Developing the Reliability and Validity of an Urdu Version-Self-Efficacy Scale for Breast Cancer Patients in Pakistan.

BACKGROUND AND PURPOSE: No suitable scale has been identified in literature that comprehensively measure self-efficacy of Pakistani breast cancer patients. The study aimed to develop a self-efficacy scale in Urdu language and determine its dimensions. METHODS: The scale was developed with input from experts and literature. It was administered, in cross-sectional phase of two pilot studies, on breast cancer patients receiving chemotherapy. Post hoc internal consistency reliability was computed and principal component analysis (PCA) was performed. RESULTS: U-SES comprised 17 questions. PCA revealed a total of five factors explaining cumulative variance of 68.7%. These factors were self-confidence, faith, coping, optimism, and decision making. Post hoc internal consistency (Cronbach's alpha) value was high (∞ = 0.87). CONCLUSION: The self-efficacy scale has acceptable validity and reliability and has potential to obtain information related to self-efficacy of cancer patients receiving chemotherapy.

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 in vitro 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.

The effect of individualized patient education, along with emotional support, on the quality of life of breast cancer patients - A pilot study.

PURPOSE: This study aimed to determine the effect of individualized patient education along with emotional support on the quality of life (QoL) of breast cancer patients undergoing chemotherapy. It also aimed to determine the intervention's feasibility in the Pakistani context. METHODS: A quasi-experimental design, with pre- and post-test, in two groups, via time block, was used. The study was conducted at a public hospital in Karachi with a sample of 50 patients; 25 patients each in the intervention and control group. The intervention was delivered over a period of six weeks. It comprised verbal and written patient education, availability of a nurse during patients' chemotherapy administration and over the telephone, and a telephone follow-up of the patients by the nurse. patients' QoL was assessed at baseline and at the sixth week of receiving chemotherapy. RESULTS: Tests indicated a significant improvement in the overall QoL, breast cancer subscale scores, and the physical and emotional well-being of the intervention group, as compared to the control group. The intervention effect size was moderate (0.655) for the QoL. CONCLUSION: The intervention was found to be effective in improving patients' QoL. However, a larger study, in a multi-center setting, is recommended to ascertain the findings of this pilot study.

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.

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.

Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

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.

Experiences and perceptions of patients living with hepatitis C in Karachi, Pakistan.

PURPOSE: Hepatitis C (HCV) is a chronic disease that affects an individual's physical, financial, social, and emotional well-being throughout the illness. Although extensive literature is available on experiences of patients with hepatitis C in developed countries, very little exists in developing countries, particularly in Pakistan. This article describes experiences of patients living with HCV in Karachi, Pakistan. METHOD: Using descriptive exploratory DESIGN: The three categories-perceptions and misperceptions, challenges and sufferings, support and shortcomings-were subsumed under the theme "deadly, dangerous, and devastating." CONCLUSION AND IMPLICATIONS FOR PRACTICE: Patients with HCV experience profound challenges that are influenced by their level of education, financial capacity, cultural norms, family support, and the sensitivity of health care professionals. Several cultural practices existing in Pakistan identify the need for public awareness. The findings on health care professionals have implications for nursing and allied health continuing education.

Characterizing Mycobacterium tuberculosis isolates from Karachi, Pakistan: drug resistance and genotypes.

OBJECTIVES: To study the prevalence, risk factors, and genotypes of drug-resistant Mycobacterium tuberculosis in Karachi. METHODS: Pulmonary tuberculosis (TB) patients were recruited in a cross-sectional study (2006-2009). Drug susceptibility testing was performed for culture-positive cases (n=1004). Factors associated with drug resistance were evaluated using logistic regression analysis. Strains were typed using spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR). The associations of genotype and drug resistance were explored using the Chi-square test. RESULTS: Resistance rates - new and previously treated - were as follows: multidrug-resistant (MDR)-TB, 2.4% and 13.9%, respectively; rifampin (RIF) monoresistance, 0.1% and 0.6%, respectively; any isoniazid (INH) resistance, 8.9% and 28.5%, respectively; and INH monoresistance, 3.0% and 6.3%, respectively. Prior TB treatment was a risk factor for MDR-TB (adjusted odds ratio (AOR) 6.8, 95% confidence interval (CI) 3.5-13.1) and INH monoresistance (AOR 2.4, 95% CI 1.1-5.2). Additional risk factors included low socioeconomic status for INH monoresistance (AOR 3.3, 95% CI 1.7-6.5), and belonging to Balouchi (AOR 9.2, 95% CI 2.5-33.4), Sindhi (AOR 4.1, 95% CI 1.2-13.5), or Pakhtun (AOR 3.4, 95% CI 1.0-11.2) ethnicity for MDR-TB. Although Central Asian strain (55.6%) was the most prevalent genotype, MDR-TB was significantly associated with Haarlem (H) genogroup (crude OR 9.2, 95% CI 3.6-23.8). CONCLUSIONS: An MDR-TB rate of 2.4% is reported in new patients. Low RIF monoresistance supports the use of RIF as a marker for MDR-TB in this population. The need to strengthen TB care in the identified at-risk groups is emphasized. Based on INH resistance rates, a review of national treatment/prevention regimens relying on INH is suggested.

Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan.

OBJECTIVE: To assess the efficacy of a line-probe assay delta (LiPA) as rapid diagnostic test for early detection of drug-resistant tuberculosis compared to conventional susceptibility methods in Pakistan. METHODS: Resistance to rifampicin (RIF) and isoniazid (INH) in 108 smear-positive pulmonary tuberculosis samples was detected using a line-probe assay [GenoType MTBDRplus (Hain Lifescience, GmbH, Nehren, Germany)] at the clinical microbiology laboratory of Aga Khan University Hospital in May, 2009. Results were compared with susceptibilities performed while using agar proportion. RESULTS: In comparison to the agar proportion method, the detection rate and specificity of resistance using MTBDR plus was 92.5% and 98.2% for rifampicin, and 76.3% and 100% for isoniazid. Mutations in codons 531 and 533 of rpoB gene (62%S531L) were responsible for 67.9% of rifampicin resistance. S315T mutation of katG gene was detected in 55.9% and inhA promoter mutation at positions -15 (C15T) in 11.9% of isoniazid resistant isolates. Four phenotypically rifampicin-resistant and 14 isoniazid-resistant strains were not detected by MTBDRplus. Sequencing these strains revealed mutations in 4 strains; 2 in rpoB gene S531W, del518 and 2 in katG genesW300L, S315N. Hence, two phenotypic rifampicin-resistant and 13 phenotypic isoniazid-resistant strains were not detected by the commercial line probe assay. CONCLUSION: The study showed that MTBDRplus had a high detection rate for rifampicin resistance. However, additional probes need to be included in the assay to improve the detection of isoniazid-resistant mycobacterium tuberculosis strains in Pakistan.

Characterization of mutations conferring extensive drug resistance to Mycobacterium tuberculosis isolates in Pakistan.

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.

Extensively drug-resistant tuberculosis, Pakistan.

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis.

M. tuberculosis Central Asian Strain 1 MDR isolates have more mutations in rpoB and katG genes compared with other genotypes.

Pakistan ranks eighth globally among TB burden countries, with a MDR rate of 2-5%. The most prevalent MTB genotype is Central Asian Strain1 (CAS1) followed by the Beijing genogroup. We investigated common mutations in multidrug resistance encoding genes rpoB, katG and inhA of CAS1 and Beijing strains using DNA sequencing and fluorescent resonance energy transfer (FRET) probe based real-time PCR methods. 30 CAS1, 12 Beijing and 20 unclustered spoligotypes, and 10 susceptible MTB strains were tested. The most common mutations in the rpoB gene were at codons 531 (60%), 526 (23%) and 516 (5%). CAS1 strains had a higher frequency of mutations at codon 526 (p<0.001), with more concurrent mutations (p<0.05) compared with Beijing and orphan types. Mutations at codon 315 of the katG gene were higher in CAS1 than Beijing strains (p=0.052). Only 1/62 MDR strain, which belonged to CAS1, had a mutation in the inhA gene. Sensitivity and specificity of probe based assay was 93% and 100% for rpoB, and 95% and 100% for katG, respectively. The FRET probes method detected 84% and 60% of rpoB and katG mutations and can therefore be used as a rapid method of screening MTB strains including CAS1.

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

BACKGROUND: The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of Mycobacterium tuberculosis 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 M. tuberculosis isolates from different regions of the country. METHODS: M. tuberculosis strains isolated (2003-2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped. RESULTS: Of 926 M. tuberculosis 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 (M. tuberculosis specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types.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. CONCLUSION: Our results show variation of prevalent M. tuberculosis 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.

Characterization of Mycobacterium tuberculosis Central Asian Strain 1 using mycobacterial interspersed repetitive unit genotyping.

BACKGROUND: The Central Asian Strain 1 (CAS1) genogroup of Mycobacterium tuberculosis (MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates. METHODS: Twelve loci based MIRU- VNTR typing was used to type 178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS6110 restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns. RESULTS: A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: >or=0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3-0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21). CONCLUSION: We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory.