Fluconazole-resistant Candida parapsilosis complex candidemia and analysis of mutations in the ERG11 gene from Pakistan

Background: Recent reports of the emergence of fluconazole resistance in Candida parapsilosis species complex poses a challenge, more specifically in settings where echinocandin-based treatment regime is not feasible.Objective: This study reported emergence of fluconazole resistance in C. parapsilosis species complex strains isolated from blood cultures.Materials and methods: This retrospective observational study was conducted from 2018 to 2020 at a tertiary care laboratory from Pakistan. Fluconazole-resistant C. parapsilosis species complex fungemia cases were identified from laboratory database and clinical details were collected. Identification of C. parapsilosis species complex was done using API 20C AUX and Cornmeal Tween80 agar morphology. Minimum inhibitory concentrations (MICs) were determined using Sensititre YeastONE and interpretation was done with CLSI M60 ED1:2017. ERG11 gene region was amplified and sequenced by Sanger sequencing and analysed by MEGA 11 Software.Results: A total of 13 (8.5%) fluconazole-resistant isolates were identified from 152 C. parapsilosis species complex candidemia cases. Fluconazole MICs of resistant isolates ranged between 8 and 256 μg/mL. Analysis of ERG11 gene revealed nonsynonymous mutations at position Y132F in 86% of the fluconazole-resistant isolates. Diabetes and hospitalization were important risk factors for candidemia with fluconazole-resistant C. parapsilosis complex.Conclusion: This is the first report of the emergence and molecular mechanisms of fluconazole resistance in C. parapsilosis species complex from Pakistan. Y132F mutation in the ERG11 gene was the most common mutation in fluconazole-resistant strains. These findings are concerning and necessitate better diagnostics, newer antifungals, ongoing surveillance and further insights on resistance mechanisms in the country

Pneumocystis pneumonia in HIV-positive and non-HIV patients: A retrospective comparative study from a lower-middle income country

In this study, we compared the predisposing factors, key demographic and clinical characteristics, clinical outcomes, and factors associated with poor prognosis in pneumocystis pneumonia (PCP) infection among the human immunodeficiency virus (HIV)-positive and non-HIV patient populations. This retrospective analysis was conducted at the Aga Khan University Hospital, Karachi, via the collection and analysis of patient records with a diagnosis of pneumocystosis between January 2015 and October 2020. Additionally, the laboratory database was evaluated, and patients with a laboratory-confirmed diagnosis of PCP were included. During the study period, 52 laboratory-confirmed hospitalized PCP patients were identified. Of these, 23 and 29 patients were diagnosed using microscopy and polymerase chain reaction, respectively. 34.6% of our patients were HIV positive, with a median CD4 count of 20.5 cells/mm3 (range: 10.7-50.5). Other conditions identified were corticosteroid use, autoimmune diseases, malignancy, radiation, and chemotherapy. On chest imaging, consolidation was found in 30%, ground-glass opacities in 24%, and nodular infiltrates in 20% of the cases. HIV-positive patients had a lower hemoglobin level and a higher level of β-D-glucan at the time of admission, whereas non-HIV patients were found to have more co-morbid conditions than HIV patients. We observed no difference in clinical outcomes between the two populations. Factors associated with a poor prognosis among our patients included concomitant infections at the time of diagnosis, the need for invasive mechanical ventilation, and a longer duration of stay in the hospital as well as the intensive care unit

Adapting international clinical trials during COVID-19 and beyond

Background: The COVID-19 pandemic and resulting restrictions, particularly travel restrictions, have had significant impact on the conduct of global clinical trials. Our clinical trials programme, which relied on in-person visits for training, monitoring and capacity building across nine low- and middle-income countries, had to adapt to those unprecedented operational challenges. We report the adaptation of our working model with a focus on the operational areas of training, monitoring and cross-site collaboration.The new working model: Adaptations include changing training strategies from in-person site visits with three or four team members to a multi-pronged virtual approach, with generic online training for good clinical practice, the development of a library of study-specific training videos, and interactive virtual training sessions, including practical laboratory-focused training sessions. We also report changes from in-person monitoring to remote monitoring as well as the development of a more localized network of clinical trial monitors to support hybrid models with in-person and remote monitoring depending on identified risks at each site. We established a virtual network across different trial and study sites with the objective to further build capacity for good clinical practice-compliant antimalarial trials and foster cross-country and cross-study site collaboration.Conclusion: The forced adaptation of these new strategies has come with advantages that we did not envisage initially. This includes improved, more frequent engagement through the established network with opportunities for increased south-to-south support and a substantially reduced carbon footprint and budget savings. Our new approach is challenging for study sites with limited prior experience but this can be overcome with hybrid models. Capacity building for laboratory-based work remains difficult using a virtual environment. The changes to our working model are likely to last, even after the end of the pandemic, providing a more sustainable and equitable approach to our research

Placental inflammation and pregnancy outcomes: A prospective, observational study in South Asia: The PURPOSe study

Objective: To examine inflammatory lesions in placentas of stillbirths, preterm neonatal deaths and term controls in India and Pakistan. Design: Prospective, observational study. Setting: Three hospitals in India and a large maternity hospital in Pakistan. Population: The enrolled participants with placentas available for histology evaluation included stillbirths (n = 814), preterm live births who died within 28 days of birth (n = 618) and term live birth controls (n = 201). From this same population, polymerase chain reaction (PCR) analysis for pathogens was performed on 809 stillbirth placentas, 614 neonatal death placentas and the placentas of 201 term controls. Placentas from preterm infants who lived beyond day 28 (n = 1432) were only available from India. Methods: A prospective observational study of placental inflammatory lesions defined by the Amsterdam criteria and on the same placentas, multiplex PCR evaluation for 75 pathogens using TaqMan Array Cards. Main outcome measures: Any placental inflammatory lesions, including chorioamnionitis, funisitis, villitis and intervillitis and their association with various pathogens. Results: In the Indian liveborn preterm infants, placental inflammation of any kind was present in 26.2% of those who died versus 16.6% of those who lived (p = 0.0002). Chorioamnionitis was present in 25.8% of those who died versus 16.3% of those who lived (p = 0.0002) and funisitis was present in 4.1% of those who died versus 1.5% of those who lived, (p = 0.005). Across all three sites, in the placentas of the 201 term controls, 18.9% had any inflammation, 16.9% had chorioamnionitis, 5.5% had funisitis, 0.5% had intervillitis and none had villitis. Overall, for stillbirths, any inflammation was observed in 30.2%, chorioamnionitis in 26.9%, funisitis in 5.7%, intervillitis in 6.0% and villitis in 2.2%. For the neonatal deaths, any inflammation was present in 24.9%, chorioamnionitis in 23.3%, funisitis in 8.1%, intervillitis in 1.9% and villitis in 0.5%. Compared with the placentas of term controls, in neonatal deaths, only chorioamnionitis was significantly increased (23.3% versus 16.9%, p = 0.05). Among stillbirths, the rates of any inflammation, chorioamnionitis, intervillitis and villitis were similar across the birthweight groups. However, funisitis was more common in the placentas of stillborn fetuses weighing 2500 g or more (13.8%) compared with 1.0% for those weighing less than 1000 g and 4.8% for stillborn fetuses weighing 1000-2499 g. In the PCR studies, Ureaplasma spp. were by far the most common pathogens found and generally were more commonly found in association with inflammatory lesions. Conclusions: Chorioamnionitis was the most common type of placental inflammatory lesion regardless of whether the placentas evaluated were from term controls, stillbirths or neonatal deaths. For stillbirths, inflammation in each inflammation category was more common than in the term controls and significantly more so for any inflammation, chorioamnionitis, intervillitis and villitis. For neonatal deaths, compared with the placentas of term controls, all inflammation categories were more common, but only significantly so for chorioamnionitis. Ureaplasma spp. were the most common organisms found in the placentas and were significantly associated with inflammatio

The PURPOSe cause of death study in stillbirths and neonatal deaths in India and Pakistan: A review

The PURPOSe study was a prospective, observational study conducted in India and Pakistan to determine the cause of death for stillbirths and preterm neonatal deaths, using clinical data together with minimally invasive tissue sampling (MITS) and the histologic and polymerase chain reaction (PCR) evaluation of fetal/neonatal tissues and the placenta. After evaluating all available data, an independent panel chose a maternal, a placental and a fetal/neonatal cause of death. Here, we summarise the major results. Among the most important findings were that most stillbirths were caused by fetal asphyxia, often preceded by placental malperfusion, and clinically associated with pre-eclampsia, placental abruption and a small-for-gestational-age fetus. The preterm neonatal deaths were primarily caused by birth asphyxia, followed by various infections. An important finding was that many of the preterm neonatal deaths were caused by a nosocomial infection acquired after neonatal intensive care (NICU) admission; the most common organisms were Acinetobacter baumannii, followed by Klebsiella pneumoniae, Escherichia coli/Shigella and Haemophilus influenzae. Group B streptococcus was less commonly present in the placentas or internal organs of the neonatal deaths

Maternal infection and stillbirth: A review

Introduction: Maternal infections likely are an important cause of stillbirths, especially in sub-Saharan Africa and south Asia, where the burden is highest. Due to the lack of routine testing for infection, which can be complex and often expensive, the prevalence of infection during pregnancy and the association of many infections with stillbirth are not well-documented, especially in low-resource countries. Methods: Following an extensive literature review of infection and stillbirth initially published in 2010, we conducted a review of literature in the last 10 years to identify infections associated with stillbirth, focused on those in low-resource settings. Results: During the last 10 years, over 40 bacterial, viral and other pathogens have been associated with stillbirth. Newly emerging viral infections such as Denge as well as several well-established, but not yet eliminated infections such as rubella have been associated with stillbirth. Two of the maternal infections most strongly associated with stillbirth, each with about a 2-fold risk, are malaria and syphilis but others have been associated with risk in a range of studies. With a lack of routine antenatal screening, many pathogens are identified as associated with stillbirth only through case reports. Infection remains an important, yet understudied, cause of stillbirth. Conclusion: Research studies to determine definitive associations between various infections and stillbirth are important to better understand the role of infections and strategies to reduce infection-related stillbirth. Summary This review explores the association between infections and stillbirths focusing on low-income country studies published in the last 10 years. Much information about these relationships comes from case reports. Research resulting in a better understanding of the causes and strategies to reduce infection-related stillbirth is necessary

Tracking SARS-CoV-2 variants through pandemic waves using RT-PCR testing in low-resource settings

COVID-19 resulted in extensive morbidity and mortality worldwide. SARS-CoV-2 evolved rapidly, with increasing transmission due to Variants of Concern (VOC). Identifying VOC became important but genome submissions from low-middle income countries (LMIC) remained low leading to gaps in genomic epidemiology. We demonstrate the use of a specific mutation RT-PCR based approach to identify VOC in SARS-CoV-2 positive samples through the pandemic in Pakistan. We selected 2150 SARS-CoV-2 PCR positive respiratory specimens tested between April 2021 and February 2022, at the Aga Khan University Hospital Clinical Laboratories, Karachi, Pakistan. Commercially available RT-PCR assays were used as required for mutations in Spike protein (N501Y, A570D, E484K, K417N, L452R, P681R and deletion69_70) to identify Alpha, Beta, Gamma, Delta, and Omicron variants respectively. Three pandemic waves associated with Alpha, Delta and Omicron occurred during the study period. Of the samples screened, VOC were identified in 81.7% of cases comprising mainly; Delta (37.2%), Alpha (29.8%) and Omicron (17.1%) variants. During 2021, Alpha variants were predominant in April and May; Beta and Gamma variants emerged in May and peaked in June; the Delta variant peaked in July and remained predominant until November. Omicron (BA.1) emerged in December 2021 and remained predominant until February 2022. The CT values of Alpha, Beta, Gamma and Delta were all significantly higher than that of Omicron variants (p\u3c0.0001). We observed VOC through the pandemic waves using spike mutation specific RT-PCR assays. We show the spike mutation specific RT-PCR assay is a rapid, low-cost and adaptable for the identification of VOC as an adjunct approach to NGS to effectively inform the public health response. Further, by associating the VOC with CT values of its diagnostic PCR we gain information regarding the viral load of samples and therefore the level of transmission and disease severity in the population