Pregnancy suppresses Mycobacterium tuberculosis-specific Th1, but not Th2, cell-mediated functional immune responses during HIV/latent TB co-infection.

Immunity to tuberculosis (TB) is suppressed due to HIV coinfection and this suppression could further be enhanced by pregnancy. However, the effect of pregnancy on Mycobacterium tuberculosis (M. tuberculosis)-specific immune response during HIV/latent TB co-infection is not well understood. Here we investigated the changes in M. tuberculosis-specific functional Th1, Th2 and antibody responses in pregnant women with HIV/latent TB co-Infection. Pregnancy, concurrent with HIV infection, triggers a substantial suppression of M. tuberculosis-specific IFN-γ responses in a CD4+ T cell count dependent manner with an insignificant change in IL-4 and IgG responses. Conversely, M. tuberculosis-specific IL-10 production was markedly augmented in latent TB infected pregnant women with a lesser extent during HIV co-infection. These findings reveal that pregnancy suppresses anti-mycobacterial protective immune response in a CD4+ T cell count dependent manner during HIV/latent TB co-infection, suggesting a higher risk of developing active TB during pregnancy as a result of failing to control TB infection.

A viral genome wide association study and genotypic resistance testing in patients failing first line antiretroviral therapy in the first large countrywide Ethiopian HIV cohort.

BACKGROUND: Antiretroviral therapy (ART) was rolled-out in Ethiopia in 2005, but there are no reports on outcome of ART and human immunodeficiency virus drug resistance (HIVDR) at national level. We described acquired drug resistance mutations in pol gene and performed a viral genome wide association study in virologic treatment failure patients who started first line ART during 2009-2011 in the first large countrywide HIV cohort in Ethiopia. METHODS: The outcome of tenofovir (TDF)- and zidovudine (ZDV)-based ART was defined in 874 ART naïve patients using the on-treatment (OT) and intention-to-treat (ITT) analyses. Genotypic resistance testing was done in patients failing ART (> 1000 copies/ml) at month 6 and 12. Near full-length genome sequencing (NFLG) was used to assess amino acid changes in HIV-1 gag, pol, vif, vpr, tat, vpu, and nef genes between paired baseline and month 6 samples. RESULTS: High failure rates were found in ITT analysis at month 6 and 12 (23.3%; 33.9% respectively). Major nucleoside and non-nucleoside reverse transcriptase (NRTI/NNRTI) drug resistance mutations were detected in most failure patients at month 6 (36/47; 77%) and month 12 (20/30; 67%). A high rate of K65R was identified only in TDF treated patients (35.7%; 50.0%, respectively). No significant difference was found in failure rate or extent of HIVDR between TDF- and ZDV- treated patients. All target regions of interest for HIVDR were described by NFLG in 16 patients tested before initiation of ART and at month 6. CONCLUSION: In this first Ethiopian national cohort, a high degree of HIVDR was seen among ART failure patients, independent on whether TDF- or ZDV was given. However, the major reason to ART failure was lost-to-follow-up rather than virologic failure. Our NFLG assay covered all relevant target genes for antiretrovirals and is an attractive alternative for HIVDR surveillance.

Serum chemokine profiles in visceral leishmaniasis, HIV and HIV/ visceral leishmaniasis co-infected Ethiopian patients.

