Critical Care, Critical Labs

Sepsis is a medical emergency and a global public health concern. The Surviving Sepsis Campaign started in 2012 and has since issued International Guidelines for Management of Sepsis and Septic Shock. These Guidelines have been updated several times, and the 4th edition of the 2016 guideline have been issued. The Guidelines are written from the perspective of developed (“resource-rich”) countries, where critical care settings are equipped with tools for managing these patients. Yet, the developing world carries the greatest burden of sepsis-related mortality. Unfortunately, the developing world lacks access to many of the necessary tools for managing the critically ill patient – including basic laboratory testing.

Laboratory values are a significant part of the management of the septic patient. Take a look at the sepsis screening tool. Analytes and lab tests included in screening patients for sepsis include: lactate, creatinine, bilirubin, INR, and blood gases. The Surviving Sepsis bundles require a lactate concentration within 3 hours of presentation, and a subsequent lactate within 6 hours. The care bundle also requires a blood culture within 3 hours of presentation and prior to administration of antibiotics. Early-goal directed therapy for sepsis requires administration of crystalloid based on lactate concentrations. Basics of laboratories in the US, lactate and blood cultures are both difficult to obtain and far from routine in the resource-poor care settings.

Blood gases and lactate are particularly difficult to find and to maintain in the developing world. While there are a number of point-of-care or small benchtop devices – like the iStat (Abbott), the Piccolo (Abaxis), and the Stat Profile pHOx (Nova), it is often cost-prohibitive to maintain these devices.  The iStat and the Piccolo are examples of cartridge-based devices. All of the chemistry takes place in single-use cartridges and the device itself is basically a timer. In my experience, cartridge based devices hold up in environmental extremes better than open reagent systems. However, they are not cheap and this can be prohibitive. Cost of a single cartridge can range from $3-10 USD. In countries where patients and their families are expected to pay upfront or as they go for even inpatient medical care, and the income for a family is $2USD/day, routine monitoring of blood gases and lactate by cartridge is just not feasible. Reagent based devices like the Stat Profile use cartons of reagent for many uses. This is much cheaper – if all the reagent is used before it expires! Some healthcare settings can accommodate only 1-3 critical patients, and might not be able to use a whole carton before the expiry, even when adhering to Surviving Sepsis guidelines.

Blood cultures and subsequent treatment with appropriate antibiotics is a large part of the surviving sepsis campaign. Microbiology in the developing world is often limited to a few reference laboratories in country. Also, the number of potential infectious agents is larger in the developing world where diseases like malaria and dengue fever are common. Multiplexed nucleic acid tests might fill the gap here. Again, the cost is a major factor. Just reagents alone for a single multiplexed NAT can be over $250 USD.

In short, if the surviving sepsis guidelines really do help decrease sepsis mortality, the developing world doesn’t have a chance unless it has a greater laboratory capacity. Basic labs that we don’t think twice about can be very hard to come by in resource-poor environments. The tests already exist in forms that can be used in resource-poor settings – they just need to be cheaper, at least for those in limited resource settings. Are you listening, Abbott?


Sarah Brown Headshot_small

Sarah Riley, PhD, DABCC, is an Assistant Professor of Pediatrics and Pathology and Immunology at Washington University in St. Louis School of Medicine. She is passionate about bringing the lab out of the basement and into the forefront of global health.  

Microbiology Case Study: A 75-Year-Old Man with Polymicrobial Bacteremia After Hemicolectomy

Case History

A 75-year-old male with a past medical history of hypertension, hyperlipidemia, and benign prostatic hyperplasia underwent an elective right hemicolectomy at an outside hospital after a cecal polypectomy demonstrated an intramucosal adenocarcinoma (in situ) arising in a background of a sessile serrated adenoma. On post-op day 6, he was transferred to our institution for management of an ST-elevation myocardial infarction that was treated with placement of a drug-eluting stent to the right coronary artery. After the cardiac catheterization, he complained of acute-onset abdominal pain and was tachypneic (49/min), hypotensive (72/48 mmHg), and febrile (39.4°C). He was emergently intubated, given vasopressors, and started on vancomycin and piperacillin/tazobactam empirically for septic shock. A chest X-ray showed atelectasis but no pulmonary consolidation. An abdominal X-ray did not show definitive evidence of pneumoperitoneum and abdominal CT showed some free fluid but no acute abdominal pathology. The WBC count was 3,640/cm3 with an absolute neutrophil count (2,880/cm3) within normal limits. The anaerobic bottle in one of two blood culture sets drawn on post-op day 7 became positive at 27 hours and Gram staining (Image 1) demonstrated gram negative bacilli. Subsequently, the bacilli detected in the anaerobic blood culture bottle were identified by MALDI-TOF as Clostridium clostridioforme, requiring a laboratory corrected report. On post-op day 8, two sets of repeat blood cultures were both positive with Clostridium tertium (Images 2 and 3) and Escherichia coli, consistent with bowel flora. Therapy for the patient’s polymicrobial bacteremia, thought to arise from an ileocolic anastomotic leak, was switched to piperacillin/tazobactam and Metronidazole. Blood cultures on post-op days 10 and 14 were negative. Meanwhile, the patient developed diarrhea, secondary to Clostridium difficile colitis, treated with oral vancomycin and oral thrush treated with micafungin. His hospital course was further complicated by formation of intra-abdominal abscesses, containing E. coli, C. tertium, and C. albicans, that required percutaneous drain placement.


Image 1. Gram stain of Clostridium clostridioforme from a positive anaerobic blood culture bottle demonstrates thin gram negative bacilli with pointed ends arranged in pairs (100x, oil immersion).


Image 2. Gram stain of Clostridium tertium from a positive anaerobic blood culture bottle demonstrates gram variable bacilli arranged in short chains (100x, oil immersion).


Image 3. Clostridium tertium colonies are β-hemolytic on an anaerobic (Schaedler) blood agar plate and appear circular with slightly irregular margins, matte, and grey-white.


The genus Clostridium contains approximately 200 species, of which approximately 32 have been associated with human pathologies (1). These organisms are normal members of the human gastrointestinal and cervical-vaginal microflora. Clostridia are also ubiquitously present in nature within soil. Thus, human infection may occur via endogenous or exogenous means. They are classified as gram positive rods and, as such, they do not grow on media that inhibit the growth of gram positive organisms (ie. MacConkey agar). However, upon gram staining, Clostridia may appear gram positive, gram variable, or gram negative. Due to the gram stain variability, inconsistent presence of spores, and atypical colony morphologies, laboratory identification of Clostridum species is problematic.

Clostridium clostridioforme was initially detected in the anaerobic blood culture bottle at 27 hours. Gram staining (Image 1) demonstrates gram negative long, thin bacilli with pointed ends, described as “elongated football shaped” that are arranged in pairs but may also lie singly or in short chains. Oval spores may not be seen but they can be central or subterminal. As obligate anaerobes, C. clostridioforme may be cultured on anaerobic blood agar plates where the gamma-hemolytic colonies appear small, convex to slightly peaked, translucent to opaque, and grey-white. They possess peritrichous flagella that confer motility. It is believed that C. clostridioforme may represent three different species that are frequently isolated anaerobically from blood cultures, particularly in association with mixed cultures, typical of colonic flora (2).

Subsequent blood cultures one day later were positive for both Escherichia coli (detected at 18 hours) and Clostridium tertium (detected at 21 hours). The anaerobic blood culture bottle gram stain (Image 2) demonstrates C. tertium staining as gram variable bacilli arranged in short chains. Terminal spores, only produced under anaerobic conditions, are not seen in Figure 2. C. tertium is one of the aerotolerant clostridia and was cultured on an anaerobic blood agar plate (Figure 3). Colonies appear circular with slightly irregular margins, low convex, matte, and grey-white. Hemolysis can be beta, alpha, or gamma. It was likely overgrown by the E. coli on the aerobic plates. This species is generally considered a weak human pathogen but it has been implicated as a cause of bacteremia in immunocompromised patients. In non-neutropenic patients, C. tertium bacteremia can occur in the setting of gastrointestinal mucosal injury due to gastrointestinal tract pathology or surgery (3).


  1. Tille PM. Bailey & Scott’s Diagnostic Microbiology, 13th ed. Elsevier Health Sciences; 2014. pp458-479.
  2. Finegold SM, Song Y, Liu C, et al. Clostridium clostridioforme: a mixture of three clinically important species. Eur J Clin Microbiol Infect Dis. 2005;24(5):319-24.
  3. Miller DL, Brazer S, Murdoch D, Reller LB, Corey GR. Significance of Clostridium tertium bacteremia in neutropenic and nonneutropenic patients: review of 32 cases. Clin Infect Dis. 2001;32(6):975-8.


-Adina Bodolan, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.


-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.



Microbiology Case Study: A 62 Year Old Male with Coronary Artery Disease

A 62 year old male with a past medical history of CAD, CABG x 4, HTN, DMII, OSA on CPAP, and GERD was admitted for acute onset of chest pressure that radiated to his back. He also complained of nausea, vomiting. He had a similar episode of pain two weeks ago which resolved with nitroglycerin. The patient was found to have Type 1A aortic dissection on CTA. Decision was made to proceed to the OR emergently. Status post the operation, he continued to have hemodynamic instability and evidence of pneumonia. He had been intermittently febrile with leukocytosis (WBC=12.66). Blood cultures were drawn and were positive for gram negative bacilli in one bottle.

