New Delhi metallo-ß-lactamases among extensively drug-resistant clinical isolates from Lahore, Pakistan.

Aim: The study determines rates of carbapenem resistance (CR) and frequency of blaNDM in multidrug-resistance (MDR) or extensive drug resistance (XDR), and evaluates the potential of phenotypic tests for detecting NDM production. Materials & methods: Singleplex PCR was used to detect blaNDM. Phenotypic tests, including combination disc test (CDST) and modified Hodge test (MHT), were evaluated for NDM production. Results: Among 338 CR isolates, 47.63% were MDR, whereas 52.36% were XDR with 53.25% carrying blaNDM. MHT was found to be discriminative for detecting NDM production, whereas no significant association was observed for CDST. Conclusion: The high incidence of CR and MDR and XDR isolates possessing blaNDM presents an impending threat in therapeutics. Limitations of phenotypic tests suggest better testing, including molecular detection of the enzyme.

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Cervical cancer screening practices in HIV positive females - a missing link in health care delivery in Pakistan.

Objectives: To probe cervical cancer screening practices in local women positive for human immunodeficiency virus, and to determine the cervical cytological changes in them. METHODS: The serial cross-sectional study was conducted at the Jinnah Hospital and Services Hospital, Lahore, Pakistan, from April 2019 to October 2020, and comprised female patients aged 18-45 years who were positive for human immunodeficiency virus or acquired immunodeficiency syndrome and were registered with the relevant programme being run by the provincial government in Punjab. Blood samples of all the patients were collected for the determination of human immunodeficiency virus viral load and cluster of differentiation 4+ count. Cervical smears were taken for cytopathological analysis, while the swabs were analysed for culture sensitivity. The same individuals were subjected to the same testing one year later, and the status of the disease and clinical stability or disease progression was explored. Data was analysed using SPSS 25. RESULTS: There were 150 women with mean age 32.08±7.13 years (range: 21-45 years). Age at marriage/sexual activity was 17.33±4.73 years in 15(10%) subjects. Cytological examination showed atypical squamous cells of undetermined significance in 6(4%) of the cases whereas 3(2%) cases showed atypical squamous cells, which cannot rule out high grade squamous intraepithelial lesion on cytology, while the rest were classified as negative for intraepithelial lesion or malignancy. Cervical microbial changes revealed methicillin-resistant staphylococcus aureus infection in 9(6%) cases, extended-spectrum beta-lactamase in 15(10%) cases, whereas fungal infection and trichomonas vaginalis infection were found in 30(20%) smears. There was a significant association between cluster of differentiation 4+ cell count and stability of high-risk patients (p<0.001). After one year, 84(56%) patients remained clinically stable, while 51(34%) developed some chronic illness. There was a significant association between cluster of differentiation 4+ cell count <200/mm3 and the risk of developing a chronic illness (p<0.001). CONCLUSIONS: There was a dire need to educate healthcare workers to offer regular cervical screening to patients with high-risk sexually-transmitted infections to prevent them from the morbidity and mortality related to cervical cancer.

Prevalence of Human Papilloma virus in potentially malignant oral disorders is a risk factor for development of early dysplasia-A cytological investigation.

Objective: The proposed study was planned to screen Human Papilloma Virus (HPV) status in potentially malignant oral disorders (PMOD) and correlated HPV positivity with cytological changes in oral smears. Methods: This descriptive cross-sectional study was conducted at University of Health Sciences Lahore, Pakistan from April 2020 to April 2021. Oral smears from N=162 patients with PMODs were taken by the Cytobrush and Manual Liquid Based Cytology was performed followed by p16 antibody detection on immunohistochemistry and HPV-DNA detection by conventional polymerase chain reaction (PCR). The cytological changes were categorized according to the updated Bethesda Classification system 2014. SPSS was used to analyze data and p-Value of <0.05 was considered as statistically significant. Results: Out of total N = 162 patients, the most prevalent lesion [39% (n=63)] was lichen planus. Fifty six percent (n=90) of the patients were habitual chewers and 43% (n=70) were smokers. Pap staining of oral smears revealed atypical squamous cells of undetermined significance (ASCUS) in 45% (n=69) cases and in 2 % (n=4) of the samples diagnosis of atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H) was made. A total of 37% cases showed HPV positivity by polymerase chain reaction (PCR) while positive p16 expression was observed in 24% of the cases. ASC-H and ASCUS category showed significant association with HPV positivity (p=0.003). Conclusion: Early detection of PMODs by adopting minimally invasive cytological techniques and screening for HPV infection in local population is pivotal to reduce the morbidity and mortality associated with the advanced disease and carcinoma.