BACKGROUND: The search for a correlation between chemokine levels in plasma or serum and protection from HIV infection or progression to AIDS has been attempted by a number of workers. Chemokines are also suggested to play a role in immunity to Leishmania and Leishmania co-infection with HIV. OBJECTIVE: To assess plasma level of alpha chemokine (CXCL12, formerly known as SDF-1alpha) and beta chemokines (CCL3, CCL4 and CCL5, formerly known as MIP-1alpha, MIP-1beta and RANTES, respectively) in HIV Visceral Leishmaniasis (VL) and HIV/VL coinfection. METHODS: Frozen serum samples from a cross sectional study were used. The samples (n = 80) were comprised of healthy controls (n = 20), HIV patients (n = 20); Visceral Leishmaniasis (VL) patients (n = 22), and HIV/VL coinfected patients (n = 18). Chemokine levels of MIP-1alpha, MIP-1beta, RANTES, and SDF-1alpha of the serum samples were determined using ELISA. RESULTS: MIP-1alpha and MIP-1beta expression were significantly elevated in Leishmania infected (p < 0.001) and in HIV/ VL co-infected individuals (p < 0.001) as compared to the control groups, while no significant difference was seen between HIV infected patients p > 0.05, implying that VL alone might modulate the production of these two chemokines in the case of co-infection In RANTES, however, its expression was significantly higher in HIV patients compared to controls (p = 0.002). Further assessment of serum RANTES concentration in HIV patients has shown a tendency of negative association with viral load. Higher amount of the alpha chemokine, SDF-1alpha, was detected in the HIV patients (p = 0.001) than the control group. Also a trend of positive association between SDF-1alpha and CD4 count was observed CONCLUSION: From our data we can speculate that RANTES and SDF-1alpha might be involved in the regulation of HIV; and MIP-1alpha and MIP-1beta in VL. Therefore, enhancing or suppressing the production of these chemokines might help in therapeutic intervention of VL or HIV.

HIV-1 Recent Infection Testing Algorithm With Antiretroviral Drug Detection to Improve Accuracy of Incidence Estimates.

BACKGROUND: HIV-1 incidence calculation currently includes recency classification by HIV-1 incidence assay and unsuppressed viral load (VL ≥ 1000 copies/mL) in a recent infection testing algorithm (RITA). However, persons with recent classification not virally suppressed and taking antiretroviral (ARV) medication may be misclassified. SETTING: We used data from 13 African household surveys to describe the impact of an ARV-adjusted RITA on HIV-1 incidence estimates. METHODS: HIV-seropositive samples were tested for recency using the HIV-1 Limiting Antigen (LAg)-Avidity enzyme immunoassay, HIV-1 viral load, ARVs used in each country, and ARV drug resistance. LAg-recent result was defined as normalized optical density values ≤1.5. We compared HIV-1 incidence estimates using 2 RITA: RITA1: LAg-recent + VL ≥ 1000 copies/mL and RITA2: RITA1 + undetectable ARV. We explored RITA2 with self-reported ARV use and with clinical history. RESULTS: Overall, 357 adult HIV-positive participants were classified as having recent infection with RITA1. RITA2 reclassified 55 (15.4%) persons with detectable ARV as having long-term infection. Those with detectable ARV were significantly more likely to be aware of their HIV-positive status (84% vs. 10%) and had higher levels of drug resistance (74% vs. 26%) than those without detectable ARV. RITA2 incidence was lower than RITA1 incidence (range, 0%-30% decrease), resulting in decreased estimated new infections from 390,000 to 341,000 across the 13 countries. Incidence estimates were similar using detectable or self-reported ARV (R2 > 0.995). CONCLUSIONS: Including ARV in RITA2 improved the accuracy of HIV-1 incidence estimates by removing participants with likely long-term HIV infection.

Baseline predictors of antiretroviral treatment failure and lost to follow up in a multicenter countrywide HIV-1 cohort study in Ethiopia.

BACKGROUND: Antiretroviral therapy (ART) has been rapidly scaled up in Ethiopia since 2005, but factors influencing the outcome are poorly studied. We therefore analysed baseline predictors of first-line ART outcome after 6 and 12 months. MATERIAL AND METHODS: 874 HIV-infected patients, who started first-line ART, were enrolled in a countrywide prospective cohort. Two outcomes were defined: i) treatment failure: detectable viremia or lost-to-follow-up (LTFU) (confirmed death, moved from study sites or similar reasons); ii) LTFU only. Using stepwise logistic regression, four multivariable models identified baseline predictors for odds of treatment failure and LTFU. RESULTS: The treatment failure rates were 23.3% and 33.9% at 6 and 12 months, respectively. Opportunistic infections (OI), tuberculosis (TB), CD4 cells <50/µl, and viral load >5 log10 copies/ml increased the odds of treatment failure both at 6 and 12 months. The odds of LTFU at month 6 increased with baseline functional disabilities, WHO stage III/IV, and CD4 cells <50/µl. TB also increased the odds at month 12. Importantly, ART outcome differed across hospitals. Compared to the national hospital in Addis Ababa, patients from most regional sites had higher odds of treatment failure and/or LTFU at month 6 and/or 12, with the exception of one clinic (Jimma), which had lower odds of failure at month 6. CONCLUSIONS: In this first countrywide Ethiopian HIV cohort, a high ART failure rate was identified, to the largest extent due to LTFU, including death. The geographical region where the patients were treated was a strong baseline predictor of ART failure. The difference in ART outcome across hospitals calls the need for provision of more national support at regional level.

