Open Online Courses for Strengthening Laboratory-Based Detection of Antimicrobial Resistance in Pakistan.

Introduction: Quality-assured antimicrobial susceptibility testing (AST) depends upon the knowledge and skills of laboratory staff. In many low- and middle-income countries (LMICs), including Pakistan, such types of knowledge and skills are limited. Therefore, the objective of this study was to use openaccess online courses to improve the knowledge of laboratory staff involved in the detection and reporting of antimicrobial resistance (AMR). Methodology: Seven online modules comprising 22 courses aimed at strengthening the laboratory detection of Antimicrobial resistance (AMR) were developed. The courses were uploaded onto the website www.parn.org.pk. Participants had an option of selecting courses of their interest. Online registration and completion of a pre-course assessment (pre-test) were essential for enrolment. However, participation in post-course assessment (post-test) was optional. The number of registered participants and the proportion of participants who completed each course were computed. A paired t-test was used to assess the increase between mean pre- and post-test scores. The association between the participants working in public vs. private laboratories and course completion rates were determined using the chi-square test. Results: A total of 227 participants from Pakistan (March 2018 to June 2020) were registered. The largest number of registered participants and the highest completion rate were noted for AST and biosafety courses, while quality-related courses attracted a lower interest. A comparison of pre- and post-test performance using the paired mean score for the individual courses showed a statistically significant (the value of p < 0.05) improvement in 13/20 assessed courses. A higher course completion rate was observed in participants from public vs. private sector laboratories (56.8 vs. 30.8%, the value of p = 0.005). Conclusions: Our study suggests a promising potential for open online courses (OOCs) toward addressing knowledge gaps in laboratory practice in resource limited settings.

Female genital tuberculosis in Pakistan - A retrospective review of 10-year laboratory data and analysis of 32 cases.

Background: Female genital tuberculosis (FGTB) is an underobserved clinical entity owing to diagnostic challenges stemming from difficulty of obtaining diagnostic specimens and paucibacillary nature of the disease. Yet, FGTB is a cause of infertility, pelvic pain, or menstrual irregularities in high-burden countries. To assess laboratory and microbiology diagnostic utilization for FGTB in Pakistan, we have collected data from 2007 to 2016 to inform the need for improved laboratory diagnostics. The objectives of this study were to determine the proportion of FGTB as culture-confirmed extrapulmonary tuberculosis (EPTB) and to describe the characteristics of women with culture-confirmed FGTB in a nationwide laboratory network in Pakistan. Method: A retrospective database was established by accessing laboratory archives and analyzed by sex and source to determine extrapulmonary cases among women. Data were checked for quality, and after removing patient identifiers and duplicate samples, frequencies were calculated in MS Excel. Clinical characteristics of patients were derived from a linked hospital database for those patients who were diagnosed and managed at the affiliated university hospital in Karachi, Pakistan. Results: Over 10 years, 410,748 mycobacterial cultures were received from multiple geographic sites throughout Pakistan and processed at the study laboratory. The overall mean culture positivity rate was 5.9% ± 3.5%, while the mean culture positivity rate among females was 2.8% ± 0.8%. Among female culture-confirmed tuberculosis cases, the pulmonary-to-EPTB ratio of infection was 5. Over 10 years, a total of 32 FGTB cases were reported on the basis of positive cultures for Mycobacterium tuberculosis; 3 (9.4%) were rifampin resistant. Conclusions: FGTB currently constitutes a small but significant proportion of culture-confirmed EPTB. A fewer number of laboratory requisitions suggest the need to increase awareness and testing. The advent of high-sensitivity molecular testing on extrapulmonary specimens has the potential to improve diagnostic accuracy and improved detection of FGTB cases in high-burden regions.

Bacterial aetiology of otitis media in children in Pakistan aged 0-59 months; laboratory surveillance data from 2004 to 2013: comparison between before and after the introduction of Hib vaccination.

