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Objective: Group B streptococcus (GBS) has been associated with adverse pregnancy outcomes, but few prospective studies have assessed its prevalence in low‐ and middle‐income country settings. We sought to evaluate the prevalence of GBS by polymerase chain reaction (PCR) in internal organ tissues and placentas of deceased neonates and stillbirths. Design: This was a prospective, observational study. Setting: The study was conducted in hospitals in India and Pakistan. Population: Pregnant women with stillbirths or preterm births were recruited at delivery, as was a group of women with term, live births, to serve as a control group. Methods: A rectovaginal culture was collected from the women in Pakistan to assess GBS carriage. Using PCR, we evaluated GBS in various tissues of stillbirths and deceased neonates and their placentas, as well as the placentas of live‐born preterm and term control infants. Main outcome measures: GBS identified by PCR in various tissues and the placentas; rate of stillbirths and 28‐day neonatal deaths. Results: The most obvious finding from this series of analyses from India and Pakistan was that no matter the country, the condition of the subject, the tissue studied or the methodology used, the prevalence of GBS was low, generally ranging between 3% and 6%. Among the risk factors evaluated, only GBS positivity in primigravidae was increased. Conclusions: GBS diagnosed by PCR was identified in <6% of internal organs of stillbirths and neonatal deaths, and their placentas, and control groups in South Asian sites. This is consistent with other reports from South Asia and is lower than the reported GBS rates from the USA, Europe and Africa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To examine internal organ tissues and placentas of stillbirths for various pathogens. Design: Prospective, observational study. Settings: Three study hospitals in India and a large maternity hospital in Pakistan. Population: Stillborn infants delivered in a study hospital. Methods: A prospective observational study. Main outcome measures: Organisms identified by pathogen polymerase chain reaction (PCR) in internal organs and placental tissues of stillbirths. Results: Of 2437 stillbirth internal tissues, 8.3% (95% CI 7.2–9.4) were positive. Organisms were most commonly detected in brain (12.3%), cerebrospinal fluid (CSF) (9.5%) and whole blood (8.4%). Ureaplasma urealyticum/parvum was the organism most frequently detected in at least one internal organ (6.4% of stillbirths and 2% of all tissues). Escherichia coli/Shigella was the next most common (4.1% one or more internal organ tissue sample and 1.3% of tissue samples), followed by Staphylococcus aureus in at least one internal organ tissue (1.9% and 0.9% of all tissues). None of the other organisms was found in more than 1.4% of the tissue samples in stillbirths or more than 0.6% of the internal tissues examined. In the placenta tissue, membrane or cord blood combined, 42.8% (95% CI 40.2–45.3) had at least one organism identified, with U. urealyticum/parvum representing the most commonly identified (27.8%). Conclusions: In about 8% of stillbirths, there was evidence of a pathogen in an internal organ. Ureaplasma urealyticum/parvum was the most common organism found in the placenta and in the internal tissues, especially in the fetal brain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To the Editor: Naegleria fowleri. a free-living ameba, causes acute, fulminant, fatal primary amebic meningoencephalitis (PAM) in persons with history of recreational activities in warm freshwater (1,2). During 2008-2009, thirteen [...]

Abstract Background The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2). Methods Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC. Results A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC). Conclusions This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.

