Molecular epidemiology of tuberculosis in Sicily, Italy: what has changed after a decade?

BACKGROUND: We aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) isolates in the province of Palermo, Sicily, Italy, by characterizing 183 isolates identified in the years 2004-2012. A comparison with 104 MTBC strains identified in the same geographic area in the years 1994-2000 was also carried out. METHODS: One hundred eighty-three MTBC isolates identified in Palermo, Italy, in the years 2004-2012 were analyzed by spoligotyping and the 24 mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem-repeat (VNTR) method typing. Susceptibility testing to streptomycin, isoniazid, rifampin and ethambutol was also performed. Furthermore, the spoligotyping dataset obtained from 104 MTBC isolates identified from 1994 to 2000 was reanalyzed. Distribution into lineages and clustering of isolates in the two periods was compared. RESULTS: One hundred seventy-seven out of the 183 isolates of MTBC submitted to molecular typing were fully characterized. Of these, 108 were from Italian-born and 69 from foreign-born individuals. Eleven different lineages and 35 families-subfamilies were identified with the most represented lineages being Haarlem (26.5%), T (19.2%), LAM (13.6%) and S (8.5%). Except for the Haarlem lineage, where isolates from foreign-born patients were overrepresented, the distribution of isolates in the families belonging to the Euro-American clone reflected the proportions of the two subpopulations. A total of 27 (15.2%) strains were clustered and three clusters were mixed. Approximately 25% of the 183 MTBC isolates under study proved to be resistant to at least one antiTB drug, with only three isolates categorized as multidrug resistant (MDR). When MTBC isolates identified in the years 1994-2000 were reanalyzed, lineages T (30.8%), LAM (29.8%), Haarlem (16.3%) and S (13.5%) proved to be predominant. No MTBC isolates belonging to CAM, U, CAS, Turkish and Ural lineages were identified. CONCLUSIONS: A wide heterogeneity was detected among the MTBC strains isolated in the years 2004-2012. Six lineages were not present among the isolates of the period 1994-2000. Comparison between distribution of lineages in the two consecutive periods depicts rapid and deep changes in the TB epidemiology in Palermo, Italy. An universal and continued laboratory-based surveillance of TB in Sicily is required.

First identification of Microsporidia MB in Anopheles coluzzii from Zinder City, Niger.

BACKGROUND: Malaria, a disease transmitted by Anopheles mosquitoes, is a major public health problem causing millions of deaths worldwide, mostly among children under the age of 5 years. Biotechnological interventions targeting parasite-vector interactions have shown that the microsporidian symbiont Microsporidia MB has the potential to disrupt and block Plasmodium transmission. METHODS: A prospective cross-sectional survey was conducted in Zinder City (Zinder), Niger, from August to September 2022, using the CDC light trap technique to collect adult mosquitoes belonging to the Anopheles gambiae complex. The survey focused on collecting mosquitoes from three neighborhoods of Zinder (Birni, Kangna and Garin Malan, located in communes I, II and IV, respectively). Collected mosquitoes were sorted and preserved in 70% ethanol. PCR was used to identify host species and detect the presence of Microsporidia MB and Plasmodium falciparum infection. RESULTS: Of the 257 Anopheles mosquitoes collected and identified by PCR, Anopheles coluzzii was the most prevalent species, accounting for 97.7% of the total. Microsporidia MB was exclusively detected in A. coluzzii, with a prevalence of 6.8% (17/251) among the samples. No significant difference in prevalence was found among the three neighborhoods. Only one An. coluzzii mosquito tested PCR-positive for P. falciparum. CONCLUSIONS: The results confirm the presence of Microsporidia MB in Anopheles mosquitoes in Zinder, Niger, indicating its potential use as a biotechnological intervention against malaria transmission. However, further studies are needed to determine the efficacy of Microsporidia MB to disrupt Plasmodium transmission as well as its impact on vector fitness.

Evaluation of the Diagnostic Performances of the SD-Bioline®HBeAg Rapid Test Used Routinely for the Management of HBV-Infected Individuals in Burkina Faso.

Hepatitis B e antigen (HBeAg) is a marker of wild-type hepatitis B virus replication. In resource-limited countries where access to enzyme-linked immunosorbent assay (ELISA) remains a challenge, rapid diagnostic tests (RDT) constitute a good alternative. The HBeAg status is employed to evaluate eligibility for antiviral therapy and to prevent the transmission of hepatitis B from mother to child (PMTCT). The objective of this study was to assess the diagnostic performance of the SD-Bioline®HBeAg RDT commonly used for detecting HBeAg in laboratories in Burkina Faso. The sample panel used was collected from HBsAg-positive patients received in the laboratory for the detection of HBeAg with the rapid test. The samples were retested for HBeAg using the VIDAS HBe/Anti-HBe enzyme-linked fluorescent assay (ELFA) (Gold standard). Then, the viral load (VL) of HBV DNA was determined using the GENERIC HBV CHARGE VIRLAE kit (GHBV-CV). The diagnostic performances of the SD-Bioline®HBeAg and its agreement with the gold standard were calculated with their 95% confidence intervals. Overall, 340 sera obtained from HBsAg-positive patients were included in this evaluation Compared to the VIDAS HBe/Anti-HBe ELFA test, the sensitivity (Se) and specificity (Sp) of the SD-Bioline®HBeAg test were 33.3% and 97.9%, respectively. The concordance between the two tests was 0.42. Depending on the viral load, the Se and Sp varied from 8.8% and 98.3% for a VL < 2000 IU/mL to 35.5% and 98.4% for a VL > 2,000,000 IU/mL. The results showed a low sensibility of the SD-Bioline®HBeAg RDT test, indicating that its use is inappropriate for the clinical management of HBV-infected patients. They also highlight the urgent need to develop HBeAg rapid tests with better sensitivities.

Spoligotyping of Mycobacterium africanum, Burkina Faso.

Using Ziehl-Neelsen-positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum-specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15-20 years ago.

Microbead-based spoligotyping of Mycobacterium tuberculosis from Ziehl-Neelsen-stained microscopy preparations in Ethiopia.

The worldwide dissemination of Mycobacterium tuberculosis strains has led to the study of their genetic diversity. One of the most used genotyping methods is spoligotyping, based on the detection of spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) locus. This study assessed the performance of a microbead-based spoligotyping assay using samples extracted from Ziehl-Neelsen-stained smear-microscopy preparations and described the genetic diversity of Mycobacterium tuberculosis among new TB patients in Southern Nations, Nationalities and Peoples' Region (SNNPR) in Ethiopia. Among the 91 samples analysed, 59 (64.8%) generated spoligotyping patterns. Fifty (84.7%) samples were classified into 12 clusters (mostly Lineage 4 or 3) comprising 2-11 samples and nine had unique spoligotyping patterns. Among the 59 spoligotyping patterns, 25 belonged to the T1 sublineage, 11 to the T3-ETH, 5 to the URAL, 4 to the H3 and 14 to other L4 sublineages. There was a remarkable variation in genetic distribution in SNNPR compared to other regions of the country. Microbead-based spoligotyping is an easy-to-perform, high-throughput assay that can generate genotyping information using material obtained from smear microscopy preparations. The method provides an opportunity to obtain data of the M. tuberculosis genetic epidemiology in settings with limited laboratory resources.

Mycobacterium tuberculosis complex genotypes circulating in Nigeria based on spoligotyping obtained from Ziehl-Neelsen stained slides extracted DNA.

METHODS: All State TB control programmes in Nigeria were requested to submit 25-50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013-2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays. RESULTS: Spoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations. CONCLUSION/SIGNIFICANCE: We present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB.

TB-EFI, a novel 18-Plex microbead-based method for prediction of second-line drugs and ethambutol resistance in Mycobacterium tuberculosis complex.

Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus. Basic analysis can be performed manually. An upgraded interpretation is made available in Excel 2016®. Using a reference set of 61 DNA extracts, we show that TB-EFI provides perfect concordance with pyrosequencing. Concordance between genotypic resistance and phenotypic DST was relatively good (72 to 98% concordance), with lower efficiency for fluoroquinolones and ethambutol due to some untargeted mutations. When compared to phenotypical resistance, performances were similar to those obtained with Hain MTBDRsl assay, possibly thanks to the use of automatized processing of data although some mutations involved in fluoroquinolone resistance could not be included. When applied on three uncharacterized sets, phenotype could be predicted for 51% to 98% depending on the setting and the drug investigated, detecting one extensively drug-resistant isolate in each of a Pakistan and a Brazilian set of 91 samples, and 9 XDR among 43 multi-resistant Kazakhstan samples. By allowing high-throughput detection of second-line drugs resistance and of resistance to ethambutol that is often combined to second-line treatments, TB-EFI is a cost-effective assay for large-scale worldwide surveillance of resistant tuberculosis and XDR-TB control.