Geometry-Aware Merge Tree Comparisons for Time-Varying Data With Interleaving Distances.

Merge trees, a type of topological descriptors, serve to identify and summarize the topological characteristics associated with scalar fields. They have great potential for analyzing and visualizing time-varying data. First, they give compressed and topology-preserving representations of data instances. Second, their comparisons provide a basis for studying the relations among data instances, such as their distributions, clusters, outliers, and periodicities. A number of comparative measures have been developed for merge trees. However, these measures are often computationally expensive since they implicitly consider all possible correspondences between critical points of the merge trees. In this paper, we perform geometry-aware comparisons of merge trees using labeled interleaving distances. The main idea is to decouple the computation of a comparative measure into two steps: a labeling step that generates a correspondence between the critical points of two merge trees, and a comparison step that computes distances between a pair of labeled merge trees by encoding them as matrices. We show that our approach is general, computationally efficient, and practically useful. Our framework makes it possible to integrate geometric information of the data domain in the labeling process. At the same time, the framework reduces the computational complexity since not all possible correspondences have to be considered. We demonstrate via experiments that such geometry-aware merge tree comparisons help to detect transitions, clusters, and periodicities of time-varying datasets, as well as to diagnose and highlight the topological changes between adjacent data instances.

Acinetobacter baumannii Sequence Types Harboring Genes Encoding Aminoglycoside Modifying Enzymes and 16SrRNA Methylase; a Multicenter Study from Pakistan.

INTRODUCTION: The aminoglycosides are widely used for the therapeutic management of infections caused by gram-negative bacteria, including the Acinetobacter baumannii strains. However, the resistance to the members of the aminoglycoside family, such as amikacin, gentamicin, and tobramycin, is increasingly being common among the clinical isolates. PURPOSE: This study aimed to investigate the presence of 16SrRNA methylases and aminoglycoside modifying enzymes (AMEs) genes among aminoglycoside resistant A. baumannii isolates and to study the genetic diversity of the clinical population of A. baumannii in local hospitals. MATERIAL AND METHODS: The 143 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, genetic screening for enzymes conferring aminoglycosides resistance followed by the multilocus sequence typing. RESULTS: The 133/143 (93%) isolates were non-susceptible to at least one of the tested aminoglycosides, including amikacin, gentamicin, and tobramycin. The MIC distribution has shown that 87.486.7% strains were resistant to amikacin and gentamicin, respectively. The aphA6, aadB, aacC1, and aphA1 were found in 74.1%, 59.4%, 16.1%, and 11.2% isolates, respectively, whereas the armA was found in 28% of the strains having a higher MIC value (MIC; ≥256µg/mL). The MLST data have shown that the ST589 and ST2 were the most common STs and corresponded to 51 (35.7%) and 38 (26.6%) isolates, respectively, and few of the isolates corresponding to these STs were found to harbor the armA gene with a variable genotypic profile for AMEs. DISCUSSION: The study has reported the incidence of various enzymes conferring aminoglycoside resistance among the A. baumannii clones for the first time from Pakistan. The findings suggest the possibility of transmission of aminoglycoside resistance determinants through the lateral gene transfer as well as clonal dissemination.

Emergence of Resistance to Fluoroquinolones and Third-Generation Cephalosporins in Salmonella Typhi in Lahore, Pakistan.

