Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Prevalence of Smear-Positive, Rifampicin-Resistant Mycobacterium tuberculosis and Related Factors Among Residents with Cough in Northern Ethiopian Refugee Health Facilities.

Purpose: To ascertain the prevalence of Mycobacterium tuberculosis (M.tb) among refugees suspected of tuberculosis (TB) and related risk factors, including smear-positive and Rifampicin-resistant M.tb. Methods: A cross-sectional study was conducted between January 2020 and May 2020 among 384 refugees in four refugee camps in Northwest Tigray, Ethiopia. Socio-demographic and clinical data were collected from refugees with a history of cough for more than two weeks prospectively. Spot-spot sputum samples were collected and transported in an ice box to the Shire Suhul Hospital Microbiology laboratory; and then examined using a Fluorescent Microscope. All smear-positive samples were further processed by GeneXpert to detect Rifampicin-resistant MTB. Data were analyzed using SPSS version 21 and a p-value <0.05 was considered statistically significant. Results: The overall prevalence of smear-positive PTB infection was 5.5% (21/384), but No TB case was resistant to Rifampicin detected by GeneXpert MTB/RIF assay. About 70% of the smear-positive pulmonary TB identified were females. Five (23.8%) of the smear-positive pulmonary tuberculosis cases were co-infected by HIV. Sharing of drink and food materials (AOR = 4.36, 95% CI = 1.19-15.89), active TB contact (AOR 7.24, 95% CI = 1.62-32.125), BMI (AOR = 5.23, 95% CI = 1.28-21.29), opening window practice (AOR = 4.32, 95% CI = 1.02-18.30) and HIV status (AOR = 9.36, 95% CI = 1.64-53.35) were statistically significant predisposing factors. Conclusion: The prevalence of smear-positive pulmonary TB among northwest Tigray refugee camps was still high. The prevalence of TB/HIV co-infection was also high. Minimizing close contact with active TB cases, reducing malnutrition, rapid TB/HIV screening, and establishing a ventilation system can reduce the transmission of TB among refugees.

Comparison of the Diagnostic Performances of Five Different Tests in Diagnosing Visceral Leishmaniasis in an Endemic Region of Ethiopia.

The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the present study, the diagnostic performances of rk39 RDT, the direct agglutination test (DAT), microscopy, loop-mediated isothermal amplification (LAMP), and miniature direct-on-blood polymerase chain reaction-nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) were assessed using quantitative polymerase chain reaction (qPCR) as the reference test in an endemic region of Ethiopia. In this study, 235 suspected VL cases and 104 non-endemic healthy controls (NEHCs) were recruited. Among the suspected VL cases, 144 (61.28%) tested positive with qPCR. The sensitivities for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA were 88.11%, 96.50%, 76.58%, 94.33%, and 95.80%, respectively. The specificities were 83.33%, 97.96%, 100%, 97.38%, and 98.92% for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA, respectively. In conclusion, rk39 RDT and microscopy exhibited lower sensitivities, while DAT demonstrated excellent performance. LAMP and mini-dbPCR-NALFIA showed excellent performances with feasibility for implementation in remote endemic areas, although the latter requires further evaluation in such regions.

Time to Sputum Culture Conversion and Its Predictors Among Multidrug Resistant Tuberculosis Patients in Tigray, Northern Ethiopia: Retrospective Cohort Study.

