Rift valley fever: diagnostic challenges and investment needs for vaccine development.

Rift valley fever virus (RVFV) is a causative agent of a viral zoonosis that constitutes a major clinical burden in wild and domestic ruminants. The virus causes major outbreaks in livestock (sheep, goats, cattle and camels) and can be transmitted to humans by contaminated animal products or via arthropod vectors. Human-to-human transmission has not been reported to date, but spill-over events from animals have led to outbreaks in humans in Africa and the Arabian Peninsula. Currently, there is no licensed human vaccine against RVFV and the virus is listed as a priority pathogen by the World Health Organisation (WHO) due to the high epidemic potential and the lack of effective countermeasures. Multiple large RVFV outbreaks have been reported since the virus was discovered. During the last two decades, over 4000 cases and ~1000 deaths have been reported. The lack of systematic surveillance to estimate the true burden and incidence of human RVF disease is a challenge for planning future vaccine efficacy evaluation. This creates a need for robust diagnostic methodologies that can be deployed in remote regions to aid case confirmation, assessment of seroprevalence as well as pathogen surveillance required for the different stages of vaccine evaluation. Here, we perform comprehensive landscaping of the available diagnostic solutions for detection of RVFV in humans. Based on the identified gaps in the currently available in-house and commercially available methods, we highlight the specific investment needs for diagnostics that are critical for accelerating the development of effective vaccines against RVFV.

Vitamin D Deficiency is Associated with Increased Use of Antimicrobials among Preschool Girls in Ethiopia.

Preschool children in Addis Ababa, Ethiopia, are highly exposed to influenza viruses. Factors related to infections, nutrition, and environmental conditions that might explain the burden of influenza among these children were investigated. Ninety-five preschool children, 48 girls and 47 boys, were followed clinically for 12 months. Illness and immune responses to influenza; three other respiratory viruses; five airway pathogenic bacteria; and levels of vitamins D, A, and B12 were assessed. Most of the children had antibodies to numerous respiratory viral and bacterial agents at study start, and many were infected during follow-up. Twenty-five girls and 25 boys fell ill during the study, and were treated with one or more courses of systemic antimicrobials. Ninety percent of both girls and boys had 25-hydroxyvitamin D [25(OH)D] levels below the recommended levels. While there was no overall difference in the levels of vitamins D, A, and B12 between girls and boys, treated girls had significantly lower 25(OH)D levels than non-treated girls and treated boys. There was a considerable number of short for age children, but only the short treated girls had significantly lower 25(OH)D levels than the non-treated children. Preschool girls with low 25(OH)D levels were more vulnerable to pathogenic microbes than boys.

Diagnostic applications for Lassa fever in limited-resource settings.

Lassa fever, caused by arenavirus Lassa virus (LASV), is an acute viral haemorrhagic disease that affects up to an estimated 300 000 individuals and causes up to 5000 deaths per year in West Africa. Currently available LASV diagnostic methods are difficult to operationalise in low-resource health centres and may be less sensitive to detecting all known or emerging LASV strains. To prioritise diagnostic development for LASV, we assessed the diagnostic applications for case detection, clinical management, surveillance, outbreak response, and therapeutic and vaccine development at various healthcare levels. Diagnostic development should prioritise point-of-care and near-patient diagnostics, especially those with the ability to detect all lineages of LASV, as they would allow for rapid detection in resource-limited health facilities closer to the patient.

High Prevalence of Humoral and Cellular Immunity to Influenza Viruses in Preschool Children Living in Addis Ababa, Ethiopia.

BACKGROUND: Influenza in children who reside in tropical and subtropical regions has until recently been regarded as insignificant. However, new evidence suggests that it significantly impacts hospitalization and promotes secondary bacterial coinfections. Ethiopia is situated in a subtropical area where influenza viruses are likely to circulate year round. METHODS: Clinical data were recorded in a cohort of 103 healthy preschool children recruited in Addis Ababa, Ethiopia. Humoral and cellular immune responses to influenza virus were determined by hemagglutination inhibition (HI) and interferon-γ enzyme-linked immunospot assays. RESULTS: Ninety-six percent of the children (2-5 years old) had pre-existing HI antibody responses to 1 or more of the circulating influenza A subtypes, H1N1 (51%), H3N2 (86%), or influenza B (51%) strains. At the age of 4, all children had been infected with at least 1 strain, and 75% had been infected with 2-4 different viral strains. CD4+ and CD8+ T-cell responses against conserved viral antigens increased with repeated exposures, indicating boosting of cross-reactive cellular immunity. Malnutrition did not seem to affect these immune responses to influenza. CONCLUSIONS: Influenza is highly prevalent among children in this area of Ethiopia. Due to the risk of secondary bacterial pneumonia, increased influenza awareness might benefit child health.

