Polymorphisms in CARD8 and NLRP3 are associated with extrapulmonary TB and poor clinical outcome in active TB in Ethiopia.

Innate immunity is a first line defense against Mycobacterium tuberculosis infection where inflammasome activation and secretion of the pro-inflammatory cytokine IL-1beta, plays a major role. Thus, genetic polymorphisms in innate immunity-related genes such as CARD8 and NLRP3 may contribute to the understanding of why most exposed individuals do not develop infection. Our aim was to investigate the association between polymorphisms in CARD8 and NLRP3 and active tuberculosis (TB) as well as their relationship to treatment outcome in a high-endemic setting for TB. Polymorphisms in CARD8 (C10X) and NLRP3 (Q705K) were analysed in 1190 TB patients and 1990 healthy donors (HD). There was a significant association between homozygotes in the CARD8 polymorphism and extrapulmonary TB (EPTB), which was not the case for pulmonary TB or HDs. Among TB-patients, there was an association between poor treatment outcome and the NLRP3 (Q705K) polymorphism. Our study shows that inflammasome polymorphisms are associated with EPTB and poor clinical outcome in active TB in Ethiopia. The practical implications and determining causal relationships on a mechanistic level needs further study.

Performance evaluation of malaria microscopists working at rechecking laboratories in Ethiopia.

BACKGROUND: Microscopic diagnosis of Giemsa-stained thick and thin blood films has remained the standard laboratory method for diagnosing malaria. High quality performance of microscopists that examine blood slides in health facilities remains critically important. MATERIALS AND METHODS: A cross-sectional study was conducted to assess the performance of 107 malaria microscopists working at 23 malaria rechecking laboratories in Ethiopia. A set of 12 blood film slides was distributed to each microscopist. Data was collected and exported to SPSS version 20 for analysis. Chi-square, sensitivity, specificity, percent agreement, and kappa scores were calculated to assess performance in detecting and identification of Plasmodium species. RESULTS: The mean age of the participants was 30 ± 5 yrs and most of them (54; 50.5%) were working at regional reference laboratories. Overall, the sensitivity of participants in detecting and identifying malaria parasite species was 96.8% and 56.7%, respectively. The overall agreement on detection and identification of malaria species was 96.8% (Kappa = 0.9) and 64.8% (Kappa = 0.33), respectively. The least accurately identified malaria parasite species was P. malariae (3/107; 2.8%) followed by P. ovale (35/107; 32.7%). Participants working at hospital laboratories had the highest percentage (72.3 %, Kappa=0.51) of accurate species identification. Study participants that had participated in malaria microscopy and quality assurance trainings were significantly better at quantifying parasite densities (P<0.001). CONCLUSION: The accuracy of parasite identification and quantification differed strongly between participants and expert microscopists. Therefore, regular competency assessment and training for malaria microscopists should be mandatory to assure proper diagnosis and management of malaria in Ethiopia.

Asymptomatic Helminth Infection in Active Tuberculosis Is Associated with Increased Regulatory and Th-2 Responses and a Lower Sputum Smear Positivity.

BACKGROUND: The impact of intestinal helminth infection on the clinical presentation and immune response during active tuberculosis (TB) infection is not well characterized. Our aim was to investigate whether asymptomatic intestinal helminth infection alters the clinical signs and symptoms as well as the cell mediated immune responses in patients with active TB. METHODOLOGY: Consecutive, newly diagnosed TB patients and healthy community controls (CCs) were recruited in North-west Ethiopia. TB-score, body mass index and stool samples were analyzed. Cells from HIV-negative TB patients (HIV-/TB) and from CCs were analyzed for regulatory T-cells (Tregs) and cytokine responses using flow cytometry and ELISPOT, respectively. RESULTS: A significantly higher ratio of helminth co-infection was observed in TB patients without HIV (Helm+/HIV-/TB) compared to HIV negative CCs, (40% (121/306) versus 28% (85/306), p = 0.003). Helm+/HIV-/TB patients showed significantly increased IL-5 secreting cells compared to Helm-/HIV-/TB (37 SFU (IQR:13-103) versus 2 SFU (1-50); p = 0.02, n = 30). Likewise, levels of absolute Tregs (9.4 (3.2-16.7) cells/µl versus 2.4 (1.1-4.0) cells/µl; p = 0.041) and IL-10 secreting cells (65 SFU (7-196) versus 1 SFU (0-31); p = 0.014) were significantly higher in Helm+/HIV-/TB patients compared to Helm-/HIV-/TB patients. In a multivariate analysis, a lower rate of sputum smear positivity for acid fast bacilli, lower body temperature, and eosinophilia were independently associated with helminth infection in TB patients. CONCLUSIONS: Asymptomatic helminth infection is associated with increased regulatory T-cell and Th2-type responses and a lower rate of sputum smear positivity. Further studies are warranted to investigate the clinical and immunological impact of helminth infection in TB patients.