Pretreatment drug resistance in a large countrywide Ethiopian HIV-1C cohort: a comparison of Sanger and high-throughput sequencing.

Baseline plasma samples of 490 randomly selected antiretroviral therapy (ART) naïve patients from seven hospitals participating in the first nationwide Ethiopian HIV-1 cohort were analysed for surveillance drug resistance mutations (sDRM) by population based Sanger sequencing (PBSS). Also next generation sequencing (NGS) was used in a subset of 109 baseline samples of patients. Treatment outcome after 6- and 12-months was assessed by on-treatment (OT) and intention-to-treat (ITT) analyses. Transmitted drug resistance (TDR) was detected in 3.9% (18/461) of successfully sequenced samples by PBSS. However, NGS detected sDRM more often (24%; 26/109) than PBSS (6%; 7/109) (p = 0.0001) and major integrase strand transfer inhibitors (INSTI) DRMs were also found in minor viral variants from five patients. Patients with sDRM had more frequent treatment failure in both OT and ITT analyses. The high rate of TDR by NGS and the identification of preexisting INSTI DRMs in minor wild-type HIV-1 subtype C viral variants infected Ethiopian patients underscores the importance of TDR surveillance in low- and middle-income countries and shows added value of high-throughput NGS in such studies.

Assessment of the implementation of HIV-rapid test kits at different levels of health institutions in Ethiopia.

OBJECTIVE: Evaluation and monitoring of Human Immunodeficiency Virus (HIV) testing reagents at the point of service is helpful to prevent the occurrence of problems related to testing and interpretation. To evaluate the implementation of HIV rapid test kits at the point of services in voluntarily counseling and testing (VCT) and diagnostic centers in Ethiopia. METHODS: The assessment was the third phase of evaluation of HIV rapid test kits in Ethiopia followed from phase-I and phase-II. Known proficiency testing panels, well-structured questionnaire (addressing type of tests, human resource and problems related to tests), onsite supervision and retesting of samples collected from sites were used to evaluate the performances of reagents and laboratories. RESULTS: Forty-four health institutions were included. Thirty-six (90.0%) health institutions had trained human resource on HIV testing. In 27 (61.4%) three types of HIV rapid test kits (Determine, Capillus and Unigold) were available. Serial-algorithm was used in all the laboratories. In 31 (70.4%) of them external quality control specimens were not used. Twenty two (50.0%) of the laboratories reported frequent shortage of reagents. All (100%) were able to identify negative specimens distributed. Positive proficiency panel samples were identified in 37 (94.8%) of the 39 laboratories. There was 98.3% agreement at a screening level between the sites and the central laboratory. Rate of discrepancy between screening and confirmatory assays was found to be 3.0% and 2.1% at the sites and at central laboratory, respectively. CONCLUSION: The test kits showed a good performance at the point of services in the field sites. However, continuous assessment of HIV test kits at the point of service and training of professionals on newly arrived techniques are recommended to have effective testing performance with acceptable sensitive and specific testing algorithm. Effective quality assurance program should be in place to support programs such as VCT, prevention of mother-to-child-transmission and antiretroviral therapy.

The effect of HIV coinfection, HAART and TB treatment on cytokine/chemokine responses to Mycobacterium tuberculosis (Mtb) antigens in active TB patients and latently Mtb infected individuals.

