Akbar Kanji

Spoligotyping of Mycobacterium tuberculosis isolates from Pakistan reveals predominance of Central Asian Strain 1 and Beijing isolates.

ABSTRACT The estimated incidence of tuberculosis in Pakistan is 181 per 100,000; however, there is limited information on Mycobacterium tuberculosis genotypes circulating in the country. We studied 314 M. tuberculosis clinical isolates; of these, 197 (63%) isolates grouped into 22 different clusters, while 119 (37%) had unique spoligotypes. Eighty-nine percent of the isolates were pulmonary (Pul), and 11% were extrapulmonary (E-Pul). We identified Central Asian Strain (CAS), Beijing, T1, Latin American-Mediterranean, and East African-Indian genogroups. Beijing strains, reportedly the most prevalent spoligotype worldwide, constituted 6% of our strain population. The CAS1 strain comprised 121 (39%) of the study isolates. No difference was observed between clustered isolates from cases of Pul and E-Pul tuberculosis. However, E-Pul isolates included a greater number of unique spoligotypes than Pul isolates (P = 0.005). The overall percentage of drug resistance was 54%, and that of MDR strains was 40%. While CAS1 strains were not associated with drug resistance, the relative risk of MDR was significant in Beijing strains compared to the non-Beijing groups (95% confidence interval, 1.2 to 8.9). The fact that the predominant strain, CAS1, is not associated with drug resistance is encouraging and suggests that an effective tuberculosis control program should be able to limit the high incidence of disease in this region.

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

BackgroundTuberculosis 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.MethodsSerum 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.ResultsCirculating 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.ConclusionCCL2 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.

Reduced TNF-α and IFN-γ 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 181/100000. The predominant genotypes of Mycobacterium tuberculosis are reported to be the Central Asian strain 1 (CAS1) and Beijing families.Mycobacteriumtuberculosis 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 line 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 lower 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 lower IFN-gamma secretion in the whole blood assay (P<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 [Pearsons 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.

Flaviviruses as a Cause of Undifferentiated Fever in Sindh Province, Pakistan: A Preliminary Report

Arboviral diseases are expanding worldwide, yet global surveillance is often limited due to diplomatic and cultural barriers between nations. With human encroachment into new habitats, mosquito-borne viruses are also invading new areas. The actual prevalence of expanding arboviruses is unknown in Pakistan due to inappropriate diagnosis and poor testing for arboviral diseases. The primary objective of this study was to document evidence of flavivirus infections as the cause of undifferentiated fever in Pakistan. Through a cooperative effort between the USA and Pakistan, patient exposure to dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was examined in Sindh Province for the first time in decades. Initial results from the 2015 arbovirus season consisting of a cross-sectional study of 467 patients in 5 sites, DENV NS1 antigen was identified in 63 of the screened subjects, WNV IgM antibodies in 16 patients, and JEV IgM antibodies in 32 patients. In addition, a number of practical findings were made including (1) in silico optimization of RT-PCR primers for flavivirus strains circulating in the Middle East, (2) shipping and storage of RT-PCR master mix and other reagents at ambient temperature, (3) Smart phone applications for the collection of data in areas with limited infrastructure, and (4) fast and reliable shipping for transport of reagents and specimens to and from the Middle East. Furthermore, this work is producing a group of highly trained local scientists and medical professionals disseminating modern scientific methods and more accurate diagnostic procedures to the community.

Increased expression of efflux pump genes in extensively drug-resistant isolates of mycobacterium tuberculosis