Background: In Pakistan, Plasmodium vivax and Plasmodium falciparum co-exist and usage of sulphadoxinepyrimethamine (SP) against P. falciparum exposes P. vivax to the drug leading to generation of resistant alleles. The main aim of this study was to investigate frequency distribution of drug resistance associated mutations in pvdhfr, pvdhps genes and provide baseline molecular epidemiological data on SP-associated resistance in P. vivax from southern Pakistan. Methods: From January 2008 to May 2009, a total of 150 samples were collected from patients tested slide-positive for P. vivax, at the Aga Khan University Hospital, Karachi, or its collection units located in Baluchistan and Sindh Province. Nested PCR using pvdhfr and pvdhps specific primers was performed for all samples.91.3% (137/150) of the samples were tested PCR positive of which 87.3% (131/137) were successfully sequenced. Sample sequencing data was analysed and compared against wild type reference sequences. Results: In dhfr, mutations were observed at codons F57L, S58R and S117N/T. Novel non-synonymous mutations were observed at codon positions N50I, G114R and E119K while a synonymous mutation was observed at codon position 69Y. In dhps, mutations were observed at codon position A383G and A553G while novel non-synonymous mutations were observed at codon positions S373T, E380K, P384L, N389T, V392D, T393P, D459A, M601I, A651D and A661V. Conclusion: This is the first report from southern Pakistan on SP resistance in clinical isolates of P. vivax. Results from this study confirm that diverse drug resistant alleles are circulating within this region. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the prevalent malarial species accounting for 70% of malaria burden in Pakistan; however, there is no baseline data on the circulating genotypes. Studies have shown that polymorphic loci of gene encoding antigens pvcsp and pvmsp1 can be used reliably for conducting molecular epidemiological studies. Therefore, this study aimed to bridge the existing knowledge gap on population structure on P. vivax from Pakistan using these two polymorphic genes. Methods: During the period January 2008 to May 2009, a total of 250 blood samples were collected from patients tested slide positive for P. vivax, at the Aga Khan University Hospital, Karachi, or its collection units located in Baluchistan and Sindh Province. Nested PCR/RFLP was performed, using pvcsp and pvmsp1 markers to detect the extent of genetic diversity in clinical isolates of P. vivax from southern Pakistan. Results: A total of 227/250 (91%) isolates were included in the analysis while the remainder were excluded due to negative PCR outcome for P.vivax. Pvcsp analysis showed that both VK 210 (85.5%, 194/227) and VK 247 type (14.5%, 33/227) were found to be circulating in P. vivax isolates from southern Pakistan. A total of sixteen and eighty-seven genotypes of pvcsp and pvmsp-1 were detected respectively. Conclusion: This is the first report from southern Pakistan on characterization of P. vivax isolates confirming that extensively diverse pvcsp and pvmsp1 variants are present within this region. Results from this study provide valuable data on genetic diversity of P. vivax that will be helpful for further epidemiological studies. [ABSTRACT FROM AUTHOR]

Background: The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2). Methods: Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC. Results: A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC). Conclusions: This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity. [ABSTRACT FROM AUTHOR]

Rapid tests such as ICT Malaria are an effective field tool in determining the presence of malarial parasites but do not provide an estimate of parasite load. We have evaluated the utility of ICT for providing semi-quantitative estimates of parasite load. Circulating parasite load in the blood of patients with malaria (n =54), were compared with the circulating HRP2 protein levels. Blood was serially diluted and analysed by a rapid diagnostic test (ICT® Now P.f/P.v) for assessment of endpoint PfHRP2 antigen titres. Significant correlation was observed between parasite load and PfHRP2 antigen titres (Spearman rank; rho = 0.821; p<0.001) with plasma dilutions > 1:16 corresponding to a parasite load of 0.1% parasitaemia. Variability in haematological parameters had no effect on the antibody titres obtained with the ICT test. Rapid semi-quantitative assessment of parasite load in conjunction with the Plasmodium speciation may provide a useful bedside and field aid in the diagnosis of malaria. [ABSTRACT FROM AUTHOR]

In Pakistan, contributes to major malaria burden. In this case, a pregnant woman presented with Plasmodium vivax P. vivax infection and which was not cleared by chloroquine, despite adequate treatment. This is probably the first confirmed case of chloroquine-resistant vivax from Pakistan, where severe malaria due to is already an P. vivax emerging problem. [ABSTRACT FROM AUTHOR]