Identification of Mtb specific induced cytokine/chemokine host biomarkers could assist in developing novel diagnostic, prognostic and therapeutic tools for TB. Levels of IFN-γ, IL-2, IL-17, IL-10, IP-10 and MIP-1α were measured in supernatants of whole blood stimulated with Mtb specific fusion protein ESAT-6/CFP-10 using xMAP technology. The study groups were HIV positive TB patients (HIV(+)TB(+)), HIV negative TB patients (HIV(-)TB(+)), HIV positive tuberculin skin test positive (TST+) (HIV(+)TST(+)), HIV negative TST+ (HIV(-)TST(+)), and HIV(-)TST(-) individuals. Compared to HIV(-)TST(-), latent TB infection led to increased levels of IP-10, IFN-γ and IL-17, while levels of IL-2 and IP-10 were increased with active TB. Levels of IFN-γ, IL-17, MIP-1α, and IL-10 were increased in HIV(-)TST(+) individuals compared to HIV(-)TB(+) patients. HIV coinfection decreased the level of IFN-γ, IL-17, IP-10 and IL-2. After six months (M6) of anti-TB treatment (ATT) in HIV(-)TB(+) patients, IFN-γ, IL-10, and MIP-1α levels normalized. After M6 and M18 of ATT plus HAART in HIV(+)TB(+) patients, levels of MIP-1α and IL-10 normalized, while this was not the case for IFN-γ, IL-2, IL-17, and IP-10 levels. In HIV(+)TST(+) patients on HAART, levels of IFN-γ, IL-17, IL-10 and MIP-1α normalized, while no change in the levels of IL-2 and IP-10 were observed. In conclusion, the simultaneous measurement of IFN-γ, IL-17 and IP-10 may assist in diagnosing LTBI; IL-2 and IP-10 may assist in diagnosing active TB; while IFN-γ, IL-17, MIP-1α, and IL-10 levels could help to discriminate LTBI and active TB. In addition, IL-10 and MIP-1α levels could help to monitor responses to TB treatment and HAART.

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.

Nine-year trends in HIV-1 prevalence among visa applicants in Urban Ethiopia.

A retrospective study was conducted to examine trends in HIV-1 prevalence among visa applicants between the years 1993 to 2001 in Urban Ethiopia. A total of 63,869 visa applicants were screened during these nine years period. The majority of them (79.5%) were females. Their mean age was 31.6 and 25.7 years for males and females, respectively. HIV-1 prevalence ranged from 6.8% in 1993 to 10.4% in 1997 (test for trend: p<0.001), while it seems stabilized at around 11% after 1997. The overall period prevalence was 9.5%. The peak prevalence was documented in the age group 25-29 for females (12.1%) while it was in the age group 30-34 for males (11.4%). This study, therefore, confirms the severity of the HIV-1 epidemic in the country. Visa applicants can be used as a sentinel population for monitoring trends in HIV-1 prevalence in the country, although additional socio-demographic information would be useful for better interpretation of such data.

Evaluation of rapid assays for screening and confirming HIV-1 infection in Ethiopia.

To evaluate a simple and rapid testing strategy to diagnose HIV infection in Ethiopia, we subjected a panel of 688 sera with known HIV serologic status (confirmed by ELISA/WB or double ELISA) to 3 rapid assays: Determine HIV-1/2, Capillus HIV-1/2 and Serocard HIV. Samples were obtained from participants in a cohort study on HIV-infection (72%), from tuberculosis patients (18%) and from participants in surveillance studies among police recruits and commercial sex workers (10%). The panel consisted of 249 HIV-1 positive samples, of which 68 were HIV-1 subtype C and 1 HIV-1 subtype A, and 439 HIV-1 negative samples. Determine and Capillus were 100% sensitive and 99.8% specific, Serocard was 100% sensitive and specific. On retrospective evaluation, both parallel (samples tested simultaneously by two rapid assays) and serial (samples tested by two consecutive rapid assays) testing algorithms were 100% sensitive and specific when compared to ELISA/WB or double ELISA testing strategy. In conclusion rapid assays have high sensitivity and specificity. HIV serodiagnosis based on rapid assays may therefore be a valuable alternative in voluntary counselling and testing centres and in facilities where sophisticated laboratories are not available.

Overall HIV-1 prevalence in pregnant women over-estimates HIV-1 in the predominantly rural population of Afar Region.

