Mycobacterium tuberculosis Central Asian Strain (CAS) lineage strains in Pakistan reveal lower diversity of MIRU loci than other strains

Mycobacterium tuberculosis (MTB) Central Asian Strain (CAS) lineage strains are predominant in South Asia. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing is an effective way of determining genetic diversity of strains. A maximum of 24 loci-based MIRU-VNTR typing can be used, however, it is important to investigate the relevance of specific MIRU loci for regional strains for more cost-effective MIRU typing. MIRU-VNTR typing was performed on MTB strains from Pakistan. Strains were comprised of CAS (n=113) and non-CAS lineages (n=87) - both multi-drug resistant (MDR) and drug susceptible. Hunter Gaston Discriminatory Index (HGDI) for each MIRU loci was interpreted as poor, moderate or highly discriminatory. Results were analyzed using Bionumerics software and miru-vntrplus database link. Clustering analysis revealed 185 different MIRU types. Eight clusters of 2 strains each were present amongst MDR (3 clusters) and drug susceptible (5 clusters) isolates. MDR clusters had orphan and Haarlem strains, whereas drug susceptible strain clusters were comprised of CAS and Beijing lineage strains. The HGDI for 15 loci-based MIRU typing of all isolates was 0.620, whereas HGDI for CAS was lower than non-CAS lineage strains (p-value: 0.023). HGDI of 8 MIRU-VNTR loci (Qub 26b, 10, 26, 4156, Mtub 04, 16, 31 and ETR-A) were all highly discriminatory. The average HGDI based on these 8 loci was significantly lower for CAS than non-CAS strains (P value: 0.03). The lower discriminatory index for CAS using both 15 and 8 MIRU loci-based analysis suggests less genetic diversity in these isolates than in other lineages. The eight highly discriminatory MIRU loci for CAS may help in monitoring the transmission of MTB strains in regions with high CAS lineage prevalence

Presence of RD149 deletions in m. tuberculosis central Asian strain 1 isolates affect growth and TNFalpha 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

Genetic diversity of Plasmodium vivax clinical isolates from southern Pakistan using pvcsp and pvmsp1 genetic markers

Background: Plasmodium vivax is the prevalent malarial species accounting for 70% of malaria burden in Pakistan; however, there is no baseline data on the circulating genotypes. Studies have shown that polymorphic loci of gene encoding antigens pvcsp and pvmsp1 can be used reliably for conducting molecular epidemiological studies. Therefore, this study aimed to bridge the existing knowledge gap on population structure on P. vivax from Pakistan using these two polymorphic genes. Methods: During the period January 2008 to May 2009, a total of 250 blood samples were collected from patients tested slide positive for P. vivax, at the Aga Khan University Hospital, Karachi, or its collection units located in Baluchistan and Sindh Province. Nested PCR/RFLP was performed, using pvcsp and pvmsp1 markers to detect the extent of genetic diversity in clinical isolates of P. vivax from southern Pakistan. Results: A total of 227/250 (91%) isolates were included in the analysis while the remainder were excluded due to negative PCR outcome for P.vivax. Pvcsp analysis showed that both VK 210 (85.5%, 194/227) and VK 247 type (14.5%, 33/227) were found to be circulating in P. vivax isolates from southern Pakistan. A total of sixteen and eighty-seven genotypes of pvcsp and pvmsp-1 were detected respectively. Conculsion: This is the first report from southern Pakistan on characterization of P. vivax isolates confirming that extensively diverse pvcsp and pvmsp1 variants are present within this region. Results from this study provide valuable data on genetic diversity of P. vivax that will be helpful for further epidemiological studies

Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan

<p>Abstract</p> <p>Background</p> <p>Scarce data are available on <it>Plasmodium falciparum </it>anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of <it>P. falciparum </it>resistance associated polymorphisms in field isolates from southern Pakistan.</p> <p>Methods</p> <p>Blood samples from 244 patients with blood-slide confirmed <it>P. falciparum </it>mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the <it>P. falciparum </it>chloroquine resistance transporter (<it>pfcrt </it>K76T), multi drug resistance (<it>pfmdr1 </it>N86Y), dihydrofolate reductase (<it>pfdhfr </it>A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (<it>pfdhps </it>A436S, G437A and E540K) genes and <it>pfmdr1 </it>gene copy numbers were determined using PCR based methods.</p> <p>Results</p> <p>The prevalence of <it>pfcrt </it>76T and <it>pfmdr1 </it>86Y was 93% and 57%, respectively. The prevalence of <it>pfdhfr </it>double mutations 59R + 108N/51R + 108N was 92%. The <it>pfdhfr </it>triple mutation (51I, 59R, 108N) occurred in 3% of samples. The <it>pfdhfr </it>(51I, 59R, 108N) and <it>pfdhps </it>(437G, 540E) quintuple mutation was found in one isolate. <it>Pfdhps </it>437G was observed in 51% and 540E in 1% of the isolates. One isolate had two <it>pfmdr1 </it>copies and carried the <it>pfmdr1 </it>86Y and <it>pfcrt </it>76T alleles.</p> <p>Conclusions</p> <p>The results indicate high prevalence of <it>in vivo </it>resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among <it>P. falciparum </it>in southern Pakistan.</p

Genetic diversity among Plasmodium falciparum field isolates in Pakistan measured with PCR genotyping of the merozoite surface protein 1 and 2

Background:The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1) and 2 (msp-2).Methods:Between October 2005 and October 2007, 244 blood samples from Patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC.Results:A total of 238/244 (98%) Patients had a positive PCR outcome in at least one genetic marker, the remaining six were excluded from analysis. A majority of Patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI, 8 MAD20, 5 RO33) and 33 msp-2 (14 FC27, 19 3D7/IC).Conclusion:This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity

Age and site of Colonic Neoplastic Lesions: Implications of screening in South Asia.

Objective : To evaluate the Age of patients and the site of Colonic Neoplastic Lesions (CNL) and to determine the appropriate screening strategy for Colorectal Carcinoma (CRC) (sigmoidoscopy versus colonoscopy) in our population. Methods : This is a cross sectional study. Data of all patients more than 16 years of age who underwent full colonoscopic examination at the Aga Khan University hospital between January 2011 till December 2013 and were diagnosed to have CRC or advanced adenomas (defined as polyp more than 1 cm and/or having villous morphology on histology) was recorded. Lesions found distal to the splenic flexure were characterized as distal lesions and while lesions found between the splenic flexure and the cecum were characterized as proximal lesions. RESULTS: During the study period colonic neoplastic lesions were found in 217 patients; 186 (85.7%) patients had CRC and 31(14.3%) patients had advanced adenomatous polyps. Mean age was 55.8±14 years and amongst them 72 (33.2%) patients were less than 50 years of age while 145 (66.8%) were more than 50 years. In 144 (66.4%) patients lesions were located in the distal colon, 65 (30%) had lesions in the proximal colon while in 8 (3.7%) patients the neoplastic lesions were found both in the proximal and distal colon. The predominant symptoms were bleeding per rectum in 39.6% of patients followed by weight loss in 31.8% of patients. Only 3 patients had familial syndromes with multiple polyps. When patients younger than 50 years of age were compared with patients more than 50 years there was no statistically significant difference between the site of neoplastic lesion as well as the presenting symptoms. (p value 0.85). CONCLUSION: Colonic Neoplastic Lesions presented at younger age in our study population and one third of the lesions were found in the right sided colon. Hence screening for CNLs should be implied at an earlier age preferably with colonoscopy. More population based data is required to further validate our results

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