Extensively drug-resistant (XDR) Salmonella Typhi has been reported in Sindh province of Pakistan since 2016. The potential for further spread is of serious concern as remaining treatment options are severely limited. We report the phenotypic and genotypic characterization of 27 XDR S. Typhi isolated from patients attending Jinnah Hospital, Lahore, Pakistan. Isolates were identified by biochemical profiling; antimicrobial susceptibility was determined by a modified Kirby-Bauer method. These findings were confirmed using Illumina whole genome nucleotide sequence data. All sequences were compared to the outbreak strain from Southern Pakistan and typed using the S. Typhi genotyping scheme. All isolates were confirmed by a sequence analysis to harbor an IncY plasmid and the CTX-M-15 ceftriaxone resistance determinant. All isolates were of the same genotypic background as the outbreak strain from Sindh province. We report the first emergence of XDR S. Typhi in Punjab province of Pakistan confirmed by whole genome sequencing.

Extended-spectrum beta-lactamases producing extensively drug-resistant Salmonella Typhi in Punjab, Pakistan.

INTRODUCTION: The multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates have been increasingly reported from the Asian and African countries. The emergence of isolates with decreased susceptibility to fluoroquinolones and cephalosporins has worsened the situation. Recently, an outbreak from Sindh, Pakistan was reported caused by extensively drug-resistant (XDR) S. Typhi strains. METHODOLOGY: In the present study, a total of 82 cases of typhoid have been investigated during 2018 from the febrile children referred to a tertiary care hospital in the population-wise largest province (Punjab) of Pakistan. S. Typhi was identified by standard microbiological techniques and isolates were characterized for antimicrobial resistance profiling and minimum inhibitory concentrations were determined. The presence of various ESBL genes in S. Typhi was confirmed by the PCR. RESULTS: Out of the 82 isolates tested, 35 (43%) were found to be XDR; resistant to the first-line drugs. The resistance to third-generation cephalosporins was mainly mediated by extended-spectrum beta-lactamases i.e. blaTEM and blaCTX-M genes. CONCLUSIONS: The higher prevalence of ESBL producing Salmonella typhi clinical strains raises the concern about transmission prevention and infection management in the community as well as clinical settings. Moreover, the study highlights the problem concerning the declining antibiotic arsenal for the therapeutic management of typhoid fever and the emergence and spread of XDR strains in Pakistan.

GeneXpert Technology for the diagnosis of HIV-associated tuberculosis: Is scale-up worth it?

Recent evaluations of the GeneXpert MTB/RIF assay for the simultaneous detection of Mycobacterium tuberculosis and drug resistance in less than 2 h have stimulated tremendous enthusiasm. This is the breakthrough that tuberculosis (TB) control has been waiting for. In this (retrospective review) case study, sputum samples from strongly suspected pulmonary tuberculosis patients were collected and assessed for the GeneXpert MTB/RIF assay for diagnosing TB and drug resistance in comparison with other tests, including Ziehl-Neelsen smear and Löwenstein-Jensen test. Of 3,784 cases, 5.7% (216/3,784) were human immunodeficiency virus (HIV)-positive and TB co-infected patients. In diagnosing HIV-positive and TB co-infected cases, the sensitivity and specificity of GeneXpert were 76.4% and 100%. While in HIV-negative and TB suspected cases, the sensitivity and specificity were 95.6% and 100%. This new test represents a major milestone for global TB diagnosis and care. It also represents new hope for the millions of people who are at the highest risk of TB and drug-resistant disease. GeneXpert is World Health Organization-endorsed technology representing the gold standard for TB testing despite attaining less sensitivity for HIV and TB co-infected patients as compared to HIV-negative patients.

Distribution of blaCTX - M , blaTEM , blaSHV and blaOXA genes in Extended-spectrum-ß-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan.