The appropriateness of sentinel sero-surveillance based upon ante-natal clinic (ANC) attendees to estimate HIV-1 prevalence in the general population has been questioned. In Ethiopia, where the population is heterogeneous and where economic and practical barriers to ANC attendance exist, problems of extrapolation may be exacerbated. We planned an unlinked anonymous sero-survey which included data on basic population characteristics to investigate whether sero-surveillance data from ANCs in Afar Region might be taken to represent the situation among the general population of the Region. 371 pregnant women attending Dubti Hospital and Assayta Health Centre were tested for HIV-1 (using a single ELISA test) and active syphilis (RPR test). Socio-demographic characteristics were collected for each woman. Of the women tested, 278 (75%) were 28 years of age or younger. Two hundred eighty (76%) were urban residents and 237 (64%) described themselves as being of Amhara ethnicity. Overall, 73 (19.7%) were HIV-1 positive, but prevalence was three times higher among the 237 women of Amhara ethnicity compared to the 112 of Afar ethnicity (24.9% vs 8.0%, p < 0.001), and almost three times higher for urban compared to rural residents (23.2% vs 8.8%, p < 0.001). Positive RPR results were strongly associated with HIV-1 infection (OR 3.37, 95% CI 1.47-7.71). According to the Demographic and Health Survey (DHS) 2000, only 4.5% of the population of Afar Region is of Amhara ethnicity, and 7.8% urban residents. We have demonstrated that basing ANC sero-surveillance in urban areas of Afar Region over-samples urban residents of Amhara ethnicity and yields a major over-estimation of overall HIV-1 prevalence for the Region. Reliable estimation of HIV-1 prevalence in Afar Region will require more flexible strategies that permit sampling of rural Afar residents.

Evaluation of rapid HIV test kits on whole blood and development of rapid testing algorithm for voluntary testing and counseling centers in Ethiopia.

Five simple and rapid HIV antibody detection assays viz. Determine, Capillus, Oraquick, Unigold and Hemastrip were evaluated to examine their performance and to develop an alternative rapid test based testing algorithm for voluntary counseling and testing (VCT) in Ethiopia. All the kits were tested on whole blood, plasma and serum. The evaluation had three phases: Primary lab review, piloting at point of service and implementation. This report includes the results of the first two phases. A total of 2,693 specimens (both whole blood and plasma) were included in the evaluation. Results were compared to double Enzyme Linked Immuno-Sorbent Assay (ELISA) system. Discordant EIA results were resolved using Western Blot. The assays had very good sensitivities and specificities, 99-100%, at the two different phases of the evaluation. A 98-100% result agreement was obtained from those tested at VCT centers and National Referral Laboratory for AIDS (NRLA), in the quality control phase of the evaluation. A testing strategy yielding 100% [95% CI; 98.9-100.0] sensitivity was achieved by the sequential use of the three rapid test kits. Direct cost comparison showed serial testing algorithm reduces the cost of testing by over 30% compared to parallel testing in the current situation. Determine, Capillus/Oraquick (presence/absence of frefrigeration) and Unigold were recommended as screening, confirmation and tiebreaker tests, respectively.

Seroprevalence and transmission of Hepatitis B virus among delivering women and their new born in selected health facilities, Addis Ababa, Ethiopia: a cross sectional study.

BACKGROUND: Hepatitis B Virus is a major public health problem worldwide. In 2012 alone, over 350 million chronic carriers and 1. 2 million annual deaths were occurred. Hepatitis B Virus causes 60 to 80% of the world's primary liver cancer and nearly 90% infants infected due to vertical transmission are at higher risk of developing chronic liver disease and cancer. Hence determining the burden of maternal and neonatal Hepatitis B Virus infection is a priority. METHODS: A cross sectional study was conducted from July - September 2012 at St. Paul's Hospital Millennium Medical College and Selam Health Center, Addis Ababa, Ethiopia. Blood samples from delivering mothers (n = 265) and their corresponding cords (n = 265) were collected. A pretested questionnaire was used to collect data. Hepatitis B Virus surface antigen was detected using Enzyme Linked Immunosorbent Assay. Frequency analysis and logistic regression test was used to identify the potential risk factors associated with Hepatitis B Virus positivity using SPSS Version -15. RESULTS: A total of 265 delivering women with the mean age of 25.8 years were enrolled in the study. Of these delivering women, 8 (3.0%) of mothers were positive for Hepatitis B Virus surface antigen, whereas 6 (2.3%) of cord bloods were positives with 75% concordance rate of exposed infants with sero-positive mothers. However, only one maternal positive case was observed for Hepatitis B e Ag test. Only 11% of the mothers know their Hepatitis B Virus status. Of the total mothers assessed for possible risk factors, 69 (26%) had only one type, while 161 (60.8%) had multiple exposure factors such as ear pricing, history of tribal marks, abortion, multiple-sexual partner and history of surgical procedures experienced from high to low frequency. The remaining 35 (13.2%) of the participants had not experienced possible risk factors. CONCLUSION: Though the maternal positivity rate was low, the rate of positivity in cord bloods was almost equal to those infected mothers. Therefore, screening of pregnant mothers and vaccination of infants could help to reduce the transmission. To minimize the higher overall risk exposure status of mothers, increasing awareness and intensive public health education is also recommended.