Introduction: Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) strains that are multidrug resistant (MDR) and also resistant to a fluoroquinolone and to one injectable aminoglycoside or capreomycin. Whilst resistance in MTB has been associated with single nucleotide polymorphisms (SNPs), efflux pumps are thought to play a role in conferring resistance to MTB but little is known about them. Methods: We studied XDR MTB (n = 10) strains characterized by whole genome sequencing (WGS; http://www.ebi.ac.uk/ena/data/view/PRJEB7798). Phenotypic susceptibility testing was performed by the MGIT 960 (Becton, Dickinson and Co., NJ, USA) method. All XDR MTB strains were resistant to at least seven drugs whilst one XDR MTB strain, X54 was resistant to isoniazid, rifampicin, pyrazinamide, streptomycin, ethambutol, fluoroquinolones, capreomycin, kanamycin, amikacin, and ethionamide. The mRNA expression of efflux candidate genes Rv0194, Rv2688c, Rv1634, drrA, and drrB was determined in XDR MTB strains as compared with the ATCC reference strain, H37Rv, and drug-susceptible (DS) MTB (n = 9) strains using the relative quantification method normalized to 16S rRNA. Results: The mRNA expression levels of efflux genes Rv2688c (p = 0.0037), Rv1634 (p = 0.0042), drrA (p = 0.0078) and drrB (p = 0.0003) were upregulated in XDR-TB strains as compared with DS MTB strains. Conclusion: The differences between XDR-TB and drug-susceptible isolates suggest that the increased expression levels of MTB efflux pump genes may contribute to drug resistance in extensively drug-resistant tuberculosis. Future studies are needed to determine whether combining efflux pump inhibitors to antitubercular drugs would be effective to treat resistant tuberculosis.

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.

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.

Effective testing for pulmonary tuberculosis using Xpert MTB/RIF assay for stool specimens in immunocompetent Pakistani children

Objective/background: Childhood tuberculosis (TB) is largely a paucibacillary disease and difficult to diagnose. It is difficult to obtain a sputum or gastric aspirate (GA) sample, and patients are often undiagnosed and treated empirically. Stool is a noninvasive specimen not usually used for TB testing in Pakistan. We investigated the value of Xpert MTB/RIF to diagnose Mycobacterium tuberculosis (MTB) in children with pulmonary TB cases, by performing comparative testing of GA and stool samples. Method: We recruited 60 children aged 1–15 years, suspected of TB, from the Department of Pediatrics, Civil Hospital, Karachi, Pakistan and The Aga Khan University Hospital, Karachi, Pakistan. All were immunocompetent. Patients had a Kenneth Jones TB score of ≥5. Paired GA/sputum and stool samples were collected for testing. All GA samples were tested by Xpert MTB/RIF assay and MTB culture, while stool was tested by Xpert MTB/RIF. Results: The study participants included 27 males and 23 females with a mean age of 6 years and a mean TB (Kenneth Jones) score of 7. Stool was received in the laboratory within 1–2 days of the GA sample for all but one participant, who expired. The rates of MTB detection were as follows: 22% (11 cases) based on Xpert MTB testing of GA, 21% (10 cases) based on MTB culture of GA, and 21% (10 cases) based on Xpert MTB testing of stool. No rifampicin resistance was detected. Overall, there was concordance between testing of GA and stool. One case had GA with low positive Xpert and positive MTB culture, but negative stool Xpert result. In another case, there was low positive GA Xpert, positive GA MTB culture, and positive stool Xpert. A positive Xpert MTB stool test was associated with a higher TB score (>5) and a greater bacillary load. All 11 cases of TB diagnosed were put on antituberculous therapy and responded well to treatment. Conclusion: Use of Xpert MTB/RIF assay for stool-based diagnosis of pulmonary TB in immunocompetent children is useful in a resource poor setting. This is a valuable and noninvasive diagnostic alternative for the diagnosis of childhood TB and can be adapted by pediatric arms of national TB programs.

Efflux pump as alternate mechanism for drug resistance in Mycobacterium tuberculosis

Tuberculosis (TB) remains an important global public health issue with an approximate prevalence of 10 million people with TB worldwide in 2015. Since antibiotic treatment is one of the foremost tools for TB control, knowledge of Mycobacterium tuberculosis (MTB) drug resistance is an important component for disease control. Although gene mutations in specific loci of the MTB genomes are reported as the primary basis for drug resistance, additional mechanisms conferring resistance to MTB are thought to exist. Efflux is a ubiquitous mechanism responsible for innate and acquired drug resistance in prokaryotic and eukaryotic cells. MTB presents a large number of putative drug efflux pumps compared to its genome size. Bioinformatics-based evidence has shown an association between drug efflux and innate or acquired resistance in MTB. This review describes the recent understanding of drug efflux 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_PGRS genes 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.