Concordance and discordance of GeneXpert MTB/RIF and conventional culture method for diagnosis of Extra-Pulmonary Tuberculosis at a tertiary care hospital in Pakistan.

Objective: To identify concordance and discordance between GeneXpert MTB/RIF assay and gold standard bacteriologic culture for the diagnosis of Mycobacterium tuberculosis (MTB) in Extra-Pulmonary tuberculosis (EPTB) specimens in our region. Methods: This is a retrospective cross-sectional study conducted at the Indus Hospital and Health Network. Data from 1st January, 2020 to 31st December, 2021 was analyzed. A total of 1499 EPTB specimens were included for which GeneXpert was requested along with acid-fast bacteria (AFB) culture from the same specimen. Specimens were processed according to specimen type following standard operating procedures of the laboratory. Fluorescent staining was performed on all specimens along with bacteriologic culture. The GeneXpert MTB/RIF assay was carried out in exact accordance with the manufacturer's instructions. Results: Out of 1499 EPTB specimens, 1370 (91.39%) specimens exhibited concordance between GeneXpert and conventional culture method, while 129 (8.60%) specimens showed discordance. GeneXpert exhibited sensitivity and specificity of 69.4% and 94.3% respectively in comparison to culture. Conclusion: GeneXpert sensitivity for the diagnosis of EPTB varied with the site involved. Lower sensitivity was observed in ascitic and pleural fluids as compared to higher sensitivity observed among urine samples and pus aspirates. However, given the quick turnaround time and ease of use, it is a helpful tool in the diagnosis of EPTB when utilized in the appropriate clinical context. Caution is advised while interpreting negative GeneXpert results in endemic settings and should be interpreted along with other supporting clinical and diagnostic features.

Determination of Inducible Clindamycin Resistance and Correlation with Vitek2 Inducible Clindamycin Resistance Test in Staphylococcus aureus Isolated from Clinical Samples.

OBJECTIVE: To compare the performance of Vitek2 with the gold standard D test in terms of inducible clindamycin resistance (ICR) detection. STUDY DESIGN: Descriptive study. Place and Duration of the Study: Indus Hospital and Health Network Karachi, Pakistan, from November 2021 to April 2022. METHODOLOGY: Standard operating procedures of the laboratory were followed for processing of clinical samples. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were included. The isolates from the same patient within two-week time period were excluded. Clinical laboratory standards institute guidelines were followed for performing and interpreting D test. The results of the D test were compared with Vitek2 results for ICR. RESULTS: A total of 313 isolates were MRSA, of which 93 isolates tested positive for ICR on both the D test and Vitek2. Nine isolates were positive for ICR on Vitek2 and negative on the Kirby-Bauer disk diffusion method. One isolate tested positive on the disk method and negative on Vitek2. CONCLUSION: Vitek 2 appeared to give false positive results. Reporting false susceptibility of clindamycin can cause therapeutic failure which can markedly affect the patient's outcome. This discordance needs to be investigated further with a large sample size and stringent observation of D-test results to pick laboratory error. KEY WORDS: Methicillin-resistant Staphylococcus aureus, Inducible clindamycin resistance, D zone, Vitek2.

From mosquitoes to menace: Unveiling the dangers of West Nile virus in Pakistan.

Pakistan, a subtropical nation, has seen a rapid rise in Arboviral transmission in the past decade. Environmental hazards such as intense monsoon rains and yearly floods in addition to unsanitary living conditions pose an increased risk of arboviral infections. In recent years, the emergence and spread of West Nile virus (WNV), which can lead to a life-threatening meningoencephalitis, in Pakistan has alarmed the health care authorities of an impending outbreak. Health professionals and policymakers should give paramount importance to prevent its transmission across Pakistan as another arboviral outbreak would wreak havoc on the already fragile health infrastructure of the country. Proactive surveillance and prompt reporting are crucial for mitigating this threat as there are no vaccines available to prevent WNV infection.

Point prevalence probing of antimicrobial prescription patterns from a developing country.