Antiretroviral treatment associated hyperglycemia and dyslipidemia among HIV infected patients at Burayu Health Center, Addis Ababa, Ethiopia: a cross-sectional comparative study.

BACKGROUND: The effects of highly active antiretroviral therapy (HAART) on glucose and lipid metabolism among sub-Saharan Africans, for whom access to antiretroviral therapy is expanding, remain largely unknown. Therefore, the aim of this study was to assess antiretroviral treatment associated hyperglycemia and dyslipidemia among HIV infected patients at Burayu health center, Addis Ababa, Ethiopia. METHODS: A cross-sectional comparative study was conducted among HIV infected adults at Burayu Health Center, Addis Ababa, Ethiopia from September, 2011 to May, 2012. Equal number of HAART naïve and HAART initiated patients (n = 126 each) were included in the study. Demographic data were collected using a well-structured questionnaire. Total cholesterol (TC), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glucose were determined. The data were analyzed using SPSS version 20 software. RESULT: Of 252 study participants, 72.2% were females; mean age was 35.3 years; mean Body Mass Index (BMI) was 21.4(kg/m2); mean time living with the virus was 20.6 months and 15.5% were TB-HIV co-infected. The prevalence of hyperglycemia, increased LDL-C hypercholesterolemia, hypertriglyceridemia and decreased HDL-C were 7.9%, 23%, 42.1%, 46.8% and 50.8% in HAART and 5.6%, 7.1%, 11.1%, 31% and 73% in non-HAART groups, respectively. First line antiretrovirals were drugs containing 2 nucleoside backbones (from Zidovudine/Stavudine/Lamivudine/Tenofovir) with either Nevirapine or Efavirenz. There was statistically significant increase in serum lipid profile levels among HAART initiated patients than HAART naïve individuals (p =0.01 for TG and <0.001 for others). CONCLUSION: First-line HAART is associated with potentially atherogenic lipid profile levels in patients with HIV infection compared to untreated patients. This indicates glucose and lipid profile levels need to be monitored regularly in HIV infected patients taking antiretroviral treatment.

Field expansion of DNA polymerase chain reaction for early infant diagnosis of HIV-1: The Ethiopian experience.

BACKGROUND: Early diagnosis of infants infected with HIV (EID) and early initiation of treatment significantly reduces the rate of disease progression and mortality. One of the challenges to identification of HIV-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identification of the HIV status of infants. METHOD: We conducted a joint site assessment and designed laboratories for the expansion of DNA polymerase chain reaction (PCR) testing based on dried blood spot (DBS) for EID in six regions of Ethiopia. Training of appropriate laboratory technologists and development of required documentation including standard operating procedures (SOPs) was carried out. The impact of the expansion of EID laboratories was assessed by the number of tests performed as well as the turn-around time. RESULTS: DNA PCR for EID was introduced in 2008 in six regions. From April 2006 to April 2008, a total of 2848 infants had been tested centrally at the Ethiopian Health and Nutrition Research Institute (EHNRI) in Addis Ababa, and which was then the only laboratory with the capability to perform EID; 546 (19.2%) of the samples were positive. By November 2010, EHNRI and the six laboratories had tested an additional 16 985 HIV-exposed infants, of which 1915 (11.3%) were positive. The median turn-around time for test results was 14 days (range 14-21 days). CONCLUSION: Expansion of HIV DNA PCR testing facilities that can provide quality and reliable results is feasible in resource-limited settings. Regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing.

