The host-directed therapeutic imatinib mesylate accelerates immune responses to Mycobacterium marinum infection and limits pathology associated with granulomas.

Infections caused by members of the mycobacterium tuberculosis complex [MTC] and nontuberculous mycobacteria [NTM] can induce widespread morbidity and mortality in people. Mycobacterial infections cause both a delayed immune response, which limits rate of bacterial clearance, and formation of granulomas, which contain bacterial spread, but also contribute to lung damage, fibrosis, and morbidity. Granulomas also limit access of antibiotics to bacteria, which may facilitate development of resistance. Bacteria resistant to some or all antibiotics cause significant morbidity and mortality, and newly developed antibiotics readily engender resistance, highlighting the need for new therapeutic approaches. Imatinib mesylate, a cancer drug used to treat chronic myelogenous leukemia [CML] that targets Abl and related tyrosine kinases, is a possible host-directed therapeutic [HDT] for mycobacterial infections, including those causing TB. Here, we use the murine Mycobacterium marinum [Mm] infection model, which induces granulomatous tail lesions. Based on histological measurements, imatinib reduces both lesion size and inflammation of surrounding tissue. Transcriptomic analysis of tail lesions indicates that imatinib induces gene signatures indicative of immune activation and regulation at early time points post infection that resemble those seen at later ones, suggesting that imatinib accelerates but does not substantially alter anti-mycobacterial immune responses. Imatinib likewise induces signatures associated with cell death and promotes survival of bone marrow-derived macrophages [BMDMs] in culture following infection with Mm. Notably, the capacity of imatinib to limit formation and growth of granulomas in vivo and to promote survival of BMDMs in vitro depends upon caspase 8, a key regulator of cell survival and death. These data provide evidence for the utility of imatinib as an HDT for mycobacterial infections in accelerating and regulating immune responses, and limiting pathology associated with granulomas, which may mitigate post-treatment morbidity.

Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS-CoV-2 infection and COVID-19 disease severity.

Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS-CoV-2 infection. Numerous clinical studies have subsequently reported that individuals with CF in North America and Europe-while susceptible to severe COVID-19-are often spared from the highest levels of virus-associated mortality. To understand features that might influence COVID-19 among patients with cystic fibrosis, we studied relationships between SARS-CoV-2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID-19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant-a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS-CoV-2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus-obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID-19.

One Health: The Role of Pathology as it Pertains to Diagnosis of Zoonoses and Discovery of Emerging Infections.

Pathologists are an integral part of One Health as they are a critical component of the multidisciplinary team that diagnoses zoonotic diseases and discovers emerging pathogens. Both human and veterinary pathologists are uniquely positioned to identify clusters or trends in patient populations that can be caused by an infectious agent and preface emerging outbreaks. The repository of tissue samples available to pathologists is an invaluable resource that can be used to investigate a variety of pathogens. One Health is an encompassing approach that focuses on optimizing the health of humans, animals (domesticated and sylvatic), and the ecosystem, including plants, water, and vectors. In this integrated and balanced approach, multiple disciplines and sectors from local and global communities work together to promote overall well-being of the 3 components and address threats such as emerging infectious diseases and zoonoses. Zoonoses are defined as infectious diseases that are spread between animals and humans through different mechanisms, including direct contact, food, water, vectors, or fomites. This review highlights examples in which human and veterinary pathologists were an integral part of the multisectoral team that identified uncommon etiologic agents or pathologies that had not been elucidated clinically. As the team discovers an emerging infectious disease, pathologists develop and validate tests for epidemiologic and clinical use and provide surveillance data on these diseases. They define the pathogenesis and pathology that these new diseases cause. This review also presents examples that demonstrate the crucial role pathologists play in diagnosing zoonoses that have an impact on the food supply and the economy.

Emerging Antimicrobial Resistance.

The burden of emerging antimicrobial resistance (AMR) in the United States is significant and even greater worldwide. Mitigation efforts have decreased the incidence and deaths from antimicrobial-resistant organisms in the United States. Yet more than 2.8 million antimicrobial-resistant infections occur every year and more than 35,000 patients die as a result. Infection prevention and control, data tracking, antimicrobial stewardship, vaccines, therapeutics, diagnostics, and sanitation are all required to decrease AMR threats. In 2019, in the second version of the Centers for Disease Control and Prevention (CDC) report on antibiotic-resistant threats, the agency categorized AMR threats as urgent, serious, concerning, or to be watched. This review will discuss the following aspects of each bacterium in the CDC report: estimated numbers of cases and deaths, identify the better known and impactful mechanisms of resistance, diagnostic testing and its limitations, and current and possible future therapies. This review also presents anatomical pathology case examples that highlight the altered morphology of antibiotic partially treated bacteria in tissues.

Nucleocapsid Antigenemia Is a Marker of Acute SARS-CoV-2 Infection.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19.

Infections Caused by Free-Living Amoebae.

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris result in a variety of clinical manifestations in humans. These amoebae are found in water and soil worldwide. Acanthamoeba spp. and B. mandrillaris cause granulomatous amoebic encephalitis (GAE), which usually presents as a mass, while N. fowleri causes primary amoebic meningoencephalitis (PAM). Acanthamoeba spp. can also cause keratitis, and both Acanthamoeba spp. and B. mandrillaris can cause lesions in skin and respiratory mucosa. These amoebae can be difficult to diagnose clinically as these infections are rare and, if not suspected, can be misdiagnosed with other more common diseases. Microscopy continues to be the key first step in diagnosis, but the amoeba can be confused with macrophages or other infectious agents if an expert in infectious disease pathology or clinical microbiology is not consulted. Although molecular methods can be helpful in establishing the diagnosis, these are only available in referral centers. Treatment requires combination of antibiotics and antifungals and, even with prompt diagnosis and treatment, the mortality for neurological disease is extremely high.

Comparison of Antibody Class-Specific SARS-CoV-2 Serologies for the Diagnosis of Acute COVID-19.

Accurate diagnosis of acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical for appropriate management of patients with this disease. We examined the possible complementary role of laboratory-developed class-specific clinical serology in assessing SARS-CoV-2 infection in hospitalized patients. Serological tests for immunoglobulin G (IgG), IgA, and IgM antibodies against the receptor binding domain (RBD) of SARS-CoV-2 were evaluated using samples from real-time reverse transcription-quantitative PCR (qRT-PCR)-confirmed inpatient coronavirus disease 2019 (COVID-19) cases. We analyzed the influence of timing and clinical severity on the diagnostic value of class-specific COVID-19 serology testing. Cross-sectional analysis revealed higher sensitivity and specificity at lower optical density cutoffs for IgA in hospitalized patients than for IgG and IgM serology (IgG area under the curve [AUC] of 0.91 [95% confidence interval {CI}, 0.89 to 0.93] versus IgA AUC of 0.97 [95% CI, 0.96 to 0.98] versus IgM AUC of 0.95 [95% CI, 0.92 to 0.97]). The enhanced performance of IgA serology was apparent in the first 2 weeks after symptom onset and the first week after PCR testing. In patients requiring intubation, all three tests exhibit enhanced sensitivity. Among PCR-negative patients under investigation for SARS-CoV-2 infection, 2 out of 61 showed clear evidence of seroconversion IgG, IgA, and IgM. Suspected false-positive results in the latter population were most frequently observed in IgG and IgM serology tests. Our findings suggest the potential utility of IgA serology in the acute setting and explore the benefits and limitations of class-specific serology as a complementary diagnostic tool to PCR for COVID-19 in the acute setting.

Therapeutic plasma exchange for COVID-19-associated hyperviscosity.

BACKGROUND: Recent data suggests an association between blood hyperviscosity and both propensity for thrombosis and disease severity in patients with COVID-19. This raises the possibility that increased viscosity may contribute to endothelial damage and multiorgan failure in COVID-19, and that therapeutic plasma exchange (TPE) to decrease viscosity may improve patient outcomes. Here we sought to share our experience using TPE in the first 6 patients treated for COVID-19-associated hyperviscosity. STUDY DESIGN AND METHODS: Six critically ill COVID-19 patients with plasma viscosity levels ranging from 2.6 to 4.2 centipoise (cP; normal range, 1.4-1.8 cP) underwent daily TPE for 2-3 treatments. RESULTS: TPE decreased plasma viscosity in all six patients (Pre-TPE median 3.75 cP, range 2.6-4.2 cP; Post-TPE median 1.6 cP, range 1.5-1.9 cP). TPE also decreased fibrinogen levels in all five patients for whom results were available (Pre-TPE median 739 mg/dL, range 601-1188 mg/dL; Post-TPE median 359 mg/dL, range 235-461 mg/dL); D-dimer levels in all six patients (Pre-TPE median 5921 ng/mL, range 1134-60 000 ng/mL; Post-TPE median 4893 ng/mL, range 620-7518 ng/mL); and CRP levels in five of six patients (Pre-TPE median 292 mg/L, range 136-329 mg/L; Post-TPE median 84 mg/L, range 31-211 mg/L). While the two sickest patients died, significant improvement in clinical status was observed in four of six patients shortly after TPE. CONCLUSIONS: This series demonstrates the utility of TPE to rapidly correct increased blood viscosity in patients with COVID-19-associated hyperviscosity. Large randomized trials are needed to determine whether TPE may improve clinical outcomes for patients with COVID-19.

Adenovirus causing hepatic abscess formation and unexplained fever in adult liver transplant recipients.

Adenovirus infection is commonly associated with self-limited respiratory and gastrointestinal illnesses. However, infection in immunocompromised individuals, such as transplant recipients, can cause severe life-threatening illness including pneumonitis, hemorrhagic cystitis, nephritis, hepatitis, and enterocolitis. In orthotopic liver transplant recipients, adenovirus viremia can cause hepatitis leading to marked transaminitis, allograft loss, and death. Although hepatic abscesses mediated by adenovirus have been described in other immunosuppressed patient populations, it has very rarely been described in liver transplant recipients. Here, we report two adult cases of hepatic abscesses following liver transplantation secondary to adenovirus infection and describe the successful treatment of these patients. Adenovirus should be considered as an uncommon etiology of hepatic abscess and unexplained fevers in adults following liver transplantation.

Electronic charting of transfusion medicine consults: implementation, challenges and opportunities.

BACKGROUND: The Joint Commission lists improving staff communication (handoffs) as part of several National Safety Goals. In this study, we developed an electronic web-based charting system for clinical pathology handoffs, which primarily consist of transfusion medicine calls, and evaluated the advantages over a paper-based handwritten call log. MATERIALS AND METHODS: A secure online web browser application using Research Electronic Data Capture (REDCap) was designed to document on-call pathology resident consults. A year after implementation, an online survey was administered to our pathology residents in order to evaluate and compare the usability of the electronic application (e-consults) to the previous handwritten call log, which was a notebook where trainees hand wrote different components of the consult. RESULTS: The REDCap web-based application includes discrete fields for patients' information, requesting physician contact, type of consult, action items for follow-up and faculty responses, as well as other information. These components have eventually progressed to be an online consult call catalog. With approximately 1079 consults per year, transfusion medicine-related calls account for ~90% of the encounters, while clinical chemistry, microbiology and immunology calls constitute the remainder. The overall response rate of the survey was 96% (29 of 30 participants). Of the 16 respondents who experienced both call log systems, 100% responded that REDCap was an improvement over the handwritten call log (P < 0·0001). CONCLUSION: E-consult documentation entered into a web-based application was a user-friendly, secure clinical information access and effective handoff system as compared to a paper-based handwritten call log.

Circulating inflammation-related markers and advanced gastric premalignant lesions.

BACKGROUND AND AIM: Chronic Helicobacter pylori infection causes gastric mucosal inflammation as an important antecedent of gastric cancer. We aimed to evaluate associations of blood markers of inflammation with gastric intestinal metaplasia and dysplasia in H. pylori-infected individuals. METHODS: We compared pre-treatment serum levels of immune-related and inflammation-related markers between 99 individuals with intestinal metaplasia or dysplasia and 75 control individuals with non-atrophic gastritis within an H. pylori eradication trial in Mexico. Serum levels of 28 markers measured with Luminex bead-based assays were categorized in tertiles as low (T1), middle (T2), and high (T3). Logistic regression models were used to calculate age-adjusted and sex-adjusted odds ratios and 95% confidence intervals. All statistical tests were two-sided, and significance values were adjusted for multiple comparisons using false discovery rate methods. RESULTS: Five markers were nominally associated (Ptrend  < 0.05) with the presence of advanced premalignant gastric lesions. Adjusted odds ratios (95% confidence interval) of T2 and T3 versus T1 were 4.09 (1.65-10.17) and 3.08 (1.23-7.68) for CCL3/MIP1A, 3.21 (1.33-7.75) and 2.69 (1.10-6.57) for CCL20/MIP3A levels, 1.79 (0.77-4.18) and 2.39 (1.02-5.60) for IL-1ß, 1.34 (0.56-3.19) and 3.02 (1.29-7.12) for IL-4, and 1.07 (0.44-2.59) and 3.07 (1.32-7.14) for IL-5, respectively. Two (IL-4 and IL-5) of the five markers had false discovery rate adjusted Ptrend  < 0.2. CONCLUSIONS: Our results suggest that certain Th2 and other cytokines may have a role in promoting carcinogenesis in the setting of H. pylori infection. Additional research is needed to replicate these findings, extend to pre-diagnostic samples, and elucidate the underlying mechanisms.

Chagas disease as example of a reemerging parasite.

Trypanosoma cruzi, the protozoan that causes Chagas disease, is primarily transmitted by three main Triatomine vectors in endemic areas. However, the infection has become a potential emerging disease because the vector is found in non-endemic areas, there is migration of infected asymptomatic people that can infect the vector, become blood donors, or pass the disease vertically (congenital infections). Lastly, the disease can be acquired through contaminated food (oral transmission). This review will present the different transmission pathways, clinical manifestations, diagnostic modalities and treatment considerations of Chagas disease.

Introduction: One health and emerging infectious diseases.

Those infectious diseases that have increased in the past two decades or that threaten to emerge are considered emerging infectious diseases. Many of these diseases are acquired through contact with plants, insects and animals. "One health" acknowledges the interconnectedness of environment, plant, animal and human health. The introduction to this issue of Seminars in Diagnostic Pathology gives the definition of emerging and reemerging infectious diseases, explores the concept of "One Health", explains the evolution of testing for infectious agents as it relates to emerging infections, and considers the difficulties in predicting which and where the next emerging infection will occur.

Four human diseases with significant public health impact caused by mosquito-borne flaviviruses: West Nile, Zika, dengue and yellow fever.

In this review we will discuss the epidemiology, clinical characteristics, diagnostic tests, pathologic features, treatment and disease prevention strategies for four mosquito-borne flaviviruses. West Nile and Zika viruses, once thought to be restricted geographically, emerged on the American continent in the first part of the 21st century. They have been constantly in the lay press and have caused a heightened awareness of emerging infections by the public, particularly given the manifestation of microcephaly in congenital Zika syndrome. Yellow fever and dengue viruses, with mosquito vectors similar to West Nile and Zika viruses, are endemic in many tropical areas of the world and produce frequent outbreaks. The global distribution of yellow fever and dengue viruses could also change and has great potential to do so. Factors that could contribute to reemergence of the diseases in areas of the world where they are currently only seen in travelers, include the presence of yellow fever and dengue virus vectors in temperate climates and growing urbanization. These two factors increase potential contact between vectors and naïve human hosts, thus could result in reemergence of yellow fever or dengue virus infections.

Emerging and reemerging fungal infections.

Fungal infections throughout the world appear to be increasing. This may in part be due to the increase in the population of patients that are susceptible to otherwise rare fungal infections resulting from the use of immune modulating procedures such as hematopoietic stem cell transplants and drugs like tissue necrosis factor antagonists. Histoplasma capsulatum, an endemic fungus throughout North and South America, is reemerging among HIV+ patients in Central and South America and among patients taking tissue necrosis factor antagonists and other biologics in North America. Fusarium species, a relatively rare fungal infection, is reemerging worldwide in the immunocompromised populations, especially those who are neutropenic like hematopoietic stem cell transplant recipients. A new yeast species is currently emerging worldwide: Candida auris, unknown just a decade ago. It is causing large healthcare-associated outbreaks on four continents and is spreading throughout the world through patient travel. In this review the epidemiology, pathology, detection and treatment of these three emerging and reemerging fungi will be discussed.

Buruli Ulcer: Review of a Neglected Skin Mycobacterial Disease.

Buruli ulcer is caused by Mycobacterium ulcerans This neglected disease occurs in scattered foci around the world, with a higher concentration of cases in West Africa. The mycobacteria produce mycolactones that cause tissue necrosis. The disease presents as a painless skin nodule that ulcerates as necrosis expands. Finding acid-fast bacilli in smears or histopathology, culturing the mycobacteria, and performing M. ulcerans PCR in presumptive cases confirm the diagnosis. Medical treatment with oral rifampin and intramuscular streptomycin or oral treatment with rifampin plus clarithromycin for 8 weeks is supported by the World Health Organization. This review summarizes the epidemiology, pathogenesis, clinical presentation, diagnostic tests, and advances in treatment.

Moxifloxacin target site concentrations in patients with pulmonary TB utilizing microdialysis: a clinical pharmacokinetic study.

Background: Moxifloxacin is a second-line anti-TB drug that is useful in the treatment of drug-resistant TB. However, little is known about its target site pharmacokinetics. Lower drug concentrations at the infection site (i.e. in severe lung lesions including cavitary lesions) may lead to development and amplification of drug resistance. Improved knowledge regarding tissue penetration of anti-TB drugs will help guide drug development and optimize drug dosing. Methods: Patients with culture-confirmed drug-resistant pulmonary TB scheduled to undergo adjunctive surgical lung resection were enrolled in Tbilisi, Georgia. Five serum samples per patient were collected at different timepoints including at the time of surgical resection (approximately at Tmax). Microdialysis was performed in the ex vivo tissue immediately after resection. Non-compartmental analysis was performed and a tissue/serum concentration ratio was calculated. Results: Among the seven patients enrolled, the median moxifloxacin dose given was 7.7 mg/kg, the median age was 25.2 years, 57% were male and the median creatinine clearance was 95.4 mL/min. Most patients (71%) had suboptimal steady-state serum Cmax (total drug) concentrations. The median free moxifloxacin serum concentration at time of surgical resection was 1.23 µg/mL (range = 0.12-1.80) and the median free lung tissue concentration was 3.37 µg/mL (range = 0.81-5.76). The median free-tissue/free-serum concentration ratio was 3.20 (range = 0.66-28.08). Conclusions: Moxifloxacin showed excellent penetration into diseased lung tissue (including cavitary lesions) among patients with pulmonary TB. Moxifloxacin lung tissue concentrations were higher than those seen in serum. Our findings highlight the importance of moxifloxacin in the treatment of MDR-TB and potentially any patient with pulmonary TB and severe lung lesions.

Essential Diagnostics for the Use of World Health Organization Essential Medicines.

BACKGROUND: There are numerous barriers to achieving high-quality laboratory diagnostic testing in resource-limited countries. These include inconsistent supply chains, variable quality of diagnostic devices, lack of human and financial resources, the ever-growing list of available tests, and a historical reliance on syndromic treatment algorithms. A list of essential diagnostics based on an accepted standard like the WHO Essential Medicines List (EML) could coordinate stakeholders in the strengthening of laboratory capacity globally. METHODS: To aid in the creation of an essential diagnostics list (EDL), we identified laboratory test indications from expert databases for the safe and effective use of WHO EML medicines. In all, 446 EML medicines were included in the study. We identified 279 conditions targeted by these medicines, spanning communicable and noncommunicable diseases (e.g., HIV, diabetes mellitus). RESULTS: We found 325 unique diagnostic tests, across 2717 indications, associated with the identified conditions or their associated medicines. The indications were divided into 10 categories: toxicity (865), diagnosis (591), monitoring (379), dosing/safety (325), complications (217), pathophysiology (154), differential diagnosis (97), comorbidities (53), drug-susceptibility testing (22), and companion diagnostic testing (14). We also created a sublist of 74 higher-priority tests to help define the core of the EDL. CONCLUSIONS: An EDL such as we describe here could align the global health community to solve the problems impeding equitable access to high-quality diagnostic testing in support of the global health agenda.

Lung Tissue Concentrations of Pyrazinamide among Patients with Drug-Resistant Pulmonary Tuberculosis.

Improved knowledge regarding the tissue penetration of antituberculosis drugs may help optimize drug management. Patients with drug-resistant pulmonary tuberculosis undergoing adjunctive surgery were enrolled. Serial serum samples were collected, and microdialysis was performed using ex vivo lung tissue to measure pyrazinamide concentrations. Among 10 patients, the median pyrazinamide dose was 24.7 mg/kg of body weight. Imaging revealed predominant lung lesions as cavitary (n = 6 patients), mass-like (n = 3 patients), or consolidative (n = 1 patient). On histopathology examination, all tissue samples had necrosis; eight had a pH of ≤5.5. Tissue samples from two patients were positive for Mycobacterium tuberculosis by culture (pH 5.5 and 7.2). All 10 patients had maximal serum pyrazinamide concentrations within the recommended range of 20 to 60 µg/ml. The median lung tissue free pyrazinamide concentration was 20.96 µg/ml. The median tissue-to-serum pyrazinamide concentration ratio was 0.77 (range, 0.54 to 0.93). There was a significant inverse correlation between tissue pyrazinamide concentrations and the amounts of necrosis (R = -0.66, P = 0.04) and acid-fast bacilli (R = -0.75, P = 0.01) identified by histopathology. We found good penetration of pyrazinamide into lung tissue among patients with pulmonary tuberculosis with a variety of radiological lesion types. Our tissue pH results revealed that most lesions had a pH conducive to pyrazinamide activity. The tissue penetration of pyrazinamide highlights its importance in both drug-susceptible and drug-resistant antituberculosis treatment regimens.

Improving Gram stain proficiency in hospital and satellite laboratories that do not have microbiology.

BACKGROUND: Consolidation of laboratories has left many hospitals and satellite laboratories with minimal microbiologic testing. In many hospitals and satellite laboratories, Gram stains on primary specimens are still performed despite difficultly in maintaining proficiency. METHODS: To maintain Gram stain proficiency at a community 450-bed hospital with an active emergency room we designed bimonthly challenges that require reporting Gram staining and morphology of different organisms. The challenges consist of five specimens prepared by the reference microbiology laboratory from cultures and primary specimens. Twenty to 23 medical laboratory scientists participate reading the challenges. Results from the challenges are discussed with each medical laboratory scientists. In addition, printed images from the challenges are presented at huddle to add microbiology knowledge. RESULTS: On the first three challenges, Gram staining was read correctly in 71%-77% of the time while morphology 53%-66%. In the last six challenges correct answers for Gram stain were 77%-99% while morphology 73%-96%. CONCLUSIONS: We observed statistically significant improvement when reading Gram stains by providing frequent challenges to medical laboratory scientists. The clinical importance of Gram stain results is emphasized during huddle presentations increasing knowledge and motivation to perform the test for patients.