Comparative Proteomic Analysis of Mycobacterium tuberculosis Lineage 7 and Lineage 4 Strains Reveals Differentially Abundant Proteins Linked to Slow Growth and Virulence.

In order to decipher the nature of the slowly growing Mycobacterium tuberculosis (M.tuberculosis) lineage 7, the differentially abundant proteins in strains of M. tuberculosis lineage 7 and lineage 4 were defined. Comparative proteomic analysis by mass spectrometry was employed to identify, quantitate and compare the protein profiles of strains from the two M. tuberculosis lineages. Label-free peptide quantification of whole cells from M. tuberculosis lineage 7 and 4 yielded the identification of 2825 and 2541 proteins, respectively. A combined total of 2867 protein groups covering 71% of the predicted M. tuberculosis proteome were identified. The abundance of 125 proteins in M. tuberculosis lineage 7 and 4 strains was significantly altered. Notably, the analysis showed that a number of M. tuberculosis proteins involved in growth and virulence were less abundant in lineage 7 strains compared to lineage 4. Five ABC transporter proteins, three phosphate binding proteins essential for inorganic phosphate uptake, and six components of the type 7 secretion system ESX-3 involved in iron acquisition were less abundant in M. tuberculosis lineage 7. This proteogenomic analysis provided an insight into the lineage 7-specific protein profile which may provide clues to understanding the differential properties of lineage 7 strains in terms of slow growth, survival fitness, and pathogenesis.

Deciphering the recent phylogenetic expansion of the originally deeply rooted Mycobacterium tuberculosis lineage 7.

BACKGROUND: A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (M. tuberculosis) termed lineage 7 was discovered in Ethiopia. Whole genome sequencing of 30 lineage 7 strains from patients in Ethiopia was performed. Intra-lineage genome variation was defined and unique characteristics identified with a focus on genes involved in DNA repair, recombination and replication (3R genes). RESULTS: More than 800 mutations specific to M. tuberculosis lineage 7 strains were identified. The proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) in 3R genes was higher after the recent expansion of M. tuberculosis lineage 7 strain started. The proportion of nsSNPs in genes involved in inorganic ion transport and metabolism was significantly higher before the expansion began. A total of 22346 bp deletions were observed. Lineage 7 strains also exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion. CONCLUSIONS: We have identified unique genomic signatures of the lineage 7 strains. The high frequency of nsSNP in 3R genes after the phylogenetic expansion may have contributed to recent variability and adaptation. The abundance of mutations in genes involved in inorganic ion transport and metabolism before the expansion period may indicate an adaptive response of lineage 7 strains to enable survival, potentially under environmental stress exposure. As lineage 7 strains originally were phylogenetically deeply rooted, this may indicate fundamental adaptive genomic pathways affecting the fitness of M. tuberculosis as a species.

Qualitative Assessment of Challenges in Tuberculosis Control in West Gojjam Zone, Northwest Ethiopia: Health Workers' and Tuberculosis Control Program Coordinators' Perspectives.

Background. Weak health systems pose many barriers to effective tuberculosis (TB) control. This study aimed at exploring health worker's and TB control program coordinator's perspectives on health systems challenges facing TB control in West Gojjam Zone, Amhara Region, Ethiopia. Methods. This was a qualitative descriptive study. Eight in-depth interviews with TB control program coordinators and two focus group discussions among 16 health workers were conducted. Purposive sampling was used to recruit study participants. Thematic analysis was used to identify and analyse main themes. Results. We found that intermittent interruptions of laboratory reagents and anti-TB drugs supplies, absence of trained and motivated health workers, poor TB data documentation, lack of adherence to TB treatment guideline, and lack of access to TB diagnostic tools at peripheral health institutions were challenges facing the TB control program performance in the study zone. Conclusions. Ensuring uninterrupted supply of anti-TB drugs and laboratory reagents to all health institutions is essential. Continuous refresher training of health workers on standard TB care and data handling and developing and implementing a sound retention strategy to attract and motivate health professionals to work in rural areas are necessary interventions to improve the TB control program performance in the study zone.