Background:Cytokine-mediated endothelial activation pathway is a known mechanism of pathogenesis employed by Plasmodium falciparum to induce severe disease symptoms in human host. Though considered benign, complicated cases of Plasmodium vivax are being reported worldwide and from Pakistan. It has been hypothesized that P.vivax utilizes similar mechanism of pathogenesis, as that of P.falciparum for manifestations of severe malaria. Therefore, the main objective of this study was to characterize the role of cytokines and endothelial activation markers in complicated Plasmodium vivax isolates from Pakistan. Methods and Principle Findings:A case control study using plasma samples from well-characterized groups suffering from P.vivax infection including uncomplicated cases (n=100), complicated cases (n=82) and healthy controls (n=100) were investigated. Base line levels of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1(VCAM-1) and E-selectin were measured by ELISA. Correlation of cytokines and endothelial activation markers was done using Spearman’s correlation analysis. Furthermore, significance of these biomarkers as indicators of disease severity was also analyzed. The results showed that TNF-α, IL-10, ICAM-1and VCAM-1 were 3-fold, 3.7 fold and 2 fold increased between uncomplicated and complicated cases. Comparison of healthy controls with uncomplicated cases showed no significant difference in TNF-α concentrations while IL-6, IL-10, ICAM-1, VCAM-1 and E-selectin were found to be elevated respectively. In addition, significant positive correlation was observed between TNF-α and IL-10/ ICAM-1, IL-6 and IL-10, ICAM-1 and VCAM-1.A Receiver operating curve (ROC) was generated which showed that TNF-α, IL-10, ICAM-1 and VCAM-1 were the best individual predictors of complicated P.vivax malaria. Conclusion:The results suggest that though endothelial adhesion molecules are inducible by pro-inflammatory cytokine TNF-α, however, cytokine-mediated endothelial activation pathway is not clearly demonstrated as a mechanism of pathogenesis in complicated P.vivax malaria cases from Pakistan. [ABSTRACT FROM AUTHOR]

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., (© 2023 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Objective: To examine internal organ tissues and placentas of stillbirths for various pathogens., Design: Prospective, observational study., Settings: Three study hospitals in India and a large maternity hospital in Pakistan., Population: Stillborn infants delivered in a study hospital., Methods: A prospective observational study., Main Outcome Measures: Organisms identified by pathogen polymerase chain reaction (PCR) in internal organs and placental tissues of stillbirths., Results: Of 2437 stillbirth internal tissues, 8.3% (95% CI 7.2-9.4) were positive. Organisms were most commonly detected in brain (12.3%), cerebrospinal fluid (CSF) (9.5%) and whole blood (8.4%). Ureaplasma urealyticum/parvum was the organism most frequently detected in at least one internal organ (6.4% of stillbirths and 2% of all tissues). Escherichia coli/Shigella was the next most common (4.1% one or more internal organ tissue sample and 1.3% of tissue samples), followed by Staphylococcus aureus in at least one internal organ tissue (1.9% and 0.9% of all tissues). None of the other organisms was found in more than 1.4% of the tissue samples in stillbirths or more than 0.6% of the internal tissues examined. In the placenta tissue, membrane or cord blood combined, 42.8% (95% CI 40.2-45.3) had at least one organism identified, with U. urealyticum/parvum representing the most commonly identified (27.8%)., Conclusions: In about 8% of stillbirths, there was evidence of a pathogen in an internal organ. Ureaplasma urealyticum/parvum was the most common organism found in the placenta and in the internal tissues, especially in the fetal brain., (© 2023 John Wiley & Sons Ltd.)

Background Malarial infection is a major cause of concern, both worldwide and in Pakistan. Gametocytes are the sexual forms of the parasite that are essential for transmission. They fuse inside the mosquito to develop sporozoites. Gametocytes of the plasmodium parasites, which cause the infection, differentiate into male and female gametocytes. These gametocytes constitute the sexual stage of the malaria parasite and are essential in transmission of the disease from human to vector Anopheles. Gametocytes are affected by factors such as host immunity, drug treatment, reticulocytemia, anemia, low levels of asexual parasitemia and stress to the parasite. The aim of this study was to observe the hematological parameters, age and gametocyte carriage in an area of seasonal malaria transmission. Methods The study was conducted at Aga Khan University Hospital (AKUH) Laboratory over the period of one year and 294 patients with uncomplicated malaria were recruited. Patients infected with Plasmodium falciparum (P. falciparum) or Plasmodium vivax (P. vivax) malaria and no co-morbidities were included in the study. Results Gametocytemia was highest during the period of July to November, with P. vivax, 267 (90.8%), predominating compared to P. falciparum, 27 (9.2%). P. vivax gametocytes were observed from May to October and P. falciparum gametocytes were observed from July to December. Low hemoglobin in females and low platelet levels were observed. The mean platelet count was significantly lower in cases of P. vivax having gametocytes compared to P. falciparum with gametocytes. Higher parasitic index was associated with lower platelet count. The most significantly altered parameters were hemoglobin, hematocrit, white blood cell (WBC), and platelet count. Hemoglobin and platelets were significantly lower during the malaria season in study participants, both male and female. Conclusion In conclusion, infection with P. falciparum and P. vivax modulates significant changes in hematological parameters in populations living in malaria endemic regions. In the malaria season males were more frequently affected by malaria with thrombocytopenia. Gametocyte carriage remains unaffected by seasonal changes thus ensuring parasite transmission during the dry season., Competing Interests: The authors have declared that no competing interests exist.

Background Intestinal parasites cause significant morbidity and impact human development with an enormous global burden. Diagnosis of intestinal parasites by conventional methods has several limitations. The gauze filtration technique is a relatively simple method that has been shown to identify intestinal parasites with a high sensitivity and specificity. The aim of this study was to determine the diagnostic value of this technique as compared to more conventional methods in a large acclaimed laboratory within Pakistan. Methods A total of 50 stool samples collected for routine diagnostic workup from patients age between 2-70 years were collected from the parasitology section of the Aga Khan University Hospital Clinical Laboratory. A direct wet mount, sedimentation technique, and gauze filtration technique were performed on all of the stool samples, and the sensitivity, specificity, negative predictive value, and positive predictive value were analyzed. Results It was observed that the number of organisms observed by gauze filtration as compared to direct wet mount and sedimentation technique was higher for B. hominis , G. lamblia cysts and trophozoites, and I. bütschlii . Also, the detection rate was significantly higher for B. hominis and G. lamblia cysts using the gauze filtration technique. The sensitivity and specificity of the gauze filtration technique were found to be 95.8% and 100%, respectively. Conclusion There is a significantly better stool sample parasite detection rate using the gauze filtration technique as compared to the conventional sedimentation techniques. The utility of the gauze filtration technique seems economically and technically feasible for diagnostic laboratories in resource-limited settings., Competing Interests: The authors have declared that no competing interests exist.

A cross-sectional survey using structured questionnaire was conducted to assess practices of microbiological laboratories working with pathogens. Forty-eight laboratory workers (50%) agreed that laboratory methods to detect antimicrobial resistance are not standardized in Pakistan, and 6% of the laboratory workers were not aware of the standardization of antimicrobial susceptibility testing in Pakistan. Reported rates of awareness regarding the role of waste disposal, disinfection, and handwashing in limiting the spread of antimicrobial resistance were 75%, 42%, and 81%, respectively. Our results provide baseline data for planning programs to train, supervise, and improve the operational quality of microbiological laboratories nationwide to prevent the spread of superbugs., (Copyright © 2014 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)

Background: Cytokine-mediated endothelial activation pathway is a known mechanism of pathogenesis employed by Plasmodium falciparum to induce severe disease symptoms in human host. Though considered benign, complicated cases of Plasmodium vivax are being reported worldwide and from Pakistan. It has been hypothesized that P.vivax utilizes similar mechanism of pathogenesis, as that of P.falciparum for manifestations of severe malaria. Therefore, the main objective of this study was to characterize the role of cytokines and endothelial activation markers in complicated Plasmodium vivax isolates from Pakistan., Methods and Principle Findings: A case control study using plasma samples from well-characterized groups suffering from P.vivax infection including uncomplicated cases (n=100), complicated cases (n=82) and healthy controls (n=100) were investigated. Base line levels of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1(VCAM-1) and E-selectin were measured by ELISA. Correlation of cytokines and endothelial activation markers was done using Spearman's correlation analysis. Furthermore, significance of these biomarkers as indicators of disease severity was also analyzed. The results showed that TNF-α, IL-10, ICAM-1and VCAM-1 were 3-fold, 3.7 fold and 2 fold increased between uncomplicated and complicated cases. Comparison of healthy controls with uncomplicated cases showed no significant difference in TNF-α concentrations while IL-6, IL-10, ICAM-1, VCAM-1 and E-selectin were found to be elevated respectively. In addition, significant positive correlation was observed between TNF-α and IL-10/ ICAM-1, IL-6 and IL-10, ICAM-1 and VCAM-1.A Receiver operating curve (ROC) was generated which showed that TNF-α, IL-10, ICAM-1 and VCAM-1 were the best individual predictors of complicated P.vivax malaria., Conclusion: The results suggest that though endothelial adhesion molecules are inducible by pro-inflammatory cytokine TNF-α, however, cytokine-mediated endothelial activation pathway is not clearly demonstrated as a mechanism of pathogenesis in complicated P.vivax malaria cases from Pakistan.