Analysis of immune responses against a wide range of Mycobacterium tuberculosis antigens in patients with active pulmonary tuberculosis.

Characterizing host immune responses to molecular targets of Mycobacterium tuberculosis is essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series of M. tuberculosis antigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n = 34) was stimulated in vitro with M. tuberculosis antigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classical M. tuberculosis antigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to several M. tuberculosis antigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenic M. tuberculosis antigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets of M. tuberculosis antigens may be promising for the development of immunodiagnostics.

Sero-prevalence of latent Toxoplasma gondii infection among HIV-infected and HIV-uninfected people in Addis Ababa, Ethiopia: A comparative cross-sectional study.

BACKGROUND: Toxoplasmosis in immuno-compromised hosts manifests primarily as a life threatening condition, toxoplasmic encephalitis. However, there is scarce information about the magnitude of Toxoplasma gondii infection among HIV-infected people in Ethiopia. This study was, therefore, conducted to determine the sero-prevalence of T. gondii infection among HIV-infected and HIV-uninfected subjects. FINDINGS: Sera were collected from people with and without HIV infection for the purpose of studying hepatitis B virus (HBV) at St. Paul Hospital, Addis Ababa, Ethiopia from 24 January 2007 to 15 February 2007. Among these sera, the first 330 consecutive sera, 165 from each HIV sero-group, were selected and tested for anti-T. gondii IgG antibodies using Enzyme Linked Immunosorbent Assay. The seroprevalence of Toxoplasma infection was assessed against socio-demographic characteristics, HIV and HBV serostatus and HBV-related risk factors. The overall sero-prevalence of latent T. gondii infection among the study subjects was 90.0%. Toxoplasma infection was observed with respective prevalence of 93.3% and 86.7% among HIV-infected and HIV-uninfected people. Though Toxoplasma infection seems to be influenced by age, gender and HIV serostatus, only HBV serostatus was significantly associated (OR 2.71, CI 1.12 to 6.57) in multivariate logistic regression analysis. CONCLUSION: The seroprevalence of latent T. gondii infection is high and similar by HIV status. Educating people to prevent acquisition of new Toxoplasma infection and minimizing the risk of disease manifestations among HIV-Toxoplasma co-infected individuals is important.

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 laboratory-based 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.

Tuberculin skin test conversion and reactivity rates among adults with and without human immunodeficiency virus in urban settings in Ethiopia.

To investigate whether low CD4+ T-cell counts in healthy and human immunodeficiency virus (HIV)-infected Ethiopians influence tuberculosis (TB) immunological memory, tuberculin skin test (TST) conversion and reactivity rates were investigated among adults with and without HIV infection in urban settings in Ethiopia. Reaction to the TST was analyzed with purified protein derivative by the Mantoux technique. A total of 1,286 individuals with TST results of > or = 5-mm (n = 851) and < or = 4-mm (n = 435) induration diameters were included. Individuals with < or = 4-mm induration sizes were followed up for 21.4 +/- 9.5 months (mean +/- standard deviation) to observe skin test conversion. The overall TST reactivity (> or = 5-mm induration diameter) was 66.2% (n = 851). Reactivity was significantly lower among HIV-positive persons (40.5%) than among HIV-negative persons (68.7%) (P < 0.001). Of the above persons, 32 incident TB patients were checked for their TST status 13.05 +/- 11.1 months before diagnosis and reactivity was found among 22 (68.7%) of them. Of the TST-negative persons with 0- to 4-mm indurations who were followed up for 3 years, the conversion rate to positivity was 17.9/100 person-years of observation (PYO) (14.4/100 PYO and 18.3/100 PYO in HIV-positive and -negative persons, respectively). Despite lower absolute CD4+ T-cell numbers in Ethiopians, higher TST conversion and reactivity rates show the presence of a higher rate of latent TB infection and/or transmission. The lower TST positivity rate before a diagnosis of TB disease showed the lower sensitivity of the test. This indicates the need for other sensitive and specific diagnostic and screening methods to detect TB infection, particularly among HIV-positive persons, so that they can be given prophylactic isoniazid therapy.