Background: Sputum culture conversion status is a cardinal index of treatment response and patient outcome for MDR TB patients on longer anti-TB drugs. But, there is limited information on time to sputum culture conversion of MDR TB patients on a longer anti-TB treatment regimen. Therefore, this study aimed to evaluate time to sputum culture conversion and its predictors among MDR TB patients in Tigray, Northern Ethiopia. Methods: A retrospective cohort study was conducted from January 2017 through September 2020 among MDR TB patients in Tigray, Northern Ethiopia. Demographic and clinical characteristics including bacteriological data were extracted from the TB registration book and electronic database in Tigray Health Research Institute. Statistical analysis was performed using SPSS version 25. The time to initial sputum culture conversion was analyzed using the Kaplan-Meier method. Bivariate and multivariate Cox proportional hazards regression analyses were used to identify predictors for culture conversions. P <0.05 was considered statistically significant. Results: A total of 294 eligible study participants with a median age of 30 years (IQR: 22.75-40) were included. The participants were followed for a total of 1066.7 person months. Sputum culture conversion was achieved in 269 (91%) of the study participants. The median time of sputum culture conversion was 64 days (IQR: 49-86). In our multivariate model, HIV-positive (aHR=1.529, 95% CI: 1.096-2.132, P=0.012), patients new to anti-TB treatment (aHR=2.093, 95% CI: 1.100-3.982, P=0.024) and baseline AFB smear grading of +1 (aHR=1.982, 95% CI: 1.428-2.750, P=0.001) significantly affected time to initial sputum culture conversion. Conclusion: The median time of culture conversion was 64 days. Moreover, the majority of the study participants achieved culture conversion within the first six months of treatment commencement, which supports predefined standard treatment durations.

Performance of rapid rk39 tests for the diagnosis of visceral leishmaniasis in Ethiopia: a systematic review and meta-analysis.

BACKGROUND: Visceral Leishmaniasis (VL) is a severely neglected disease affecting millions of people with high mortality if left untreated. In Ethiopia, the primary laboratory diagnosis of VL is by using an antigen from a 39-amino acid sequence repeat of a kinesin-related (rK39) of leishmania donovani complex (L. donovani), rapid diagnostic tests (RDT). Different rk39 RDT brands are available with very variable performance and studies from Ethiopia showed a very wide range of sensitivity and specificity. Therefore, a systematic review and meta-analysis were conducted to determine the pooled sensitivity and specificity of rk39 RDT in Ethiopia. METHOD: PUBMED, EMBASE, and other sources were searched using predefined search terms to retrieve all relevant articles from 2007 to 2020. Heterogeneity was assessed by visually inspecting summary receiver operating curves (SROC), Spearman correlation coefficient (rs), Cochran Q test statistics, inconsistency square (I2) and subgroup analysis. The presence and statistical significance of publication bias were assessed by Egger's test at p < 0.05, and all the measurements showed the presence of considerable heterogeneity. Quality assessment of diagnostic accuracy studies (QUADAS-2) checklists was used to check the qualities of the study. RESULTS: A total of 664 articles were retrieved, and of this 12 articles were included in the meta-analysis. Overall pooled sensitivity and specificity of the rk39 RDT to diagnose VL in Ethiopia were 88.0% (95% CI 86.0% to 89.0%) and 84.0% (95% CI 82.0% to 86.0%), respectively. The sensitivity and specificity of the rk39 RDT commercial test kits were DiaMed: 86.9% (95% CI 84.3% to 89.1%) and 82.2% (95% CI 79.3% to 85.0%), and InBios: 80.0% (95% CI 77.0% to 82.8%) and 97.4% (95% CI 95.0% to 98.8%), respectively. CONCLUSION: Referring to our result, rk39 RDT considered an essential rapid diagnostic test for VL diagnosis. Besides to the diagnostic accuracy, the features such as easy to perform, quick (10-20 min), cheap, equipment-free, electric and cold chain free, and result reproducibility, rk39 RDT is advisable to remains in practice as a diagnostic test at least in the remote VL endemic localities till a better test will come.

Utility of the Loop-Mediated Isothermal Amplification Assay for the Diagnosis of Visceral Leishmaniasis from Blood Samples in Ethiopia.

Rapid and accurate diagnosis of visceral leishmaniasis (VL) is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69-98.66) and a specificity of 98.0% (95% CI: 94.34-99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61-98.34) in HIV-negatives and 100% (95% CI: 85.18-100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10-98.11) of the microscopy+ cases and was negative in 11/27 (40.7%, 95% CI: 22.39-61.20) of the microscopy- cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35-97.62) of the rK39+ cases and was negative in 149/154 (96.8%, 95% CI: 92.59-98.94) of the rK39- cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64-98.73) of the microscopy+ cases and negative in only 12/27 (44.4%, 95% CI: 25.48-64.67) of the microscopy- cases. There was an excellent agreement between rK39 and LAMP (Kappa = 0.91, 95% CI: 0.86-0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52-99.89) and a specificity of 98.0% (95% CI: 94.34-99.59). The findings demonstrate that LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 coinfected patients, in an endemic setting.

Effect of rK39 testing in guiding treatment initiation and outcome in patients with visceral leishmaniasis in Ethiopia: A prospective cohort study.

BACKGROUND: The rapid diagnostic test (RDT) rK39 is currently being used for routine diagnosis of visceral leishmaniasis (VL) in East Africa. However, continuous monitoring of the performance of the assay, in particular its impact on the clinical decision in initiating anti-leishmanial treatment and outcomes remains needed as there are concerns about the diagnostic performance of this test. METHODS: VL patients prospectively enrolled in a diagnostic trial and with rK39 RDT were included. We evaluated the effect of rK39 testing in guiding treatment initiation and outcome. On the basis of rK39 RDT test result as well as clinical case definition for VL and microscopy examination, the clinicians decide whether to initiate VL therapy or not. Poisson regression models were used to identify factors associated with a decision to initiate VL therapy. In addition, treatment outcomes of those who received VL therapy were compared to those who received non-VL treatment. RESULTS: Of 324 VL suspects enrolled, 184 (56.8%) were rK39+ and 140 (43.2%) were rK39‒. In addition, microscopy exam was done on tissue aspirates from a sub-population (140 individuals), which is 43.2% of the suspected cases, comprising of 117 (63.6%) rK39+ and only 23 (16.4%) rK39‒ cases. Of those with microscopy examination, only 87 (62.1%) were found positive. Among 184 (56.8%) patients without microscopy, 67 (36.4%) were rK39+, of whom 83 (65.9%) were positive by microscopy, 21 (16.7%) were negative by microscopy and 22 (17.5%) had no microscopy results. On the other hand, of those who did not receive VL treatment 58/189 (30.7%) were rK39+ and 131 (69.3%) were rK39‒. Of the rK39+ cases who did not receive VL therapy, only 1 (1.7%) patient was microscopy positive, 12 (20.7%) were negative and 45 (77.6%) patients had no microscopy result. Of the rK39‒ cases (n = 131) who did not receive VL treatment, 16 were microscopy negative and 115 without microscopy exams. Whereas positive rK39 result [adjusted Relative Risk (aRR) 0.69; 95% CI: 0.49-0.96, p = 0.029] and positive microscopy results (aRR 0.03; 95% CI: 0.00-0.24, p = 0.001) were independently associated with VL treatment, having confirmed diagnosis other than VL (aRR 1.64; 95% CI: 1.09-2.46, p = 0.018) was independently associated with initiation of non-VL therapy. The proportion of rK39+ patients who received non-VL treatment with no improvement outcome was significantly higher when compared to those who received VL treatment (24.1%, 95% CI: 14.62-37.16 vs. 11.9%, 95%CI: 7.26-18.93; p<0.0001). CONCLUSION: The study shows that a significant proportion of patients with rK39+ results were undertreated. Failure to do microscopy was associated with lack of improved clinical outcome. Including an additional simple point-of-care assay in the diagnostic work-up is urgently needed to correctly identify VL cases and to prevent morbidity and mortality associated with the disease.

Prevalence and factors associated with exclusive breastfeeding among rural mothers of infants less than six months of age in Southern Nations, Nationalities, Peoples (SNNP) and Tigray regions, Ethiopia: a cross-sectional study.