Genomic characterization of Mycobacterium tuberculosis lineage 7 and a proposed name: 'Aethiops vetus'.

Lineage 7 of the Mycobacterium tuberculosis complex has recently been identified among strains originating from Ethiopia. Using different DNA typing techniques, this study provides additional information on the genetic heterogeneity of five lineage 7 strains collected in the Amhara Region of Ethiopia. It also confirms the phylogenetic positioning of these strains between the ancient lineage 1 and TbD1-deleted, modern lineages 2, 3 and 4 of Mycobacterium tuberculosis. Four newly identified large sequence polymorphisms characteristic of the Amhara Region lineage 7 strains are described. While lineage 7 strains have been previously identified in the Woldiya area, we show that lineage 7 strains circulate in other parts of the Amhara Region and also among foreign-born individuals from Eritrea and Somalia in The Netherlands. For ease of documenting future identification of these strains in other geographical locations and recognizing the place of origin, we propose to assign lineage 7 strains the lineage name 'Aethiops vetus'.

Mycobacterium tuberculosis lineage 7 strains are associated with prolonged patient delay in seeking treatment for pulmonary tuberculosis in Amhara Region, Ethiopia.

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n=240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n=138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n=60; 26.0%), the second largest to lineage 7 (n=36; 15.6%), and the third largest to the lineage Haarlem (n=35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P<0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.

Tuberculosis management time: an alternative parameter for measuring the tuberculosis infectious pool.

OBJECTIVE: To demonstrate the application of TB management time as an alternative parameter to estimate the size of the tuberculosis infectious pool in West Gojjam Zone of Amhara Region, Ethiopia. METHODS: In this study, we used the TB management time, i.e. the number of days from start of cough until start of treatment, to determine the infectious period. Patients with sputum smear-positive and smear-negative pulmonary TB, retreatment and an estimated number of undetected cases were included. The infectious pool was then estimated as the annual number of infectious person days in a defined population. RESULTS: The TB management time of presently undiagnosed TB cases and sputum smear-positive patients contributed significantly to the infectious pool with 151,840 and 128,750 infectious person days per year, respectively. The total infectious pool including sputum smear-negative TB cases and retreatment patients in the study area was estimated at 325,410 person days or 15,447 person days per 100,000 population during the study year. CONCLUSION: Recording TB management time may be used to estimate the infectious pool of TB and to monitor programme performance in the community.

Time to first consultation, diagnosis and treatment of TB among patients attending a referral hospital in Northwest, Ethiopia.

BACKGROUND: Early detection and treatment of TB is essential for the success of TB control program performance. The aim of this study was to determine the length and analyze predictors of patients', health systems' and total delays among patients attending a referral hospital in Bahir Dar, Ethiopia. METHODS: A cross-sectional study was conducted among newly diagnosed TB cases ≥ 15 years of age. Delay was analyzed at three levels: the periods between 1) onset of TB symptoms and first visit to medical provider, i.e. patients' delay, 2) the first visit to a medical provider and the initiation of treatment i.e. health systems' delay and 3) onset of TB symptoms and initiation of treatment i.e. total delay. Uni- and multi-variate logistic regression analyses were performed to investigate predictors of patients', health systems' and total delays. RESULTS: The median time of patients' delay was 21 days [(interquartile range (IQR) (7 days, 60 days)]. The median health systems' delay was 27 days (IQR 8 days, 60 days) and the median total delay was 60 days (IQR 30 days, 121 days). Patients residing in rural areas had a three-fold increase in patients' delay compared to those from urban areas [Adjusted Odds Ratio (AOR) 3.4; 95% (CI 1.3, 8.9)]. Extra-pulmonary TB (EPTB) cases were more likely to experience delay in seeking treatment compared to pulmonary (PTB) cases [(AOR 2.6; 95% (CI 1.3, 5.4)]. Study subjects who first visited health centres [(AOR) 5.1; 95% (CI 2.1, 12.5)], private facilities [(AOR) 3.5; 95% (CI 1.3, 9.7] and health posts [(AOR) 109; 95% (CI 12, 958], were more likely to experience an increase in health systems' delay compared to those who visited hospitals. CONCLUSIONS: The majority of TB patients reported to medical providers within an acceptable time after the onset of symptoms. Rural residence was associated with patients' and total delays. Providing the population with information about TB symptoms and the importance of early health seeking may be an efficient way to decrease TB transmission, morbidity and mortality. Establishing efficient TB diagnostic and treatment facilities at the periphery level is imperative to reduce diagnostic delay and expedite TB treatment.

