Occurrence of RD149 and RD152 deletions in Mycobacterium tuberculosis strains from Pakistan

Introduction: Central Asian Strain 1 (CAS1) is the predominant Mycobacterium tuberculosis genotype in Pakistan. The occurrence of deletions in regions of differences (RDs) among CAS1 and other predominant genogroups in the country were investigated. Methodology: Using stratified random sampling, 235 M. tuberculosis (185 pulmonary, 50 extrapulmonary) strains were selected from 926 previously spoligotyped isolates, including 171 CAS strains (133 CAS1 (ST26), 38 CAS subfamily), 8 Beijing isolates, 47 isolates belonging to other previously defined ( Other ) clusters, and 9 previously undefined Unique isolates. Commonly reported RD deletions, RD1, RD750, RD207, RD149, RD152, RD105, RD150, RD142 and RD181, were investigated using a PCR - based method. Results: Deletions in RDs 750, 149 and 152 were identified among CAS strains, and in RDs 207, 149, 152, 105, 150, 142 and 181 in Beijing isolates. CAS1 strains showed more frequent RD149 deletions compared with CAS subfamily strains (p=0.036), and more frequent RD152 deletions compared with Other clusters (p=0.003). RD149 and RD152 deletions were more frequent in Beijing isolates compared with CAS1 strains (p \u3c 0.001). Concurrent RD149 and RD152 deletions were more frequent in CAS1 compared with Other clusters (p \u3c 0.001) and in Beijing strains compared with CAS1 (p \u3c 0.001). No significant difference was detected in RD deletion patterns between pulmonary and extra pulmonary isolates. Conclusion: Higher frequencies of RD149 and RD152 deletions and of concurrent RD149 and RD152 deletions were found in CAS1 and Beijing strains compared with CAS subfamilies, Other clusters and Unique strains. No association between these deletions and disease presentation, pulmonary or extrapulmonary tuberculosis, was observed

Elevated ex vivo monocyte chemotactic protein-1 (CCL2) in pulmonary as compared with extra-pulmonary tuberculosis

BACKGROUND: Tuberculosis causes 3 million deaths annually. The most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. Restriction of Mycobacterium tuberculosis depends on effective recruitment and subsequent activation of T lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. Tumor necrosis factor (TNF)-α is essential for granuloma formation and is a potent activator of monocyte chemotactic protein (MCP-1, CCL2). CCL2 is essential for recruitment of monocytes and T cells and has been shown to play a role in protection against tuberculosis. Interleukin -8 (CXCL8) is a potent activator of neutrophils. Increased levels of CCL2, CXCL8 and TNFα are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. We have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis. METHODS: Serum levels of CCL2, CXCL8 and TNFα were measured in patients with pulmonary tuberculosis (N = 12), extra-pulmonary tuberculosis (N = 8) and BCG-vaccinated healthy volunteers (N = 12). Whole blood cells were stimulated with non-pathogenic Mycobacterium bovis bacille-Calmette Guerin (BCG) vaccine strain or bacterial lipopolysaccharide (LPS) and cyto/chemokines were monitored in supernatants. RESULTS: Circulating serum levels of CXCL8 and TNFα were raised in all tuberculosis patients, while CCL2 levels were not. There was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. M. bovis BCG-induced ex vivo CCL2 secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. In response to LPS stimulation, patients with pulmonary tuberculosis showed increased CCL2 and TNFα responses as compared with the extra-pulmonary group. BCG-, and LPS-induced CXCL8 secretion was comparable between patients and controls. CONCLUSION: CCL2 is activated by TNFα and is essential for recruitment of monocytes and T cells to the site of mycobacterial infection. Increased CCL2 activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site

A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

Background Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.info:eu-repo/semantics/publishedVersio

Alternate efflux pump mechanism may contribute to drug resistance in extensively drug-resistant isolates of mycobacterium tuberculosis

