Human brucellosis in the southern West Bank, 2015–17: a seroprevalence and molecular characterisation study

Abstract

BackgroundBrucellosis is a global zoonotic disease ranked by WHO as being among the top seven neglected zoonoses. After implementing a livestock vaccination strategy in 1998, the incidence rate of human brucellosis in Palestine dropped significantly. However, in 2015 the disease re-emerged with alarming incidence rates similar to those in the period before 1998. The aim of this study was to characterise the strains associated with this episode using genetic and genomic tools. MethodsAll patients were referred to the department of preventive medicine from several public or private clinics in the southern West Bank due to undulant fever. After a careful analysis of a patients’ history to evaluate exposure to Brucella, blood samples from patients suspected of brucellosis were collected. Samples positive for the Rose Bengal test with an antibody titre of 1:80 IU/mL or more by Brucella standard agglutination test, and not under antibiotic treatment, were further cultured using the BACTEC 9240 blood culture system. DNA was extracted from the positive culture samples and analysed using three biomarkers to validate the presence of the Brucella genus, establish the species, and identify the Brucella melitensis Rev 1 vaccine strain. Nine randomly chosen isolates were sequenced using targeted next generation sequencing technology to screen for the rpoB gene mutations. Multiple-locus variable number of tandem repeats analysis (MLVA) with 16 variable number of tandem repeats markers was used to genotype the isolates and BioNumerics version 7.6 software was used to analyse the data using the categorical coefficient and unweighted pair group method using the arithmetic averages algorithm. FindingsBlood samples were obtained from a total of 1324 patients during 2015–17. 704 (53·2%) of 1324 serum samples showed agglutination in the Rose Bengal test and 559 (79·4%) of the 704 were considered seropositive based on a standard agglutination test titre of 1:80 IU/mL or more. Only 130 (23·3%) of the 559 seropositive samples were from patients not under antibiotic treatment. Upon culturing, only 72 (12·9%) of the seropositive samples showed growth signals and were positive by PCR for the Brucella genus marker, and all isolates were B melitensis. None of the samples were associated with the B melitensis Rev 1 vaccine strain. The results of sequencing the rpoB gene by next generation sequencing showed four different single nucleotide variations that were not located within the rifampicin resistance-determining region. Using MLVA-16 analysis and the MLVA bank benchmark, the studied isolates were divided into 22 unique genotypes. A hierarchical clustering analysis for the 72 isolates and 18 B melitensis global isolates representing four major regions in the Middle East, Europe, Africa, and America, clustered the 73 isolates in the Middle East clade, providing a similarity cutoff value of more than 97% for genotype and more than 75% for clade. InterpretationThis study shows that the brucellosis episode of 2015–17 was due to B melitensis local isolates and was not associated with the Rev 1 vaccine stain. The results show that MLVA-16 is a useful tool for the public health surveillance of human brucellosis. The present study analysed samples from the Hebron governorate only. It is recommended to include more human and animal samples from different areas in Palestine to understand the molecular epidemiology of brucellosis in Palestine and to make more effective decisions regarding control and treatment programmes. FundingPalestine Polytechnic University.