Background: Frequency of extended-spectrum-ß-lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-ß-lactamase-producing Clinical isolates in Pakistan. Methods: A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results: A total of 497/995 (50%) isolates including Escherichia coli 65% (n = 321), Klebsiella spp. 25% (n = 124) and Pseudomonas. 5% (n = 24), Enterobacter spp. 4% (n = 20) and Acinetobacter spp. 2% (n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% (n = 278) followed by pus 20% (n = 99) and wound swab 6% (n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% (n = 392). PCR revealed blaCTX - M (76%) gene followed by blaOXA (52%), blaTEM (28%) and blaSHV (21%) were most prevalent among ESBL-producers detected by CDST. blaCTX - M - 1(65%), blaOXA (78%) and blaTEM (57%) genes were carried on plasmids. Amplicon sequencing revealed blaCTX - M - 15 (75%), blaOXA - 1 (49%) and blaTEM - 1B (34%) and 21 (n = 28) isolates carried three genes in them. Conclusion: Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding blaCTXM - 15 gene and narrow spectrum blaOXA - 1 and blaTEM - 1B were also prevalent.

Acinetobacter Spp: Resistance and therapeutic decisions at the turn of the novel millennium.

This study was planned to evaluate sample wise isolation and antimicrobial resistant trends of Acinetobacter spp in different departments of a tertiary care hospital. This was a transversal descriptive study, carried out in the clinical microbiology laboratory of the Allama Iqbal Medical College/ Jinnah Hospital, Lahore, Pakistan, during the period of January 2015 to December 2016. Every clinical specimen was processed for bacterial culture and antimicrobial susceptibly testing. A total of 3590 (2015=1780, 2016=1810) clinical specimens were processed. Of the total, only 54.7% were gram-negative, among these Acinetobacter spp were isolated from 10.1% and 16.5% samples respectively in 2015-16 with an overall rate of 24.3%. The highest occurrence of Acinetobacter spp isolates was reported from Intensive care units (ICU) (54%) followed by surgical units (25%) and medical units (16%). It is noteworthy that ICU and internal medicine showed the highest resistance rates, whereas, lower resistance rate was observed for the outdoor patients (OPD). Although collistin showed 0% resistant while ceftriaxone, ciprofloxacin, gentamicin, and tigecycline showed 90%, 68%, 66%, 66% and 62% resistance against Acinetobacter spp. respectively. An alarming increase in the resistance rate of meropenem, cefoperazone/sulbactam, piperacillin/ tazobactam, ciprofloxacin, and imipenem was observed from the year 2015 to 2016. This startling resistance acquired by Acinetobacter spp. within a period of one year, represent very limited therapeutic options left for the infections caused by Acinetobacter spp. Unavailability of effective drugs and limited therapeutic options enforce the health care practitioners to prescribe expensive and broad range antibiotics, which may cause harm to the patient. Therefore, it is need of an hour to better understand the antimicrobial patterns and optimize antimicrobial prescription policies for the control of multidrug-resistant Acinetobacter spp.

High frequency and molecular epidemiology of metallo-ß-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan.

Background: Metallo-ß-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the ß-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem). Methods: A total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of ß-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection. Results: The rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of bla TEM, bla SHV, bla OXA, bla IMP-1, and bla VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla MBL (bla IMP and bla VIM) and bla ESBL (bla TEM, bla SHV, bla OXA) was also detected through multiplex and singleplex PCR. bla OXA, bla TEM and bla SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion: To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla OXA, bla TEM and bla SHV with bla IMP-1 and bla VIM in MBL-producing gram-negative bacilli.

Pseudomonas aeruginosa: Evaluation of Pathogen Burden and Drug-Resistance Trends in a Tertiary Care Hospital.

