Raman spectroscopy and machine learning-based optical probe for tuberculosis diagnosis via sputum.

Tuberculosis (TB) is a contagious disease that causes 1.5 million deaths per year globally. Early diagnosis of TB patients is critical to control its spread. However, standard TB diagnostic tests such as sputum culture take days to weeks to produce results. Here, we demonstrate a quick, portable, easy-to-use, and non-invasive optical sensor based on sputum samples for TB detection. The probe uses Raman spectroscopy to detect TB in a patient's sputum supernatant. We deploy a machine-learning algorithm, principal component analysis (PCA), on the acquired Raman data to enhance the detection sensitivity and specificity. On testing 112 potential TB patients, our results show that the developed probe's accuracy is 100% for true-positive and 93.4% for true-negative. Moreover, the probe correctly identifies patients on TB medication. We anticipate that our work will lead to a viable and rapid TB diagnostic platform.

Process evaluation of chest camps for increased tuberculosis case finding in Punjab, Pakistan.

BACKGROUND: To contribute to the World Health Organization's End TBStrategy, the active tuberculosis (TB) case-finding approach has been proven effective. METHODS: A total of 66 chest camps were organised for patients in 15 selected districts in Punjab, Pakistan, in 2017. A mixed-method process evaluation was conducted in four randomly selected districts to evaluate the use of chest camps for active TB case finding to reach the maximum number of people with TB and to assess the implementation outcomes, such as effectiveness, feasibility, fidelity, and costs. RESULTS: Results indicated that 1458 attendees visited 24 chest camps in four selected districts. Among attendees, 297 presumptive cases were found and smear-tested; and 34 of the smear-tested were diagnosed as smear-positive TB patients. The prevalence of smear-positive TB patients among the chest camp participants was found to be 2.3%. The findings from interviews showed that preparation of chest camp activities, especially the involvement of community leaders, was found to be effective in achieving the desired level of attendance. The respondents found attending the chest camps for TB symptoms feasible and acceptable. The chest camp costs approximately US$280, including the pre-camp mobilisation events, whereas the cost per TB-positive patient was found to be US$197.64. CONCLUSIONS: The higher number of attendees without TB symptoms, the low proportion of smear-negative case registrations; and relatively high unit cost (per patient detected) were the areas identified for further attention. The study supports the continuation of chest camp activity, with further attention required for quality and efficiency concerns.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

Fluoroquinolone resistance and mutational profile of gyrA in pulmonary MDR tuberculosis patients.

BACKGROUND: Fluoroquinolones (FQs) are potential drugs that inhibit DNA synthesis and are used in the treatment of multidrug-resistant tuberculosis (TB) and short-term anti-TB regimens. In recent years, a high proportion of FQ resistance has been observed in Mycobacterium tuberculosis isolates. The development of FQ resistance in multidrug-resistant TB negatively impacts patient treatment outcome and is a serious threat to control of TB. METHODS: The study included a total of 562 samples from patients with pulmonary TB that had been on anti-tuberculosis therapy. MTBDRsl assays were performed for the molecular detection of mutations. Sequence analysis was performed for the characterization and mutational profiling of FQ-resistant isolates. RESULTS: FQ resistance was observed in 104 samples (18.5%), most of which were previously treated and treatment failure cases. A total of 102 isolates had mutations in DNA gyrase subunit A (gyrA), while mutations in gyrB were observed in only two isolates. Mutational analysis revealed that the mutations mostly alter codons 94 (replacing aspartic acid with glycine, D94G) and 90 (replacing alanine with valine, A90V). In MDR and treatment failure cases, resistance to FQs was most commonly associated with the D94G mutation. In contract, a high proportion of A90V mutations were observed in isolates that were newly diagnosed. CONCLUSION: The findings suggest that genotypic assays for FQ resistance should be carried out at the time of initial diagnosis, before starting treatment, in order to rule out mutations that impact the potential use of FQs in treatment and to control drug resistance.

Seroprevalence of Crimean-Congo haemorrhagic fever among three selected risk human groups in disease-endemic region of Pakistan.