BACKGROUND: Irrational use of antibiotics intensifies resistance and jeopardizes advances made in modern medicine. We aimed to conduct a baseline gap analysis survey on antibiotic prescription practices across Pakistan. RESEARCH DESIGN AND METHODS: This multi-centered cross-sectional survey was conducted at six public sector tertiary care hospitals from February 2021 to March 2021. Data related to various variables including hospital infrastructure, policies and practices, monitoring and feedback, and epidemiological, clinical, and antibiotic prescription for surveyed patients was collected using World Health Organization (WHO) Point Prevalence Survey (PPS) methodology. RESULTS: In a survey of 837 inpatients, 78.5% were prescribed antibiotics. Most commonly prescribed antimicrobial was ceftriaxone (21.7%), followed by metronidazole (17.3%), cefoperazone-sulbactam (8.4%), amoxicillin-clavulanate (6.3%), and piperacillin/tazobactam (5.9%). Surgical prophylaxis (36.7%) and community-acquired infections (24.7%) were the main reasons for antibiotic prescriptions. Single antibiotics were given to 46.7% of patients, 39.9% received a combination of two antibiotics, and 12.5% were prescribed three or more antibiotics. Among six hospitals surveyed, two had drug and therapeutic committees, three had infection prevention and control committees, and one had an antibiotic formulary. CONCLUSION: Findings demonstrate high consumption of broad-spectrum antimicrobials and emphasize the importance of expanding antimicrobial stewardship programs among hospitals. Mentoring clinical teams could help rationalize antimicrobial use.

Cholera outbreak in 2022 among children in Karachi: Study of cases attending to a Tertiary Care Hospital.

Objective & Background: Repeated outbreaks of cholera have occurred in Karachi. Changing patterns in seasonality, serotypes and antibiotic resistance have been observed in these outbreaks. Recently, in the year 2022, a surge of cholera cases has been reported from Karachi during the months of April-June. This study aimed to identify clinical features, antibiotic susceptibility, complications, and response to treatment of V. cholerae infection among children attending Indus hospital, Karachi. Methods: A retrospective chart review of pediatric patients was conducted for children aged 0-16 years. All children treated for culture-proven cholera infection at Indus Hospital from March to June 2022 were included. Details of clinical features, complications, antibiotic susceptibility, and response to treatment were retrieved from the health management information system (HMIS) of the hospital. Results: Twenty children were included. The median age was 01 (0.50-3.75) years. There were 9 (45%) males and 11 (55%) females. All the culture isolates belonged to serogroup O1 Ogawa of the Vibrio cholerae. Vomiting and diarrhea were the most common symptoms. Dehydration, acute kidney injury, and shock were seen in 19 (95%), 6 (30%), and 2 (10%) children respectively. Eleven children were admitted with an average hospital stay of 5 (Median-IQR 3-6) days. The isolates were completely susceptible to tetracycline, ciprofloxacin, and azithromycin. Different antibiotics were given which included cefotaxime, ceftriaxone, doxycycline, and ciprofloxacin. All children responded completely to the antibiotics. Conclusion: In present study all V. cholerae isolates belonged to the O1 Ogawa serotype that showed complete susceptibility to tetracycline, ciprofloxacin, and azithromycin. Dehydration, electrolyte imbalance, and renal impairment were the most common complications observed. Drinking unboiled water was identified as a potential source of cholera in most children. Therefore, advocacy of hygienic practices and disinfection of water supplies is recommended to prevent future cholera outbreaks.

Cholera Outbreak 2022 in Karachi: A Report on Serotype and Antibiotic Susceptibility Pattern.

OBJECTIVE: To identify the bacterial agent responsible for the surge of cases of acute diarrhoeal disease in Karachi, Pakistan, and assess the antimicrobial susceptibility pattern of the isolates. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Department of Microbiology, Indus Hospital & Health Network, Karachi, Pakistan, from 1st March to 31st May 2022. METHODOLOGY: Vibrio cholerae (V. Cholerae) isolates from stool samples received in the laboratory in the months of March, April, and May were identified and antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion method. Serology was performed to identify the serotype causing a surge in cases of acute watery diarrhoea. RESULTS: A total of three-hundred and seventy-eight stool samples were received during the study period, out of which seventy-eight were positive for V. cholerae serogroup O1 serotype Ogawa. The isolate tested 100% sensitive for Ciprofloxacin, Tetracycline, Doxycycline, and Azithromycin, while 74% resistance was observed in Trimethoprim/Sulfamethoxazole and 25% resistance in Ampicillin. CONCLUSION: V. cholerae serogroup O1 serotype Ogawa was the causative agent responsible for cholera outbreak in Karachi during the early summer of 2022. Ciprofloxacin, Tetracycline, Doxycycline, and Azithromycin are appropriate antimicrobials for treatment; whereas resistance to Trimethoprim/Sulfamethoxazole, and Ampicillin was higher. KEY WORDS: Vibrio cholerae, Outbreak, Acute diarrhoeal disease.

Changing Trends Of Multidrug Resistant Bacteria: A Six Year Experience From A Tertiary Care Hospital.

BACKGROUND: There is a continuous increase in the number of bacteria showing resistance to various antibiotics, limiting the treatment options for infections. The objective of this study was to assess the trend in resistance pattern of multi drug resistant organisms over a period of 6 years. METHODS: A retrospective study was conducted in Indus Hospital and Health Network, Karachi, Pakistan from January 2014 to December 2019. Multidrug resistant organisms were isolated from various samples and the data of corresponding patients were extracted from electronic medical record. The patients of all age groups and either gender was included. Specimens were inoculated on Sheep Blood Agar, chocolate agar and MacConkey agar. Organisms were identified and antibiotic susceptibility testing was performed according to Clinical Laboratory Standard Institute guidelines. RESULTS: In 34628 cases, 5159 (14.8%) were isolated as MDR organisms. Out of these 44.2% were Gram negative, while 55.7% were Gram-positive bacteria. The highest MDR trend was observed for A. baumannii (0-70%) followed by MRSA (0-64%) P. aeruginosa (0-16%) Enterococcus (0-10%) CRE (2.8-5.8%). CONCLUSIONS: The continuous rising trend of multidrug resistant organisms has been observed during the period of our study. Therefore, there is an imperative need of constant monitoring and firm adherences to infection control strategies to avoid spread of MDR organisms.

Disseminated cryptococcal infection in a lymphoma suspected patient.

Cryptococcus neoformans is an opportunistic pathogen, mainly responsible for meningitis in immunodeficient individuals. We report a rare case of dessiminated cryptococcosis in a six years old boy, patient was being evaluated for lymphoma. In the present case the causative agent was Cryptococcus neoformans. It was diagnosed through Bactec, aerobic blood culture bottle. The cause of hospitalization of the patient was fever with abdominal pain. Blood and CSF culture revealed the presence of Cryptococcus neoformans which was further confirmed by urease test and corn meal tween agar (CMT). In the present case fungus was unusually isolated earlier from blood culture rather than cerebrospinal fluid.

Susceptibility pattern of Mycobacterium tuberculosis over a period of five years at Indus Hospital and Health Network, Karachi, Pakistan.

Objective: To determine the susceptibility pattern and frequency of isolation of multidrug, pre-extensively drug and extensively drug resistant TB in a tertiary care hospital in Karachi, Pakistan. Method: A cross-sectional study was designed. Samples received in the lab were processed for growth and sensitivity testing of Mycobacterium tuberculosis. Isolation of MTB was done on Mycobacteria growth indicator tube (MGIT) followed by identification using MPT64. Samples were than evaluated for drug sensitivity against first and second-line antimycobacterial drugs. Statistical analysis was performed using SPSS version 24.0. Results: Of the 20014 samples received, 23.1% were identified as Mycobacterium tuberculosis. Drug sensitivity testing was performed on 95.9% isolates. Fifty-two percent samples were from males and 48% female patients. The study found statistically non-significant relationship between gender and likelihood of disease with drug-resistant (DR)-MTB organisms. The rate of isolation of MDR-TB was highest (43%) among ages 25-55 years and previously treated patients compared to newly diagnosed patients (62% vs 36%). Among MTB positive samples, 91.5% were pulmonary while 8.5% were extrapulmonary samples. Extrapulmonary samples were more likely to be sensitive to antimycobacterial drugs. The highest resistance was observed against Isoniazid (pulmonary=58%; extrapulmonary=12.7%), Rifampicin (pulmonary=58.7%; extrapulmonary=8.2%), and Levofloxacin (pulmonary=29.2%; extrapulmonary=20%). Conclusion: A considerable number of drug resistant tuberculosis cases were identified in the present study. It is essential to develop further strategies to reduce the spread of this disease.

Sensitivity and Specificity of Anti-SARS-CoV-2 Detection Kits - Comparison and Agreement between Fifteen Different Assays.

Accurate and rapid diagnosis of coronavirus disease 2019 (COVID-19) is critical for proper care and identification of affected individuals. This led to early availability of many serological assays in the market, but with limited validation. In this study, we aimed to validate the serological assays based on different techniques. We evaluated 15 different assays based on four immunoassay techniques in 235 patients. The most sensitive kits employed were as follows: immunochromatography (Zybio severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] IgM/IgG Antibody Assay Kit - 83%), ELISA (Aeskulisa SARS-CoV-2 NP IgG -88.1%), chemiluminescence (Alinity SARS-CoV-2 IgG - 82.2%), and immunofluorescence (Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit [IgG]) - 88.9%). The kits by Uniper (Singuway Biotec COVID-19 IgM/IgG Presumptive Kit), Genrui 2019-nCoV IgM/IgG Test Kit, Wondfu SARS-CoV-2 Antibody Test, and Aeskulisa SARS-CoV-2 NP IgG exhibited 100% specificity, whereas IgG assay using Lifotronic FA160 (Shenzhen SARS-CoV-2 Assay Kit) exhibited the lowest specificity at 58%. Maximum agreement was observed between Aeskulisa SARS-CoV-2 NP IgG and Alinity SARS-CoV-2 IgG at 94%. Serological tests are practical alternatives, but their reliability depends on critical validation. The COVID-19 pandemic warranted investment in healthcare research at both the national and international levels.

Disc Diffusion Testing of Azithromycin Against Clinical Isolates of Typhoidal Salmonellae: A Diagnostic Conundrum.

INTRODUCTION:  Typhoid remains a major healthcare problem in low and middle-income countries. The emergence of extremely drug-resistant (XDR) typhoid strains from the Indian subcontinent has led to very limited therapeutic options. Azithromycin being the only oral option for XDR typhoid faces a threat of rapid resistance due to its overuse after the COVID-19 pandemic. OBJECTIVE:  To evaluate the reliability of azithromycin disc diffusion testing against clinical isolates of typhoidal salmonellae in comparison with E-test minimum inhibitory concentrations (MICs). STUDY DESIGN:  This is a cross-sectional validation study. Place and duration of the study: The Department of Microbiology, Pakistan Navy Ship Shifa hospital, Karachi from June 1 to December 31, 2020. METHODOLOGY:  Antimicrobial susceptibility was performed by Kirby Bauer disc diffusion method for 60 isolates including Salmonella enterica ser. Typhi and Paratyphi A using Clinical Laboratory Standard Institute (CLSI) guidelines. MICs by the E-test method were determined for Azithromycin only. RESULTS:  A significant proportion of the isolates (55%) had high azithromycin MIC in the wild-type distribution range (8-16 µg/ml). Ten (16.6%) isolates showed false resistance, i.e., zone diameter <13 mm by disc diffusion method when compared to E-test MIC results. Isolates with MICs close to breakpoint, i.e., 16 µg/ml were more likely to show discordant results. The sensitivity, specificity, negative predictive value, positive predictive value, and diagnostic accuracy of the disc diffusion method versus E-test were 100%, 83%, 100%, 9%, and 83%, respectively. CONCLUSIONS:  Disc diffusion method as recommended by CLSI is not reliable for azithromycin susceptibility testing particularly for isolates with high MICs in the susceptible range. The E-test method may be a better alternative to disc diffusion provided appropriate training is done prior to its application.

Frequency of isolation and susceptibility pattern of E. coli from in and outpatients over a period of five years at The Indus Hospital Network, Karachi Pakistan.

OBJECTIVE: To determine the frequency of isolation and susceptibility pattern of Escherichia coli at a urban health centre. METHODS: The retrospective study was conducted at the Indus Hospital Network, Karachi, and comprised Enterobacteriaceae isolates obtained from urine, blood and routine culture from patients presenting between 2013 and 2017. The samples were evaluated for resistance patterns against a range of antibiotics and frequency of isolation was determined. Data was analysed using SPSS version 21.0. RESULTS: Of the 10,667 isolates analysed, 6380(60%) were Escherichia coli. Outpatient and inpatient isolates constituted 4184(65.6%) and 2196(34.4%) of the total E. coli isolates respectively. Of the 1446(22.66%) isolates obtained from urine, 1007(59.64%) had E. coli which was the highest isolation rate among all types of samples. There was a significant difference in resistance rates between inpatient and outpatient isolates for first-line and second-line injectable and oral antibiotics (p<0.05). CONCLUSIONS: Escherichia coli isolates showed high resistance towards co-amoxiclav, ampicillin, ceftriaxone and ciprofloxacin, leaving little empirical options for treating outpatients.

