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Since 2007, national public health laboratories in the WHO Eastern Mediterranean Region (EMR) have participated in a regional external quality assessment scheme in bacteriology to improve testing proficiency.To assess laboratory performance in bacteriology in the EMR between 2011 and 2019 using the regional external quality assessment scheme.We analysed the accuracy of participant-reported data in bacterial identification, Gram stain microscopy, and antimicrobial susceptibility testing. For each category, we assessed the performance over time, the performance on multiple organisms, and whether a laboratory repeatedly failed to attain satisfactory results.Between 2011 and 2019, 70% of laboratories achieved satisfactory performance for bacterial identification and antimicrobial susceptibility testing, and 85% performed satisfactory Gram stain microscopy. Testing did not improve on multiple organisms and results were consistently low for some pathogens and test categories. Twenty-nine percent of laboratories underperformed throughout the study period.The unchanged performance over time and underperformance of laboratories highlight the need for improvements in the regional external quality assessment scheme. Participating laboratories and WHO need to work more actively to strengthen the problem areas.التقييم الخارجي لجودة أداء المختبرات المتخصصة في علم الجراثيم في إقليم شرق المتوسط، 2011–2019.همايون أصغر، كارين نهابتيان، أماني غنيم، فرانك كونينجز، أمينة الجرداني.منذ عام 2007 ، شاركت مختبرات الصحة العامة الوطنية في إقليم شرق المتوسط في مخطط إقليمي للتقييم الخارجي للجودة في علم الجراثيم لتحسين كفاءة الاختبارات.هدفت هذه الدراسة الى تقييم أداء المختبرات في مجال علم الجراثيم في إقليم شرق المتوسط بين عامَي 2011 و 2019 ، باستخدام النظام الإقليمي للتقييم الخارجي للجودة.حللنا دقة البيانات التي أبلغ بها المشاركون فيما يخص تحديد الجراثيم، والفحص المجهري لصبغة جرام، واختبار الحساسية لمضادات الميكروبات. وبالنسبة إلى كل فئة، قيمنا الأداء مع مرور الزمن، والأداء على كائنات حية متعددة، وما إذا كان المختبر قد فشل مرارًا في تحقيق نتائج مُرضية.بين عامَي 2011 و 2019 ، حقق 70 ٪ من المختبرات أداءً مُرضيًا في التعرف على الجراثيم وتحديدها واختبار الحساسية لمضادات الميكروبات، ونفذ 85 ٪ منها الفحصَ المجهري لصبغة جرام تنفيذًا مُرضيًا. ولم يتحسن مستوى الاختبار على كائنات حية متعددة، وكانت النتائج منخفضة باستمرار فيما يخص بعض مسببات الأمراض وفئات الاختبارات. وكان أداء 29 ٪ من المختبرات دون المستوى خلال فترة الدراسة.إن عدم تغيُُّّر الأداء بمرور الوقت والأداء القاصر للمختبرات يبرزان الحاجة إلى إدخال تحسينات على الخطة الإقليمية للتقييم الخارجي للجودة. لذا، يتعيََّّن على المختبرات المشارِكة و المختبرات المشاركة ومنظمة الصحة العالمية بحاجة إلى العمل بشكل أكثر نشاطا لتقوية مناطق المشاكل.Évaluation externe de la qualité des performances des laboratoires en bactériologie dans la Région de la Méditerranée orientale, 2011-2019.Depuis 2007, les laboratoires de santé publique nationaux de la Région OMS de la Méditerranée orientale ont participé à un système régional d'évaluation externe de la qualité en bactériologie afin d'améliorer la bonne exécution des analyses.Évaluer les performances des laboratoires en bactériologie dans la Région de la Méditerranée orientale entre 2011 et 2019 à l'aide du système régional d'évaluation externe de la qualité.Nous avons analysé l'exactitude des données communiquées par les participants concernant l'identification bactérienne, la microscopie après coloration de Gram et les tests de sensibilité aux antimicrobiens. Pour chaque catégorie, nous avons évalué la performance au fil du temps, la performance sur plusieurs micro-organismes et avons vérifié si un laboratoire n'a pas obtenu des résultats satisfaisants à plusieurs reprises.Entre 2011 et 2019, 70 % des laboratoires ont obtenu des résultats satisfaisants pour l'identification bactérienne et les tests de sensibilité aux antimicrobiens, et 85 % ont effectué une microscopie après coloration de Gram satisfaisante. Les tests ne se sont pas améliorés sur plusieurs micro-organismes et les résultats étaient systématiquement faibles pour certains agents pathogènes et certaines catégories de tests. Vingt-neuf pour cent des laboratoires ont eu des résultats insuffisants tout au long de la période d'étude.Les performances inchangées au cours du temps et les résultats insuffisants des laboratoires soulignent la nécessité d'améliorer le système régional d'évaluation externe de la qualité. Les laboratoires participants et l'OMS doivent collaborer plus activement pour renforcer les domaines qui posent problème.

Arboviruses continue to pose serious public health threats in the World Health Organization (WHO) Western Pacific Region. As such, laboratories need to be equipped for their accurate detection. In 2011, to ensure test proficiency, the WHO Regional Office for the Western Pacific piloted an external quality assessment (EQA) programme for arbovirus diagnostics. By 2016, it had grown into a global programme with participation of 96 laboratories worldwide, including 25 laboratories from 19 countries, territories and areas in the Region. The test performance of the 25 laboratories in the Region in 2016 was high with 23 (92%) reporting correct results in all specimens for dengue and chikungunya viruses. For Zika virus, 18 (72%) of the 25 laboratories reported correct results in all specimens, while seven (28%) demonstrated at least one error. When comparing iterations of this EQA programme in the Region between 2013 and 2016, the number of participating laboratories increased from 18 to 25. The first round only included dengue virus, while the latest round additionally included chikungunya, Zika and yellow fever viruses. Proficiency for molecular detection of dengue virus remained high (83-94%) over the four-year period. The observed proficiency for arbovirus diagnostics between 2013 and 2016 is an indicator of laboratory quality improvement in the Region.

Arboviruses such as dengue virus, yellow fever virus, Zika virus and chikungunya virus are major threats to human health globally, including countries in the Eastern Mediterranean Region (EMR).This study aimed to assess laboratory proficiency in EMR countries for detection of dengue virus, yellow fever virus, Zika virus and chikungunya virus.A global external quality assessment programme for arbovirus diagnostics was developed and run in 2016 and 2018. National-level public health laboratories were instructed to apply the polymerase chain reaction detection method on specimen panels containing dengue virus, yellow fever virus, Zika virus and chikungunya virus.Over both rounds of the programme, 100% of participating EMR laboratories correctly detected yellow fever virus and chikungunya virus, ≥ 84.6% detected dengue fever virus and ≥ 76.9% detected Zika virus.While participating EMR countries demonstrated good proficiency in detecting arboviruses, only half of them were enrolled in the global external quality assessment programme, providing an incomplete picture of regional capacity. Effort should be put into increasing participation in subsequent rounds.أداء مختبرات إقليم شرق المتوسط في برنامج تقييم الجودة الخارجي لمنظمة الصحة العالمية لتشخيص الفيروسات المنقولة بالمفصليات.رينال سكويرز، كريستوفر أوكسنفورد، سيباستيان كوجنات، فرانك كونينجس.تشكل الفيروسات المنقولة بالمفصليات مثل فيروس حمى الضنك، وفيروس الحمى الصفراء، وفيروس زيكا، وفيروس الشيكونجونيا، تهديدات رئيسية لصحة الإنسان على الصعيد العالمي بما في ذلك بلدان إقليم شرق المتوسط.هدفت هذه الدراسة إلى تقييم الكفاءة المختبرية في بلدان إقليم شرق المتوسط للكشف عن فيروس حمى الضنك وفيروس الحمى الصفراء وفيروس زيكا وفيروس الشيكونجونيا.أُعد ونُفذ برنامج عالمي لتقييم الجودة الخارجية لتشخيص الفيروسات المنقولة بالمفصليات في عامي 2016 و 2018. وصدرت تعليمات إلى مختبرات الصحة العامة على الصعيد الوطني بتطبيق طريقة التفاعل السلسي للبوليميراز (PCR) على مجموعة عينات تحتوي على فيروس حمى الضنك، وفيروس الحمى الصفراء، وفيروس زيكا، وفيروس الشيكونجونيا.خلال جولتي البرنامج العالمي اكتشفت المختبرات المشاركة في التقييم فيروس الحمى الصفراء وفيروس الشيكونجونيا اكتشافًا صحيحًا بنسبة %100، واكتشفت فيروس حمى الضنك بنسبة أكبر من أو يساوي %84.6، واكتشفت فيروس زيكا بنسبة %76.9.برغم أن بلدان إقليم شرق المتوسط المشاركة أظهرت إتقانًا جيدًا في الكشف عن الفيروسات المنقولة بالمفصليات، فقد سُجِّل نصفها فقط في برنامج تقييم الجودة الخارجية العالمي، وهذا يعرض صورة غير مكتملة لقدرات الإقليم. وينبغي بذل الجهود لزيادة المشاركة في الجولات التالية.Performance des laboratoires de la Région de la Méditerranée orientale dans le cadre du programme d’évaluation externe de la qualité de l’Organisation mondiale de la Santé en matière de diagnostic des arbovirus.Les arbovirus tels que le virus de la dengue, le virus de la fièvre jaune, le virus Zika et le virus chikungunya constituent des menaces majeures pour la santé humaine dans le monde, y compris dans les pays de la Région de la Méditerranée orientale.La présente étude visait à évaluer l’aptitude des laboratoires dans les pays de la Région pour la détection des virus susmentionnés.Un programme mondial d’évaluation externe de la qualité pour le diagnostic des arbovirus a été mis au point et adopté en 2016 et 2018. Les laboratoires de santé publique nationaux ont reçu l’instruction d’appliquer la méthode de détection par amplification génique sur des panels d’échantillons de laboratoires contenant le virus de la dengue, le virus de la fièvre jaune, le virus Zika et le virus chikungunya.Au cours des deux cycles du programme, 100 % des laboratoires participants de la Région de la Méditerranéenne orientale ont correctement détecté le virus de la fièvre jaune et le virus du chikungunya, un pourcentage supérieur ou égal à 84,6 % ont détecté le virus de la dengue et un pourcentage supérieur ou égal à 76,9 % de ces laboratoires ont détecté le virus Zika.Alors que les pays participants de la Région de la Méditerranée orientale ont démontré une bonne maîtrise de la détection des arbovirus, seulement la moitié d’entre eux étaient inscrits au programme mondial d’évaluation externe de la qualité, fournissant une image incomplète de la capacité régionale. Des efforts devraient être déployés pour accroître la participation aux cycles suivants.