BACKGROUND: Exclusive breastfeeding (EBF) is the global recommended nutrition for infants less than 6 months of age. The prevalence of exclusive breastfeeding in Ethiopia is much lower than the recommendations of World Health Organization (WHO). This study aimed to assess the prevalence and associated factors of EBF among rural mothers of infants less than 6 months of age in two regions, Southern Nations, Nationalities and Peoples (SNNP) and Tigray Regions, of Ethiopia. METHODS: The research was based on the secondary data analysis of community-based cross-sectional study conducted in 56 rural woredas (districts) in two regions of Ethiopia for impact evaluation of Alive & Thrive multi-year project. The 24-h recall dietary data were collected from 600 mother-infant dyads where the infant was less than 6 month of age, using multistage cluster sampling technique and 584 dyads were found eligible for analysis. Bivariate and multivariable logistic regressions were applied to identify the associated factors of exclusive breastfeeding. RESULTS: The prevalence of EBF of infants less than 6 months of age was 88.0% (95% CI 84.9, 90.4%). The odds of practicing EBF were significantly higher among infants less than 2 months of age (AOR 4.47, 95% CI 2.41, 8.27), married mothers (AOR 4.35, 95% CI 1.50, 12.67), mothers who gave birth in health facilities (AOR 2.07, 95% CI 1.15, 3.73) and mothers who received breastfeeding counseling during pregnancy (AOR 2.23, 95% CI 1.26, 3.96). CONCLUSIONS: The prevalence of EBF was relatively high when compared with previous studies in Ethiopia but close to the WHO recommendations of 90 %. Infant age, marital status of mothers, delivery place and breastfeeding counseling during pregnancy were identified as factors associated with EBF practices in 24 h preceding the survey. Devising appropriate strategies on breastfeeding messaging/counseling and support in addressing poor breastfeeding practices through existing government-led health intervention packages are recommended.

Biofilm and hemagglutinin formation: a Hallmark for drug resistant uropathogenic Escherichia coli.

OBJECTIVE: Urinary tract infection (UTI) is one of the most frequent disease encounters in pregnant mothers, and the most drug resistant, biofilm and hemagglutinin producer Uropathogenic Escherichia coli (UPEC) is the major etiologic agent. Therefore, the aim of this study was to assess the association between the antimicrobial resistance, and biofilm and hemagglutinin production of Uropathogenic Escherichia coli. RESULTS: UTI among the study participants was 27.3%; and UPEC was found the major etiologic agent followed by coagulase negative staphylococcus. Risk factors, previous history of catheterization and previous history of UTI were found significantly associated with UTI, recurrent UTI, drug resistance and biofilm formation. Of the tested antibiotics, nitrofurantoin was the most effective drug for UPEC. Nearly 100% of the biofilm producers were resistant to norfloxacin, cotrimoxazole, and gentamicin.

Rifampicin resistant tuberculosis in presumptive pulmonary tuberculosis cases in Dubti Hospital, Afar, Ethiopia.

INTRODUCTION: Ethiopia stood third in drug-resistant tuberculosis (TB) in Africa, and more than 5,000 MDR-TB patients are reported each year. Greater than 90% of rifampicin (RIF) resistant strains are resistant to isoniazid (INH) and hence the objective of this study was to determine the prevalence and risk factors of RIF resistant MTB among presumptive TB cases at Dubti General Hospital, Afar, Ethiopia. METHODOLOGY: In this cross-sectional study, 384 presumptive TB cases were recruited and a structured questionnaire was used to collect socio-demographic and clinical data. Sputum samples were collected and examined using X-pertMTB/RIF assay. Bivariate, multivariate logistic regressions, and fishers' exact analysis were done to assess the associations between the prevalence of TB and MDR-TB with different socio-demographic and clinical variables. RESULTS: In the present study, the overall prevalence of pulmonary TB was 24.5% (94/384), of this 4 (4.3%) isolates were resistant to RIF. History of anti-TB treatment (AOR = 2.4, 95% CI: 1.3-4.4 and TB contact (AOR = 3.6, 95% CI: 2.1-6.2 were significantly associated with gene X-pert MTB/RIF positive TB. Moreover, resistance to rifampicin was statistically associated with the history of TB contact with multi-drug resistant TB (P = 0.027) and khat chewer cases (P = 0.04). CONCLUSIONS: The overall prevalence of TB and its drug-resistant were relatively higher than that of in the general population in Ethiopia. History of anti-TB treatment and TB contact were significantly associated with X-pert MTB/RIF positive MDR-TB.