Spoligotyping of Mycobacterium tuberculosis isolates among pulmonary tuberculosis patients in Amhara Region, Ethiopia.

The aim of this study was to characterize Mycobacterium tuberculosis isolates circulating in the Amhara Region of Ethiopia. Sputum samples were collected from new pulmonary tuberculosis (TB) patients in the Region. Genotyping of mycobacterial DNA was performed by spoligotyping and isolates were assigned to families using the SpolDB4 and the model-based program 'Spotclust'. A high level of diversity was found among the 237 isolates. Sixty-five different spoligopatterns were obtained. The T (30.8%), Central Asian (CAS; 21.1%) and U (17.7%) families were the predominant isolates comprising 69.6% of the total strains. Eighty-five per cent of the U lineage belonged to Spoligo-International-Type (SIT) 910 and SIT 1729. Only a few of these strains are included in SpolDB4 to date. Of the total strains, 41 (17.3%) were unique and have not been described in SpolDB4 to date. This study indicated that the TB epidemic in Amhara Region, Ethiopia, is characterized by the circulation of numerous M. tuberculosis strain families. The high proportion of SIT 910 and SIT 1729 strains may indicate an increase in the importance of these lineages in the transmission of TB in the study region.

Assessment of knowledge and practice of private practitioners regarding tuberculosis control in Ethiopia.

INTRODUCTION: Ethiopia has a growing private health sector. In recent years, the directly observed treatment short course (DOTS) strategy was initiated in selected private health facilities in the country. The objective of the present study was to assess knowledge and practice of private practitioners in tuberculosis (TB) control in Amhara Region, Ethiopia. METHODOLOGY: An institution-based cross-sectional study was conducted among 112 private practitioners selected from all private health facilities in the region. The study was conducted between May and August 2008 and data was collected using a semi-structured questionnaire. Group differences were analyzed using the chi-square test. RESULTS: Fifty-nine (52.7%) of the private practitioners suspected TB in patients with three weeks' duration of cough. Only 37 (33.0%) of the private practitioners were able to precisely list the correct treatment regimens for all categories as recommended in the National Tuberculosis and Leprosy Control Program guidelines. The correct frequency of TB treatment monitoring was provided by 44 (50%) of the respondents. Overall 44 (39.3%) of the private practitioners did not have satisfactory knowledge about the directly observed treatment short course (DOTS) strategy. Those who attended DOTS training during the two years prior to the survey were more likely to have satisfactory knowledge compared to those who did not receive training (OR 4.45, 95% CI: 1.33, 14.87, p < 0.02). CONCLUSION: A significant proportion of private practitioners did not have satisfactory knowledge and practice about DOTS. The provision of regular DOTS refresher courses improves TB management for patients in the region.

The perspective of private practitioners regarding tuberculosis case detection and treatment delay in Amhara Region, Ethiopia: a cross-sectional study.

BACKGROUND: Engaging all health care providers in tuberculosis (TB) control has been incorporated as an essential component of World Health Organization's Stop TB Strategy and the Stop TB Partnership's global plan 2006-2015. Ethiopia has a growing private health sector. The objective of the present study was to investigate the role of private practitioners (PPs) in TB case detection and assess their perspectives on TB treatment delay in Amhara Region, Ethiopia. RESULTS: A cross-sectional study among 112 PPs selected from private health facilities (PHF) in the region was conducted. The study was carried out between May and August 2008 and data was collected using a semi-structured questionnaire. Group differences were analyzed using one-way Anova test and a p-value of < 0.05 was considered statistically significant.In this study, PPs saw a median of 12 TB suspects and 1.5 patients a week. The mean number of TB suspects and patients seen varied significantly among the different professions with p < 0.009 and p < 0.004, respectively. Pulmonary TB patients referred by PPs were delayed up to one week before starting treatment at government health facilities. A 22% increase in the detection of smear-positive TB cases may be achieved by involving all PHFs in the TB control program in the region. Nineteen percent of the PPs indicated that TB patients' prior attendance to non medical health providers resulted in complication of disease and increased treatment delay for TB. CONCLUSION: PPs manage a substantial number of TB suspects and patients in Amhara Region, Ethiopia. The GHF delay observed among TB patients referred by PPs to GHF is unnecessary. Expanding PPM-DOTS in the region and improving the quality of TB care at both government and private health facilities reduces treatment delay and increases TB case detection.