BACKGROUND: Otitis media (OM) is a leading cause of childhood illness. In Pakistan, the estimated incidence of OM-associated hearing impairment is >40/10,000 population and OM-associated mortality is 50-79·9/10×10(6) population. No OM microbiology data are available from Pakistan since 2004. OBJECTIVES: To describe the microbiology of OM in children aged 0-59 months in Pakistan. METHODS: Laboratory data on ear pus specimens taken from children seen between 2004 and 2013 were retrieved from the Laboratory Information Systems of the Aga Khan University and entered into Microsoft Excel and SPSS version 16.0. RESULTS: Bacterial culture results from 277 specimens were analysed. Staphylococcus aureus and Pseudomonas aeruginosa were the organisms most commonly isolated, followed by Streptococcus pneumoniae and Haemophilus influenzae. Polymicrobial cultures significantly increased in the post-Hib vaccination period from 19·5% to 32·7% (P = 0·038). H. influenzae also increased significantly from 16·8% to 24·5% (P = 0·038). CONCLUSIONS: An increase in H. influenzae may reflect non-b capsular types (not determined in the study), or even capsular types from areas with low vaccine coverage. Increases in polymicrobial cultures and H. influenzae warrant further study.

Flaviviruses as a Cause of Undifferentiated Fever in Sindh Province, Pakistan: A Preliminary Report.

Arboviral diseases are expanding worldwide, yet global surveillance is often limited due to diplomatic and cultural barriers between nations. With human encroachment into new habitats, mosquito-borne viruses are also invading new areas. The actual prevalence of expanding arboviruses is unknown in Pakistan due to inappropriate diagnosis and poor testing for arboviral diseases. The primary objective of this study was to document evidence of flavivirus infections as the cause of undifferentiated fever in Pakistan. Through a cooperative effort between the USA and Pakistan, patient exposure to dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was examined in Sindh Province for the first time in decades. Initial results from the 2015 arbovirus season consisting of a cross-sectional study of 467 patients in 5 sites, DENV NS1 antigen was identified in 63 of the screened subjects, WNV IgM antibodies in 16 patients, and JEV IgM antibodies in 32 patients. In addition, a number of practical findings were made including (1) in silico optimization of RT-PCR primers for flavivirus strains circulating in the Middle East, (2) shipping and storage of RT-PCR master mix and other reagents at ambient temperature, (3) Smart phone applications for the collection of data in areas with limited infrastructure, and (4) fast and reliable shipping for transport of reagents and specimens to and from the Middle East. Furthermore, this work is producing a group of highly trained local scientists and medical professionals disseminating modern scientific methods and more accurate diagnostic procedures to the community.

Accuracy of frozen section analysis of sentinel lymph nodes for the detection of Asian breast cancer micrometastasis - experience from Pakistan.

BACKGROUND: Intraoperative sentinel lymph node biopsy has now become the standard of care for patients with clinically node negative breast cancer for diagnosis and also in order to determine the need for immediate axillary clearance. Several large scale studies confirmed the diagnostic reliability of this method. However, micrometastases are frequently missed on frozen sections. Recent studies showed that both disease free interval and overall survival are significantly affected by the presence of micrometastatic disease. The aim of this study was to determine the sensitivity and specificity of intraoperative frozen section analysis of sentinel lymph nodes (SLNs) for the detection of breast cancer micrometastasis and to evaluate the status of non-sentinel lymph nodes (non-SLNs) in those patients subjected to further axillary sampling. MATERIALS AND METHODS: We performed a retrospective study on 154 patients who underwent SLN biopsy from January 2008 till October 2011. The SLNs were sectioned at 2 mm intervals and submitted entirely for frozen sections. Three levels of each section submitted are examined and the results were compared with further levels on paraffin sections. RESULTS: Overall 40% of patients (62/154) were found to be SLN positive on final (paraffin section) histology, out of which 44 demonstrated macrometastases (>2mm) and 18 micrometastases (<2mm). The overall sensitivity and specificity of frozen section analysis of SLN for the detection of macrometastasis was found to be 100% while those for micrometastasis were 33.3% and 100%, respectively. Moreover 20% of patients who had micrometastases in SLN had positive non-SLNs on final histology. CONCLUSIONS: Frozen section analysis of SLNs lacks sufficient accuracy to rule out micrometastasis by current protocols. Therefore these need to be revised in order to pick up micrometastasis which appears to have clinical significance. We suggest that this can be achieved by examining more step sections of blocks.

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.