Prevalence and antimicrobial susceptibility patterns of methicillin-resistant Staphylococcus aureus among janitors of Mekelle University, North Ethiopia.

OBJECTIVE: The objective of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus and antimicrobial susceptibility patterns among janitors working at Mekelle University, Tigray, Northern Ethiopia. RESULT: The overall prevalence of S. aureus and MRSA in the present study were 17.97% (69/384) and 6.25% (24/384) respectively. Although not statistically significant, the prevalence of MRSA among janitors working in the medical area (9.7%, 10/103) was two times higher than the non-medical area (4.9%, 14/281). Janitors who had more service year and who were unable to read and write were found with high isolates of MRSA. Nasal carriage of MRSA among janitors who work in the hospital and who were hospitalized in the last 3 months and those who had exposure to wastes and body fluids were 13 (37.1%) and 10 (38.5%) respectively. Majority of the isolates of S. aureus were sensitive to ciprofloxacin (67; 97%), doxycycline (56; 81%), erythromycin (54; 78%), chloramphenicol (50; 72.5%) and cefoxitin (45; 65.2%). Sixty-seven of the 69 (97%) were resistant to penicillin. Of the 69 isolates of S. aureus, 22 (31.9%) showed multidrug resistant. Fourteen were resistant to three antimicrobials, 2 were resistant to four antimicrobials, and 7 were resistant to five antimicrobials.

Virological and immunological failure of HAART and associated risk factors among adults and adolescents in the Tigray region of Northern Ethiopia.

BACKGROUND: Human immunodeficiency virus/Acquired immunodeficiency syndrome associated morbidity and mortality has reduced significantly since the introduction of highly active antiretroviral therapy. As a result of increasing access to highly active antiretroviral therapy, the survival and quality of life of the patients has significantly improved globally. Despite this promising result, regular monitoring of people on antiretroviral therapy is recommended to ensure whether there is an effective treatment response or not. This study was designed to assess virological and immunological failure of highly active antiretroviral therapy users among adults and adolescents in the Tigray region of Northern Ethiopia, where scanty data are available. METHODS: A retrospective follow up study was conducted from September 1 to December 30, 2016 to assess the magnitude and factors associated with virological and immunological failure among 260 adults and adolescents highly active antiretroviral therapy users who started first line ART between January 1, 2008 to March 1, 2016. A standardized questionnaire was used to collect socio-demographic and clinical data. SPSS Version21 statistical software was used for analysis. Bivariate and multivariate logistic regression analyses were conducted to identify factors associated to virological and immunological failure. Statistical association was declared significant if p-value was ≤ 0.05. RESULT: A total of 30 (11.5%) and 17 (6.5%) participants experienced virological and immunological failure respectively in a median time of 36 months of highly active antiretroviral therapy. Virological failure was associated with non-adherence to medications, aged < 40 years old, having CD4+ T-cells count < 250 cells/µL and male gender. Similarly, immunological failure was associated with non-adherence, tuberculosis co-infection and Human immunodeficiency virus RNA ≥1000 copies/mL. CONCLUSIONS: The current result shows that immunological and virological failure is a problem in a setting where highly active antiretroviral therapy has been largely scale up. The problem is more in patients with poor adherence. This will in turn affect the global targets of 90% viral suppression by 2020. This may indicate the need for more investment and commitment to improving patient adherence in the study area.