The occurrence of Crimean-Congo haemorrhagic fever (CCHF) in humans is linked with animals living in close vicinity, and information on the incidence of CCHF at the human-animal interface is scarce. Therefore, the current study was designed to identify the high-risk groups of individuals linked with animals in the Chakwal district of Pakistan having a history of CCHF cases in humans. In subject matter, coupled with risk factor analysis, we performed a sero-based CCHF surveillance in three selected risk groups of humans including abattoir workers (n = 137), milkmen (n = 169) and animal handlers (n = 147). Sera samples and questionnaire-based data were collected from each of the participants and screened for anti-CCHFV IgG antibodies using enzyme-linked immunosorbent assay. The highest seroprevalence was observed in animal handlers (n = 14, 9.52%, 95% CI: 4.68-13.99) followed by abattoir workers (n = 9, 6.57%, 95% CI: 2.42-10.72) and milkmen (n = 3, 1.78%, 95% CI: 0.24-4.24). The risk of seropositivity was significantly associated with humans linked with tick-infested animals (OR: 11.0, 95% CI: 1.5-83.0, p = .002), old age >40 years (OR: 6.6, 95% CI: 2.7-16.0, p < .0001), illiteracy (OR: 4.3, 95% CI: 1.5-13.0, p = .004) and humans without knowledge about CCHF (OR: 7.6, 95% CI: 1.8-33.0, p = .0009). The findings of the current study highlighted the seroprevalence of CCHF in high-risk groups of humans living in a disease-endemic area of Pakistan and highlight the need for well-integrated disease surveillance in the future to better comprehend disease control interventions.

Middle East Respiratory Syndrome Coronavirus Seropositivity in Camel Handlers and Their Families, Pakistan.

A high percentage of camel handlers in Saudi Arabia are seropositive for Middle East respiratory syndrome coronavirus. We found that 12/100 camel handlers and their family members in Pakistan, a country with extensive camel MERS-CoV infection, were seropositive, indicating that MERS-CoV infection of these populations extends beyond the Arabian Peninsula.

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type 1 (HIV-1) in selected districts of Punjab province, Pakistan.

Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are now emerging worldwide. Therefore, an understanding of the genetic characteristics of prevailing strains, particularly with regard to drug-resistance-associated substitutions, is essential for devising and implementing treatments and disease control interventions in endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers in selected districts of Punjab province, Pakistan. The samples were first screened using an HIV-1 Ag/Ab Combo test followed by amplification of the pol gene (1084 bp) from samples that were positive either for the antigen or for both the antigen and antibodies simultaneously. Screening revealed that a total of 45 samples were positive (34.62%; 95% CI: 26.99-43.13) for either antigen or both antigen and antibodies (n = 18, 40%; 95% CI: 27.02-54.55) or for antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A largest number of positive samples was from the district of Lahore (n = 19/43, 44.18%; 95% CI: 30.44-58.9) followed by Faisalabad (n= 12/36, 33.33%; 95% CI: 20.21-49.66), Gujranwala (n = 05/23, 21.7%; 95% CI: 9.66-41.9) and Sargodha (n = 09/28, 32.1%; 95% CI: 17.93-50.66). The probability of occurrence of HIV infection was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). Phylogenetic analysis based on the pol gene showed that the sequences from this study clustered into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), and subtypes A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for drug-resistance-associated mutations, except for an accessory mutation (M46K) in the protease (PR) region in one subject, we found a lack of drug-resistance-associated substitutions in the PR region. On the other hand, we found two subjects (2/18) carrying a resistance-associated mutation (V106I) conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Continuous disease surveillance coupled with evaluation of drug resistance at higher resolution should be done in future studies.

Crimean-Congo Haemorrhagic Fever: Case study analysis of a sporadic outbreak from Chakwal, Pakistan.

Crimean-Congo Haemorrhagic Fever (CCHF) is a deadly viral zoonotic disease, which is endemic in Pakistan. We report a case study analysis of three cases of CCHF which occurred in Chakwal, Pakistan in 2016. The disease was suspected in three patients exhibiting clinical symptoms suggestive of CCHF; two of the three patients died. The presence of CCHF was detected by seroconversion (IgG) from the surviving patient, while the antigen was detected in Hyalomma ticks originating from animals in the vicinity. This report indicates increase threat emergence of CCHF in Pakistan and highlights its zoonotic implications requiring immediate interventions under the "One-Health" concept.

Detection of rifampicin resistance of Mycobacterium tuberculosis using multiplex allele specific polymerase chain reaction (MAS-PCR) in Pakistan.

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.

Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates in Punjab, Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However, insufficient data are available on the genetic structure of M. tuberculosis strains circulating in this country. The objective of this study was to explore the genetic diversity of multidrug-resistant M. tuberculosis isolates from Punjab, Pakistan with a combination of spoligotyping and 24-loci MIRU-VNTR typing. Among a total of 127 MDR isolates studies, 53 spoligotypes were obtained, split into 14 clusters (n = 88, 69.3%, 2-29 isolates per cluster) and 39 (30.7%) unique patterns. At the phylogenetic level, the most prevalent sublineage was CAS1_DELHI (n = 53, 41.7%), mostly represented by ST 1942 (n = 29, 22.8%), followed by T1 (n = 14, 11%) and Beijing (n = 10, 7.8%). The remaining nine sublineages (CAS, MANU2, EAI5, T2, LAM10_CAM, H1, X1, H4 and CAS2) involved altogether 24 (18.9%) isolates. Twenty-six (20.5%) isolates could not be assigned to any specific clade. MIRU-VNTR typing identified 123 (96.8%), 97 (76.4%) and 65 (51.2%) unique types with a tolerance of 0, 1, and 2 locus differences between the patterns. Upon combined spoligotyping and MIRU-VNTR typing analysis, 123 (96.8%), 108 (85%), and 91 (71.7%) unique types were identified if a tolerance of 0, 1, and 2 locus differences in the MIRU-VNTR patterns was assumed, respectively. Based on the clustering results, the transmission rate for MDR-TB cases under the study was calculated at 3.2%, 15%, and 28.3%. Overall, three clades, namely CAS1_DELHI, T1, and Beijing accounted for the majority of MDR-TB cases in Pakistan. Up to a third of the cases were clustered upon combined spoligotyping and MIRU-VNTR typing, suggesting a moderate level of active transmission.

Virulent Epidemic Pneumonia in Sheep Caused by the Human Pathogen Acinetobacter baumannii.

The human pathogen Acinetobacter baumannii has emerged as a frequent cause of hospital-acquired infections, but infection of animals has rarely been observed. Here we analyzed an outbreak of epidemic pneumonia killing hundreds of sheep on a farm in Pakistan and identified A. baumannii as the infecting agent. A pure culture of strain AbPK1 isolated from lungs of sick animals was inoculated into healthy sheep, which subsequently developed similar disease symptoms. Bacteria re-isolated from the infected animals were shown to be identical to the inoculum, fulfilling Koch's postulates. Comparison of the AbPK1 genome against 2283 A. baumannii genomes from the NCBI database revealed that AbPK1 carries genes for unusual surface structures, including a unique composition of iron acquisition genes, genes for O-antigen synthesis and sialic acid-specific acetylases of cell-surface carbohydrates that could enable immune evasion. Several of these unusual and otherwise rarely present genes were also identified in genomes of phylogenetically unrelated A. baumannii isolates from combat-wounded US military from Afghanistan indicating a common gene pool in this geographical region. Based on core genome MLST this virulent isolate represents a newly emerging lineage of Global Clone 2, suggesting a human source for this disease outbreak. The observed epidemic, direct transmission from sheep to sheep, which is highly unusual for A. baumannii, has important consequences for human and animal health. First, direct animal-to-animal transmission facilitates fast spread of pathogen and disease in the flock. Second, it may establish a stable ecological niche and subsequent spread in a new host. And third, it constitutes a serious risk of transmission of this hyper-virulent clone from sheep back to humans, which may result in emergence of contagious disease amongst humans.

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

Detection of selected arboviral infections in patients with history of persistent fever in Pakistan.

Surveillance is a valuable tool for understanding prevailing and previously undiagnosed infections in a geographic area. We examined 480 archived serum samples from patients with history of persistent fever (>40°C, 60-72h) who were referred to hospitals in Rawalpindi/Islamabad, Lahore, and Faisalabad districts for dengue antibody detection in 2014-15. Each sample was processed for detection of antigens and seroconversion, using real-time polymerase chain reaction and enzyme linked immunosorbent assay, respectively, against dengue haemorrhagic fever (DHF) virus serotypes 1-4, West Nile virus fever (WNVF), Crimean-Congo haemorrhagic fever (CCHF), and Chikungunya virus (CGV). The presence of antigens and antibodies to at least one of the studied viral haemorrhagic fevers (VHFs) was detected in 465 (96.8%, 95% CI: 94.9-98.1) and 442 samples (92.1%, 95% CI: 89.3-94.2), respectively. No sera were found positive to CCHF. There was a significant association between gender and positivity to at least one of the VHFs (χ2=8.12, df=1, p<0.005). Except for DHF serotype 2 and 3 (ττ=0.41), Goodman and Kruskal's Tau statistic revealed no significant association for occurrence of different viruses within the studied population (ττ=0-0.06). Cosinor analysis confirmed significant seasonality, with a higher number of cases of persistent fever in August through November, peaking in October. The study suggests circulation of multiple arthropod-borne viral infections and, in addition to DHF, ascertain the needs for screening patients for CGV and WNVF too. It also demonstrates the necessity of well-integrated disease surveillance in several geographic regions and at-risk populations in Pakistan to develop appropriate disease and vector control strategies.

DRUG RESISTANCE PATTERN AND MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS STRAINS IN PUNJAB, PAKISTAN.

