cases due to antihuman antibodies use of sandwich fifth generation Troponin T immunoassay. Recently the troponin T fifth generation by (Roche diagnostics) is approved by US-FDA. The cutoffs detections and positive 99th percentile of reference population cutoffs has to be established by verification of manufacturers reference cutoff s limits. The delta change value after serial measurements of suspected patients with symptoms of AMI leads to diagnosis.