Concurrent adult pulmonary tuberculosis prevalence survey using digital radiography and Xpert MTB/RIF Ultra and child interferon-gamma release assay Mycobacterium tuberculosis infection survey in Karachi, Pakistan: a study protocol.

Background: Assessment of the effectiveness of tuberculosis control strategies requires the periodic measurement of M. tuberculosis transmission in populations, which is notoriously difficult. One well-established method is to measure the prevalence of infectious pulmonary tuberculosis in the population which is then repeated at a second time point after a period of 'intervention', such as scale up of the Search-Treat-Prevent strategy of the Zero TB Cities initiative, allowing for a 'before and after' comparison.  Protocol: The concurrent adult pulmonary tuberculosis prevalence survey (using digital radiography and Xpert MTB/RIF Ultra) and child M. tuberculosis infection survey (using QuantiFERON-TB® Gold Plus) will primarily provide a baseline measure of the burden of adult infectious tuberculosis in Karachi and assess whether a large-scale interferon gamma release assay survey in children aged 2 to 4 years is feasible. The target population for the prevalence survey is comprised of a stratified random sample of all adults aged 15 years and above and all children aged 2 to 4 years resident in four districts in Karachi. The survey procedures and analyses to estimate pulmonary tuberculosis prevalence are based on the World Health Organization methodology for tuberculosis prevalence surveys. Ethics and dissemination: The study protocol has been approved by the Interactive Research Development / The Indus Hospital Research Centre Research Ethics Committee in Karachi, Pakistan and the London School of Hygiene & Tropical Medicine Research Ethics Committee. Due to non-representative sampling in this setting, where a large proportion of the population are illiterate and are reluctant to provide fingerprints due to concerns about personal security, verbal informed consent will be obtained from each eligible participant or guardian. Results will be submitted to international peer-reviewed journals, presented at international conferences and shared with participating communities and with the Provincial and National TB programme.

Molecular characterisation of Bacillus chitinase for bioconversion of chitin waste.

In this work chitin was extracted chemically from shrimp shells. Seventeen Bacillus isolates were screened for chitinolytic activity. The chitinolytic strains of Bt. were screened at different temperatures and pHs for their hydrolytic potentials. By using a pair of specific primers, endochitinase gene was amplified from SBS Bt-5 strain through PCR, and then cloned into pTZ57 TA cloning vector and transferred in Escherichia coli DH5α strain. The sequenced gene (GenBank Accession No: HE995800) consists of 2031 nucleotides capable of encoding 676 residues. The protein consisted of three functional domains with a calculated molecular mass of 74.53 kDa and a pI value of 5.83. The amino acid sequence of chi gene showed 99% similarity to the genes of Bt MR11 endochitinase, Bt serovar kurstaki chitinase (kchi), Bt strain MR21 endochitinase and Bacillus cereus B4264.

Antimicrobial susceptibility pattern of clinical isolates of methicillin resistant Staphylococcus aureus.

OBJECTIVE: To study the antimicrobial susceptibility pattern of MRSA isolates from patients in a tertiary care hospital. METHODS: This was in-vitro study on MRSA isolates received from clinical samples in the department of microbiology during one year (March 2004-February 2005). All samples were processed by conventional method using sheep blood agar, MacConkey's agar and Chocolate agar plates. Staphylococci were identified by catalase, coagulase and D'nase tests. Antimicrobial susceptibility testing of all isolates was performed on Mueller-Hinton agar plates by modified Kirby Bauer's Disc Diffusion method. The disc used Oxacillin (1 microg), Cephalexin (10 microg), Ofloxacin(5 microg), Fusidic acid (10 microg), Penicillin (10 microg), Vancomycin (30 microg), Erythromycin (15 microg), Gentamicin (10 microg), Teicoplanin (30 microg), Amikacin (30 microg) and Clindamycin (2 microg). RESULTS: Our results indicate that out of 190 positive isolates of S. aureus, 82 (43%) were found to be MRSA. These strains were also resistant to many other antistaphylococcal antibiotics. CONCLUSION: A total of 82 (43%) MRSA were isolated from various clinical samples. Pattern of first line antistaphylococcal antibiotics is changing. Antimicrobial susceptibility testing is crucial in the treatment of these patients.