Multi-drug resistant Mycobacterium tuberculosis complex genetic diversity and clues on recent transmission in Punjab, Pakistan.

Multi-Drug Resistant Tuberculosis (MDR-TB), i.e. bacilli resistant to rifampicin (RIF) and isoniazid (INH), is a major Public Health concern in Pakistan according to WHO estimates (3.5% and 32% of new and retreated cases, respectively). Previous Pakistanis reports identified a correlation between being MDR and belonging to Beijing or EAI lineages in one study, and belonging to "H4"-Ural Euro-American sublineage in another study. In addition, MDR-TB transmission was suspected in Karachi. We tested MDR characteristics on a Punjab sample of 278 clinical isolates (without selection for Multi-Drug Resistance) including new and retreated cases collected from 2008 to 2012. All samples were characterized by a new, microbead-based method named "TB-SPRINT" (molecular diagnostic including spoligotype identification, and genetic resistance determinants to first-line anti-TB drugs RIF and INH). Isolates from 2011 to 2012 (n=100) were further analyzed using 24-loci MIRU-VNTR. We detected 8.7% MDR isolates (CI95%=[5.0; 12.5]), mainly among CAS lineage that predominates in this central-East region of Pakistan. Out of 20 MDR-TB cases, 12 different TB-SPRINT profiles were identified, limiting the suspicion of MDR-TB transmission. 24 MIRU-VNTR confirmed the unrelatedness of isolates with different TB-SPRINT profiles and discriminated 3 isolates with identical TB-SPRINT profiles. In conclusion, our study did not confirm any of the correlations between Multi-Drug Resistance and lineage or sublineage in Punjab, Pakistan. MDR-TB isolates were diverse indicating that transmission is not pervasive. TB-SPRINT proved useful as a first step for detecting MDR-TB likely transmission events, before more extensive genotyping such as 15 or 24 MIRU-VNTR and thorough epidemiological investigation.

Multidrug resistant Mycobacterium tuberculosis: a retrospective katG and rpoB mutation profile analysis in isolates from a reference center in Brazil.

BACKGROUND: Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in Mycobacterium tuberculosis isolates from a national reference center. METHODS AND FINDINGS: Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical IS6110-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 rpoB mutations while codon 531 mutations prevailed in the other isolates. CONCLUSIONS: Our data suggest that there were no major multidrug resistant M. tuberculosis strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.

Insights into the population structure of Mycobacterium tuberculosis using spoligotyping and RDRio in a southeastern Brazilian prison unit.

Tuberculosis (TB) is still a serious public health problem, continuing to be an important threat for confined populations. We used spoligotyping to estimate the genotypic clades of Mycobacterium tuberculosis isolates from inmates in two blocks in a southeastern Brazilian prison unit, with TB incidence rate of 8185/100.000. The Latin American Mediterranean (LAM) clade is well represented in the country, and the LAM specific molecular markers, RD(Rio) large sequence polymorphism and the SNP on the Rv3062 [ligB(1212)], were used to characterize spoligotype signatures from prison isolates. Typing of RD(Rio) and ligB increase LAM clade from 66.7% (n=72/108) to 69.4% (n=75). The LAM2 SIT17 (n=23) and SIT179 (n=12) signatures comprised one third of all isolates, followed by Haarlem (11.5%, n=12), T (8.7%, n=9) and X (5.7%, n=6) clades. Strains with unknown signatures represented 5.5% (n=6), and four (3.7%) did not match any lineage. We observed RD(Rio) among 64 (59.2%) isolates, and 54 (50%) were of the LAM clade. In particular, the LAM2/RD(Rio) sub-lineage was significantly associated with clustering (p=0.02) and its frequency was higher (32%) when compared to that of the previous general TB cases in RJ (4.29%). Overall cluster frequency defined by spoligotyping/IS6110-RFLP was 62%. The two evolutionary markers helped to evaluate some LAM signature misconceptions and demonstrate that LAM2/RD(Rio) was found with high frequency, hitherto being unnoticed. All these data, allied to high clustering, imply that public health measures to minimize the escalation of TB in prison is essential, and both spoligotyping as well as RD(Rio) would be useful tools to monitor the effects of the measures with respect to M. tuberculosis lineage variation.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

A molecular epidemiological and genetic diversity study of tuberculosis in Ibadan, Nnewi and Abuja, Nigeria.

BACKGROUND: Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. METHODOLOGY/PRINCIPAL FINDINGS: DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other "modern" genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.