Objective: To conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013. Methods: Twenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies. Results: Among 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT-PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT-PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT-PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories. Discussion: Accurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA.

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.

Numerous early events in anthrax lethal toxin (LT)-mediated cell killing have been described, including uptake of LT and MAPKK cleavage. However, critical downstream events in LT killing remain to be identified. In this study we show that LT cause mitochondrial dysfunction in murine J774A.1 macrophages, as indicated by a continuous drop in both mitochondrial membrane potential and SDH activity over 90 min of LT exposure. This was further supported by ultrastructural analysis revealing LT-induced swelling of mitochondria. Mitochondrial impairment and cytolysis were controlled by proteasomes: Proteasome inhibitors restored mitochondrial activity and rescued cells from cytolysis, even when added immediately prior to membrane perturbation, suggesting a link between mitochondrial impairment and cytolysis. The addition of KCl delayed LT-mediated cytolysis, but could not block mitochondrial impairment or rescue cells. KCl treatment precluded events linked to cytolysis, including a precipitous drop in ATP levels ...

HIV-1-infected monocyte/macrophages located in lymph nodes and tissues are highly productive sources of HIV-1 and may function as a persistent reservoir contributing to the rebound viremia observed after highly active antiretroviral therapy is stopped. Mechanisms activating latently infected, primary monocyte/macrophages to produce HIV-1 were investigated using monocytes isolated from a transgenic mouse line carrying a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF), regulated by the endogenous long terminal repeat (LTR) (JR-CSF mice). Granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) induced infectious HIV-1 production by JR-CSF mouse monocytes over 10-fold and 100-fold higher than that stimulated by GM-CSF or LPS alone, respectively. We examined mechanisms of GM-CSF synergy with LPS and demonstrated that GM-CSF up-regulated the LPS receptor, TLR-4, and also synergized with LPS to activate mitogen-activated protein (MAP) kinase/ERK kinase and the Sp1 transcription factor. Inhibitors of either MAP kinase/ERK kinase or p38 kinase but not PI 3-kinase potently suppressed GM-CSF and LPS-induced HIV-1 production by JR-CSF mouse monocytes. Because Sp1 is activated by both the MAP kinase/ERK kinase and p38 kinase pathways, we postulate that synergistic activation of these pathways by GM-CSF and LPS induced sufficient levels of Sp1 to activate the HIV-1 LTR in a Tat-independent manner and induced HIV-1 production by JR-CSF mouse monocytes. Thus, our study delineated the pathway of HIV-1 LTR activation by GM-CSF and LPS and indicated that JR-CSF transgenic mice may provide a new in vitro and in vivo system for investigating the mechanism by which inflammatory and infectious stimuli activate HIV-1 production from latently infected monocytes.

Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.

Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent.

Numerous early events in anthrax lethal toxin (LT)-mediated cell killing have been described, including uptake of LT and MAPKK cleavage. However, critical downstream events in LT killing remain to be identified. In this study we present evidence that LT causes mitochondrial dysfunction in murine J774A.1 macrophages, as indicated by a continuous drop in both mitochondrial membrane potential and SDH activity. This was further supported by ultrastructural analysis revealing LT-induced swelling of mitochondria. Mitochondrial impairment and cytolysis were controlled by proteasomes in LT-treated macrophages: proteasome inhibitors restored mitochondrial activity and rescued cells from cytolysis, even when added immediately prior to membrane perturbation. Similar to proteasome inhibitors, KCl also efficiently blocked LT-mediated cytolysis, even after late addition. However, KCl did not prevent mitochondrial impairment, though it precluded events linked to LT-induced cytolysis. These events included a precipitous drop in ATP levels and ubiquitinated proteins, revealing that they are epiphenomena in LT killing. Our studies suggest that proteasomes and potassium control LT-induced mitochondrial dysfunction and membrane perturbation, key events in LT killing.