Genetic diversity of Plasmodium falciparum isolates based on MSP-1 and MSP-2 genes from Kolla-Shele area, Arbaminch Zuria District, southwest Ethiopia.

BACKGROUND: The genetic diversity of Plasmodium falciparum has been extensively studied in various countries. However, limited data are available from Ethiopia. This study was conducted to evaluate the extent of genetic diversity of P. falciparum in Kolla-Shele, in the southwest of Ethiopia. METHODS: A total of 88 isolates from patients with uncomplicated P. falciparum attending Kolla-Shele Health Centre was collected from September to December, 2008. After extraction of DNA by Chelex method, the samples were genotyped by using nested-PCR of msp1 (block 2) and msp2 (block 3) including their allelic families: K1, MAD20, RO33 and FC27, 3D7/IC1, respectively. RESULTS: Allelic variation in both msp1 and msp2 were identified in the 88 blood samples. For msp1 67% (59/88) and msp2 44% (39/88) were observed. K1 was the predominant msp1 allelic family observed in 33.9% (20/59) of the samples followed by RO33 and MAD20. Of the msp2 allelic family 3D7/IC1 showed higher frequency (21.5%) compared to FC27 (10.3%). A total of twenty-three alleles were detected; of which, eleven were from msp2 and twelve from msp2 genes. Fifty-nine percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 1.8 (95% CI: 1.48-2.04). The heterozygosity index was 0.79 and 0.54for msp1 and msp2, respectively. There was no statically significant difference in the multiplicity of infection by either age or parasite density (P > 0.05). CONCLUSION: This genetic diversity study showed the presence of five allelic types in the study area, with dominance K1 in the msp1 family and 3D7/IC1 in the msp2 family. Multiple infections were observed in nearly 60% of the samples.

Genotyping of human papillomavirus in paraffin embedded cervical tissue samples from women in Ethiopia and the Sudan.

Cervical cancer is the most frequent female malignancy in most developing countries. Previous studies have demonstrated a strong association of human papillomavirus (HPV) infection with dysplasia and carcinoma of the uterine cervix. The objective of this study was to identify the prevailing HPV genotypes responsible for the development of cervical cancer among women in Ethiopia and the Sudan. A molecular characterization of HPV was done on 245 paraffin embedded cervical biopsy samples collected from the two countries. Amplification of HPV and subsequent genotyping was done using SPF10 primers and Line probe assay. Of samples collected from Ethiopian patients, 93% (149/160) and 13% (21/160) had high risk and low risk HPV genotypes, respectively. Among samples collected from the Sudan, 94% (80/85) harbored high risk and 11.7% (10/85) low risk HPV genotypes. Human papillomavirus 16 was the most frequent genotype identified in samples from Ethiopia (91%, 136/149) and the Sudan (82.5%, 66/80). HPV 52, 58, and 18 were the second, third and fourth common genotypes identified in Ethiopia, whereas HPV 18, 45, and 52 were the second, third, and fourth genotypes identified in samples collected from the Sudan. Thus, individuals living in different geographical localities should receive vaccines based on the specific genotypes circulating in the area and a vaccine targeting HPV 16, 18, 45, 52, and 58 may be optimal for the control of cervical cancer in the two countries.

The impact of asymptomatic helminth co-infection in patients with newly diagnosed tuberculosis in north-west Ethiopia.

BACKGROUND: Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls. METHODOLOGY: Consecutive smear positive TB patients (n = 112), their household contacts (n = 71) and community controls (n = 112) were recruited in Gondar town, Ethiopia. Stool microscopy, HIV serology, serum IgE level, eosinophil and CD4 counts were performed and tuberculosis patients were followed up for 3 months after initiation of anti-TB treatment. RESULTS: Helminth co-infection rate was 29% in TB patients and 21% in both community control and household contacts (p = 0.3) where Ascaris lumbricoides was the most prevalent parasite. In TB patients the seroprevalence of HIV was 47% (53/112). Eosinophilia and elevated IgE level were significantly associated with asymptomatic helminth infection. During TB treatment, the worm infection rate of HIV+/TB patients declined from 31% (10/32) at week 0 to 9% (3/32) at week 2 of TB treatment, whereas HIV-/TB patients showed no change from baseline to week 2, 29% (13/45) vs. 22.2% (10/45). This trend was stable at week 8 and 12 as well. CONCLUSION: One third of smear positive TB patients were infected with helminths. Eosinophilia and elevated IgE level correlated with asymptomatic worm infection, indicating an effect on host immunity. The rate of worm infection declined during TB treatment in HIV+/TB co-infected patients whereas no decline was seen in HIV-/TB group.