OBJECTIVE: To evaluate the pathogen burden and antibiotic-resistance trends of Pseudomonas aeruginosa among hospitalised patients at a tertiary care hospital. STUDY DESIGN: Retrospective, hospital record-based, cross-sectional study. PLACE AND DURATION OF STUDY: Microbiology Laboratory, Allama Iqbal Medical College/Jinnah Hospital, Lahore, from January 2014 to December 2016. METHODOLOGY: A total of 5,960 samples were collected from clinically suspected cases of bacterial infections, admitted to the hospital. Microbial identification and antibiotic susceptibility pattern were carried out and analysed. RESULTS: Out of a total of 5,960 samples, Pseudomonas aeruginosa was isolated from 1,268 (21.2%) specimens. Department-wise isolation rate was n=600 (42.9%), n=268 (15.4%), n=201 (12.6%), and n=199 (16.0%) from intensive care unit (ICU), surgical units, medical units, and Gynae wards, respectively (p<0.0001). Sample-wise isolation rate was, wound swabs n=448 (35%), urine n=356 (28%), sputum n=187 (14 %), tracheal aspirate n=127 (10%), blood n=99 (7%), and broncho-alveolar lavage n=51 (4%) (p<0.0001). Drug-resistance pattern showed low rates for carbapenems(meropenem n=440 (35%), Imipenem n=436 (34%) and beta-lactam + beta-lactamase inhibitor combination (piperacillin+tazobactam n=437 (34%) while alarming rates were observed for cephalosporins (ceftazidime n=716 (56%), fluoroquinolones (ciprofloxacin n=690 (54%), cefoperazone+sulbactam n=685 (54%), aminoglycosides (gentamicin, n=669 (53%), amikacin n=608 (48%), and monobactams (aztreonam n=666 (52%). Decreasing trend was observed only for amikacin 63% to 37%, aztreonam showed similar pattern throughout, while there was an increasing trend of drug resistance in all groups of antibiotics. CONCLUSION: Emerging drug-resistant strains of Pseudomonas aeruginosa are probably linked to the injudicious use of antibiotics, leading to ineffective empirical therapy. Therefore, we suggest that culture and antimicrobial susceptibility testing should be done for targeted antimicrobial therapy against Pseudomonas aeruginosa.

False Negativity of Ziehl-Neelsen Smear Microscopy: Is the Scale-up the Worth It in Developing Countries?

OBJECTIVE: To evaluate the false negative results of Ziehl-Neelsen (ZN) smear microscopy. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Mycobacteriology Laboratory, Allama Iqbal Medical College (AIMC) and Jinnah Hospital, Lahore (JHL), Pakistan, from February 2014 to August 2016. METHODOLOGY: A total of 3,951 (pulmonary 2,773 and extra-pulmonary 1,178) samples were collected from strong TB suspected patients attending JHL Lahore. Follow-up cases were excluded. Every specimen was processed for ZN smear microscopy, Lowenstein Jensen (LJ) culture. SPSS 21.0 was used; false negative and positive results of ZN smear were calculated keeping LJ culture as gold standard. RESULTS: Out of total 3,951 samples, sputum was most frequently found pulmonary sample 48.4% (n=1915), extra- pulmonary samples, pleural fluid and pus samples were most commonly observed samples 12.0% (n=476) and 8.3% (n=329), respectively. Overall false negativity was 23.1% (pulmonary=19.6%, extra-pulmonary=29.2%) (p<0.001), Maximum false negative results were observed in pericardial, synovial, pleural fluids, and pus samples as 40.0%, 38.0%, 33.0% and 32.0%, respectively. CONCLUSION: ZN smear microscopy is not a very efficient tool in case of patients with the low mycobacterial load. Therefore, National TB Control programs should consider extending their diagnostic approaches from ZN microscopy to more advanced techniques.

Silent killers: Transfusion Transmissible Infections-TTI, among asymptomatic population of Pakistan.

OBJECTIVE: To analyse transfusion transmissible infections in asymptomatic population. METHODS: This study was conducted at the Allama Iqbal Medical College and Jinnah Hospital, Lahore, Pakistan, from December 2014 to November 2015, and comprised healthy asymptomatic blood donors.Every sample was screened for the presence of antibodies/antigens of hepatitis C virus, human immunodeficiency virus, treponemapallidum, hepatitis B virus and malaria parasite through rapid immunochromatographic technique. RESULTS: Of the 18,274 blood donors, 17,276(94.53%) were found healthy and 998(5.46%) were infected. Besides, 71(0.38%) had multiple infections. The overall frequency of anti-hepatitis C virus, treponemapallidum (syphilis), hepatitis B surface antigen, malaria parasite and anti-human immunodeficiency virus was 480(2.62%), 284(1.55%), 210(1.10%), 20(0.10%) and 4(0.02%), respectively. CONCLUSIONS: Blood transfusion was found to be a significant but preventable mode of spread of transfusion transmissible infections.