Tuberculosis (TB) is a cause of death from a single infectious agent Mycobacterium tuberculosis (MTB), leading to approximately 2.5 million deaths annually worldwide. Information regarding prevalence and pattern of drug resistance among TB patients in Pakistan remains inadequate due to the country's limited resources. This study compared conventional diagnostic techniques with a PCRbased assay targeting IS6110 sequence. In addition, MTB drug resistant profiles against four first-line drugs (ethambutol, isoniazid, rifampin, and streptomycin) from new and retreatment cases of TB. From 101 sputum samples microscopic examination of Ziehl-Neelsen-stained smears and culturing on Lowenstein Jensen medium resulted in 96% and 100% positives, compared to 98% by PCR. Prevalence of MDR-MTB was 41.5% and 58.5% among new (n = 51) and retreatment (n = 50) cases, but 10% of the former group were sensitive to all four first-line anti-TB drugs. Thus, MDR-MTB is highly prevalent among TB patients in Punjab Province, Pakistan (where the study was conducted) and, although PCR amplification of MTB-specific IS6110 sequence was rapid, it lacked the sensitivity of the culture assay.

Diabetes mellitus among tuberculosis patients: a cross sectional study from Pakistan.

BACKGROUND: The co-occurrence of diabetes mellitus (DM) and tuberculosis (TB) is largely associated with high frequency of morbidity. OBJECTIVE: To determine the prevalence of DM among TB patients and describe the socio-demographic and behavioral factors associated with TB-DM co-occurrence. METHODS: We enrolled 500 TB patients from September, 2014 to August 2015 at four major public sector hospitals of Lahore, Pakistan. A questionnaire was used to collect information regarding associated socio-demographic and behavioral factors of the patients. We monitored the fasting blood sugar of each patient by using a semi automated clinical chemistry analyzer followed by an HbA1c level check of all hyperglycemic patients. RESULTS: The prevalence of TB-DM co-occurrence was 14.8%. The prevalence of TB-DM was higher (62.2%) among males. The >57 year age group had the highest proportion of patients (35.1%), with co-existent TB-DM. Most were illiterate (73.0%) and unemployed (48%). Moreover, among the 74 patients positive for TB-DM had a history of smoking. Age and education level were significantly associated with DM-TB while gender, occupation and smoking were not associated. CONCLUSION: The study revealed a 14.8% prevalence of DM among TB patients. This was associated with several socio-demographic factors, including age, unemployment, literacy and polluted environment. Thus, poor and unhealthy lifestyles were the factors associated with DM among immunologically compromised individuals due to TB.

A cross-sectional study about knowledge and attitudes toward multidrug-resistant and extensively drug-resistant tuberculosis in a high-burden drug-resistant country.

OBJECTIVE/BACKGROUND: Tuberculosis (TB) is a leading cause of death worldwide, with new threats of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Pakistan is the fifth highest among high-burden TB countries and the fourth highest among high-burden drug-resistant-TB countries. Pakistan is the sixth most populous country in the world, and Pakistani youth is the highest population group in Pakistan and second in the world. This study was aimed at assessing the understanding, awareness, and mindset of university students toward TB, MDR TB, and XDR TB in Lahore. METHODS: A cross-sectional questionnaire-based study was performed on 1137 individuals from three major public-sector universities in Lahore, Pakistan. Information regarding their knowledge and attitude toward MDR and XDR TB was gathered using a structured questionnaire. Data collected was analyzed using SPSS version 20. RESULTS: Male (531) and female (606) students were asked about different aspects of MDR and XDR TB. Although 80.47% students had good knowledge about simple TB, a very small fraction had awareness and appropriate knowledge about MDR/XDR-TB. Considering TB as a stigma, only 9.3% students disclosed that they had household TB contact. Only 25% students knew about XDR TB. CONCLUSION: Our results indicated that a small fraction of people knew the exact definition and treatment duration of MDR TB and XDR TB in our society. There is a need to increase the awareness and knowledge status of university students about MDR and XDR TB.

Pattern of Drug Resistance and Risk Factors Associated with Development of Drug Resistant Mycobacterium tuberculosis in Pakistan.

BACKGROUND: Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan. OBJECTIVE: The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan. METHODOLOGY: Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18. RESULTS: Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10-25 years) and previously treated TB patients. CONCLUSION: DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.

Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan.

BACKGROUND: GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase ß subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP. MATERIAL/METHODS: The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2mg/L and 40mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions. RESULTS: In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T - in 10 strains, and T-8C - in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene were of four types: C1349T, A1304T, C1333G, and TC1324CA found in 63 (63%), 11 (11%), 8 (8%), and one (1%) strain, respectively. Of the 100 strains designated as MDR by the proportion method, GenoType MTBDRplus confirmed this phenotype in only 62 strains. The results of GenoType MTBDRplus and the conventional drug susceptibility method were consistent in 70% (70/100) for INH, and 77% (77/100) for RMP. CONCLUSIONS: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30%) strains resistant to INH and 23 (23/100; 23%) strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38%) strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan.

Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff's method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.