INTRODUCTION: Extensively drug-resistant tuberculosis (XDR-TB) has emerged as one of the biggest threats to public health and TB control programs worldwide. XDR-TB is caused by Mycobacterium tuberculosis (MTB) strains resistant to rifampin and isoniazid, as well as to a fluoroquinolone and to at least one injectable aminoglycoside. Drug resistance in MTB has primarily been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, it has also been shown that efflux pumps may play a role in resistance of MTB. Upregulation of drug efflux pumps can decrease the intracellular concentration of drugs and reduce their efficacy. METHODS: Whole genome sequencing was performed on 32 XDR-TB clinical isolates. Sequence data were used to investigate SNPs in efflux pump genes as compared with the H37Rv reference genome. RESULTS: Of the XDR MTB strains, eight (21.62%) were wild type for rpsL, rrs (500 region), and gidB genes, but had non-synonymous (ns) SNPs (aspartic acid to histidine) in the drrA efflux pump gene at position 3273138. Three of eight (37.5%) XDR MTB strains, wild type for rpsL, rrs (500 region), gidB, and gyrB genes were phenotypically streptomycin sensitive and five (62.5%) XDR MTB strains were streptomycin resistant, while all XDR MTB strains, wild type for rpsL, rrs, gidB, and gyrB genes were resistant to fluoroquinolone (ofloxacin) and ethambutol. In addition, three XDR MTB strains wild type for rpsL, rrs, gidB, and drrA genes showed nsSNPs (isoleucine to valine) in the major facilitator superfamily, Rv1634 efflux pump gene at position 1839306. CONCLUSION: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB

Single nucleotide polymorphisms in efflux pumps genes in extensively drug resistant Mycobacterium tuberculosis isolates from Pakistan

It is challenging to understand mechanisms of drug resistance in Mycobacterium tuberculosis (MTB) due to the large variability in resistance associated genes. Efflux pump genes contribute to drug resistance and thus add to this complexity. Efflux pump gene protein superfamilies have been characterized by genome analysis of drug resistant strains and through invitro transcriptional studies. However, there is limited information regarding efflux pump genes in extensively drug resistant (XDR) tuberculosis (TB) isolates. Whole genome sequencing (WGS) based analysis of 37 extensively drug resistant (XDR) and five drug sensitive (DS) MTB clinical isolates was performed. Single nucleotide polymorphisms (SNPs) in efflux pump genes Rv0194, Rv1217, Rv1218, drrA, drrB, Rv1258, Rv1634, Rv2688, Rv1273, Rv1819, Rv1458, Rv1877 and Rv1250 were determined in the clinical isolates as compared with the H37Rv reference strain. Allele frequencies of SNPs identified in XDR strains were compared with DS strains. Gene expression of Rv0194, Rv2688, Rv1634, drrA and drrB was determined in XDR -TB isolates (n=9), DS-TB strains (n=4) and H37Rv. We identified SNPs in XDR-TB isolates which were either unique or present at very low frequencies in DS strains; Rv0194 G170V; Rv1217 L151R; Rv1258 P369T and G391R; Rv1273 S118G and I175T; Rv1877 I534T; Rv1250 V318X/A and S333A, and Rv2688 P156T. The expression of Rv2688 and drrB was found to be raised in XDR-TB as compared with DS-TB strains. We identified unique SNPs in efflux pump genes which may be associated with increased drug resistance in the isolates. Increased levels of Rv2688 and drrB efflux pump gene expression observed in XDR strains even in the absence of antibiotics suggests that these clinical isolates may be more refractory to treatment. Further studies are required to directly associate these mutations with increased resistance in MTB

Characterization of genomic variations in SNPs of PE_PGRS genes reveals deletions and insertions in extensively drug resistant (XDR) M. tuberculosis strains from Pakistan.

BACKGROUND: Mycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multigene family. Although the function of PE_PGRS genes is unknown, it is hypothesized that the PE_PGRS genes may be associated with antigenic variability in MTB. MATERIAL AND METHODS: Whole genome sequencing analysis was performed on (n=37) extensively drug-resistant (XDR) MTB strains from Pakistan, which included Lineage 1 (East African Indian, n=2); Other lineage 1 (n=3); Lineage 3 (Central Asian, n=24); Other lineage 3 (n=4); Lineage 4 (X3, n=1) and T group (n=3) MTB strains. RESULTS: There were 107 SNPs identified from the analysis of 42 PE_PGRS genes; of these, 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in PE_PGRS genes - 6, 9 and 10 - were common in all EAI, CAS, Other lineages (1 and 3), T1 and X3. Deletions (DELs) in PE_PGRS genes - 3 and 19 - were observed in 17 (80.9%) CAS1 and 6 (85.7%) in Other lineages (1 and 3) XDR MTB strains, while DELs in the PE_PGRS49 were observed in all CAS1, CAS, CAS2 and Other lineages (1 and 3) XDR MTB strains. All CAS, EAI and Other lineages (1 and 3) strains showed insertions (INS) in PE_PGRS6 gene, while INS in the PE_PGRSgenes 19 and 33 were observed in 20 (95.2%) CAS1, all CAS, CAS2, EAI and Other lineages (1 and 3) XDR MTB strains. CONCLUSION: Genetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs and INDELs in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence

Presence of RD149 Deletions in M. tuberculosis Central Asian Strain1 Isolates Affect Growth and TNFα Induction in THP-1 Monocytes

Central Asian Strain 1 (CAS1) is the prevalent Mycobacterium tuberculosis genogroup in South Asia. CAS1 strains carry deletions in RD149 and RD152 regions. Significance of these deletions is as yet unknown. We compared CAS1 strains with RD149 and concurrent RD149-RD152 deletions with CAS1 strains without deletions and with the laboratory reference strain, M. tuberculosis H37Rv for growth and for induction of TNFα, IL6, CCL2 and IL10 in THP-1 cells. Growth of CAS1 strains with deletions was slower in broth (RD149; p = 0.024 and RD149-RD152; p = 0.025) than that of strains without deletions. CAS1 strains with RD149 deletion strains further showed reduced intracellular growth (p = 0.013) in THP-1 cells as compared with strains without deletions, and also as compared with H37Rv (p = 0.007) and with CAS1 RD149-RD152 deletion strains (p = 0.029). All CAS1 strains induced higher levels of TNFα and IL10 secretion in THP-1 cells than H37Rv. Additionally, CAS1 strains with RD149 deletions induced more TNFα secretion than those without deletions (p = 0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid growth and induced lower levels of TNFα and IL6 secretion in THP-1 cells than isolates from pulmonary sources. This data suggests that presence of RD149 reduces growth and increases the induction of TNFα in host cells by CAS1 strains. Differences observed for extrapulmonary strains may indicate an adaptation which increases potential for dissemination and tropism outside the lung. Overall, we hypothesise that RD149 deletions generate genetic diversity within strains and impact interactions of CAS1 strains with host cells with important clinical consequences

Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan

<p>Abstract</p> <p>Background</p> <p>The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of <it>Mycobacterium tuberculosis </it>strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of <it>M. tuberculosis </it>isolates from different regions of the country.</p> <p>Methods</p> <p><it>M. tuberculosis </it>strains isolated (2003–2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped.</p> <p>Results</p> <p>Of 926 <it>M. tuberculosis </it>strains studied, 721(78%) were grouped into 59 "shared types", while 205 (22%) were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters), 4% East African-Indian (EAI), 3% Beijing, 2% poorly defined TB strains (T), 2% Haarlem and LAM (0.2). Also TbD1 analysis (<it>M. tuberculosis </it>specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types.</p> <p>Prevalence of CAS1 clade was significantly higher in Punjab (P < 0.01, Pearsons Chi-square test) as compared with Sindh, North West Frontier Province and Balochistan provinces. Forty six percent of isolates were sensitive to five first line antibiotics tested, 45% were Rifampicin resistant, 50% isoniazid resistant. MDR was significantly associated with Beijing strains (P = 0.01, Pearsons Chi-square test) and EAI (P = 0.001, Pearsons Chi-square test), but not with CAS family.</p> <p>Conclusion</p> <p>Our results show variation of prevalent <it>M. tuberculosis </it>strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan.</p

Molecular epidemiology and deletion analysis of mycobacterium tuberculosis strains associated with pulmonary and extrapulmonary tuberculosis

Pakistan ranks 6th amongst the list of 22 high tuberculosis (TB) burden countries with an estimated incidence rate of 231 person and prevalence rate of 364 person per 100,000 population respectively (WHO, 2011). TB remains one of the leading causes of dealth due to infectious diseases in Pakistan and is responsible for 5% of the country\u27s total disease burden (WHO, 2011). Molecular epidemiological studies have shown a predominance of the Central Asian Strainl (CAS1) with Beijing strains constituting 3% of Mycobacterium tuberculosis isolates in Pakistan. M tuberculosis strains display high degree of genomic deletions called large sequence polymorphisms (LSPs) or region of differences (RDs) causing genetic variation. However, the role of LSPs in pathogenicity amongst Al tuberculosis strains is still not clear. The overall aims of this study were to explore the frequency of Region of Differences (RDs) genomic deletions amongst strain prevalent in Pakistan including CAS1, CAS subfamily and Beijing spoligotypes. The presence of commonly reported RD deletions, RD1, RD750, RD207, RD149, RD152, RD105, RD 150, RD142 and RD181 was investigated using a PCR based method. The biological relevance of RD149 and concurrent RD149-RD152 deletions on CAS1 strains was explored using growth in broth as well as growth and cytokine induction in THP-1 cell line model. RD genomic deletion analysis was performed on 235 M tuberculosis (185 pulmonary; 50 extrapulmonary) strains, including 171 CAS strains (133 CAS1 (ST26), 38 CAS subfamily), 8 Beijing isolates, 47 isolates belonging to other previously defined ( Other ) clusters, and 9 previously undefined Unique isolates. All the 133 CAST isolates studied had RD750 deletions. RDI49 and RD152 deletions were observed in 39.8% (n=53) and 21.8 % (n=29) respectively, whereas concurrent RD149-RD152 deletions were observed in 18.79% (n=25) of CAS 1 strains. All the 38 CAS subfamily isolates were characterized by a RD750 deletion; 21.05% (n=8) had RD149 deletions and 21.05% (n=8) had RD152 deletions. No deletions in RDI, RD207, RD105, RD150, RD142 and RD181 regions were observed in CAS1 and CAS subfamily isolates. All Beijing isolates (n=8) showed RD207, RD149, RD152, RD105 and RD150 deletions. RD142 and RD181 deletions however, were observed in 62.5% (n=5) and 87.5% (ip--7) of Beijing strains respectively. Of the Other cluster strains, 29.8% (n=14) had RD149 deletions, followed by 2.13% (n=1) with RD152 deletions. No deletions of studied RDs were observed in the Unique strains (n=9). CASI strains showed more frequent RD149 deletions as compared with the CAS subfamily (p=0.036) and more frequent RD152 deletions compared with Other cluster (p=0.0003). RD149 and RD152 deletions were more frequent in Beijing isolates compared with CAST strains (p\u3c0.001). Concurrent RD149 and RD152 deletions were more frequent in CAS] compared with Other clusters (p\u3c0.001) and in Beijing strains compared with CAST (p\u3c0.001). Given the fact that CAS1 is the prevalent spoligotype in this region, biological significance of RD149 and concurrent RD149 and RDI52 deletions in these strains was investigated. Growth of CAS1 strains with deletions was slower in broth (RD149, p=0.024 and RD149-RD152, p=0.025) than that of strains without deletions. CAST strains with RD149 deletions further showed reduced intracellular growth (1)=0.013) as compared with CAS 1 strains without deletions and also as compared to H37Rv (p=0.007) and CAS1 strains with concurrent RD149-RD152 deletion (1)=0.029). All CAS1 strains induced higher levels of TNFa and ILI 0 secretion in THP-1 cells than H37Rv. In addition, CAS I strains with RD149 deletions induced more TNFa secretion than CAS1 strains without deletions (p=0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid intracellular growth (p=0.015) and lower levels of TNFa (p=0.01), CCL2 (p=0.003) and IL6 (p=0.008) secretion as compared with strains from pulmonary sources. These data suggest that the presence of RD149 deletion in CAS1 strains is associated with reduced growth and increased TNFct induction in host cells possibly, reducing strain virulence. The differences observed for extrapulmonary strains may indicate an adaptation that increases their potential for dissemination and tropism outside the lung. Overall, RD149 deletions result in genetic diversity within strains and could have an important effect on interactions with host cells with important clinical consequences

Reduced TNF-alpha and IFN-gamma responses to Central Asian strain 1 and Beijing isolates of Mycobacterium tuberculosis in comparison with H37Rv strain

Pakistan ranks eighth in terms of tuberculosis burden worldwide, with an incidence of 181/100 000. The predominant genotypes of Mycobacterium tuberculosis are reported to be the Central Asian strain 1 (CAS1) and Beijing families. Mycobacterium tuberculosis down-regulates host pro-inflammatory cytokines, which are essential for protection against infection. There is currently little information regarding the interaction of the CAS1 genotype with host cells. We studied the growth rates of CAS1 and Beijing clinical isolates, and their ability to induce cytokines compared with the laboratory reference strain H37Rv. Host responses were studied using a THP-1 monocytic cell tine model and an ex vivo whole blood assay. Growth rates of CAS1 and Beijing isolates were significantly lower (P = 0.011) compared with H37Rv. All clinical isolates induced significantly tower levels of TNF-alpha secretion (P = 0.003) than H37Rv in THP-1 cells and in the whole blood assay of healthy donors (n = 8). They also induced tower IFN-gamma secretion in the whole blood assay (P \u3c 0.001). A positive correlation was observed between the growth indices (GI) of H37Rv, Beijing and CAS1 strains and the TNF-alpha responses they induced [Pearson\u27s correlation coefficient (R(2)): 0.936, 0.775 and 0.55, respectively], and also between GI and IFN-gamma production (R(2): 0.422, 0.946, 0.674). These findings suggest that reduced growth rate, together with down-modulation of pro-inflammatory cytokines, is a contributory mechanism for the predominance of the CAS genotype