Gram stain demonstrating Gram-negative rods.
Gram stain demonstrating Gram-negative rods.
Blood agar plate with dry, yellow colonies.
Blood agar plate with dry, yellow colonies.


Pseudomonas luteola was identified on the MALDI-TOF.

P. luteola was originally identified as Chryseomonas, but later changed to be a part of the Pseudomonas family. It is an opportunistic pathogen found in damp environments. It is a gram negative rod of 0.8 μm to 2.5 μm and is a motileaerobe. Its motility is created by multitrichous flagella. Colonies produce a yellow-orange pigment. P. luteola can be differentiated from most other motile yellow-pigmented nonfermenters by a negative oxidase reaction and from the Enterobacteriaceae by its strict aerobic growth. Optimal temperature for growth is 30°C, although it can grow at 42°C and not at 5°C. It grows best on heart infusion agar supplemented with 5% horse blood, but is also able to grow on TSA, Nutrient Agar, MacConkey or CASA Agar. The pathogenic form of P. luteola is a saprophyte and it can cause septicemia, peritonitis, endocarditis in patients with health disorders or with indwelling devices, and meningitis. Most strains are susceptible to broad-spectrum antibiotics, such as cephalosporins and ciprofloxacin.

Based on the history, the clinical team was unsure if it was a false positive/contaminant or truly a pathogen. The patient did have grafts and bioprosthetic material and due to the virulence of Pseudomonas, they decided to treat with cefepime and remove the central line. The patient clinically improved after removal of the line, which favored a line infection.

-Mustafa Mohammed, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.


-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Microbiology Case Study: Young Girl with Community-Acquired Pneumonia

A young girl presented to the emergency department with 6 days of cough, congestion, and intermittent fever up to 102°F. Respiratory viral testing, blood cultures, and a chest x-ray were ordered. The patient was found to be RSV positive and sent home with oral steroids. Two days later she presented the emergency department once again with worsening respiratory symptoms and a positive blood culture with the following organism (Figure 1).

Figure 1. Gram stain demonstrating Gram-positive cocci in pairs and chains.

Our patient developed a Streptococcus pneumoniae superinfection and bacteremia in conjunction with RSV pneumonia.


S. pneumoniae is a Gram-positive cocci that forms “lancet” shaped pairs on Gram stain (Figure 1). Due to pretreatment with antibiotics, our Gram stain shows some pairs, but many Gram variable chains of cocci as well. S. pneumoniae grows as alpha hemolytic colonies on 5% sheep blood, chocolate, and CAN (colistin nalidixic acid) agar in 12-18 hours, where it forms umbelicated colonies with a characteristic navel-like depression in the middle due to autolysins produced by the bacterium. Some serotypes of S. pneumoniae, primarily serotype 3, have a mucoid phenotype seen in Figure 3. S. pneumoniae is a member of the Streptococcus mitis group, but due to its pathogenic potential it has always been singled out. This is accomplished using two biochemical tests: bile solubility testing with 10% deoxycholate, which dissolves colonies of S. pneumoniae but not those of other Viridans group streptococci, and optochin disc testing, to which S. pneumoniae is sensitive while other Virdians group streptococci are resistant (Figures 2 and 3). Many molecular assays have trouble differentiating S. pneumoniae from S. mitis group due to their similarities on a nucleotide and protein level, so biochemical testing is still a mainstay of organism identification.

Figure 2. Growth of α-hemolytic bacterial colonies on 5% sheep blood agar. Zone around the disc indicates the organism is optochin susceptible.


Figure 3. Growth of mucoid, α-hemolytic bacterial colonies on 5% sheep blood agar. The mucoid colony morphology suggests this isolate is likely serotype 3.

Clinical Significance

S. pneumoniae is known to cause a variety of clinical manifestations in children, from community acquired pneumonia and acute otitis media to bacteremia and meningitis. S. pneumoniae is also a colonizer of the upper respiratory tract; approximately 21% of children in developed countries and 90% of children in developing countries are asymptotically colonized. Due to the high rates of S. pneumoniae colonization in children, direct urine antigen testing is inappropriate, as it cannot distinguish asymptomatic carriage from invasive disease. S. pneumoniae direct antigen detection from CSF has been shown to have < 30% sensitivity and offers no benefit over a routine cytospin Gram stain.

Vaccination in children

Around 2000 the first S. pneumoniae vaccine became available. PCV7 was a heptavalent conjugate vaccine which provided protection from the 7 most common S. pneumoniae serotypes known to cause invasive disease (4, 6B, 9V, 14, 18C, 19F, and 23F). Routine vaccination of children was a huge success which reduced the incidence of invasive pneumococcal disease attributed to vaccine strains by 99%. An indirect benefit of the PCV7 vaccine was that adults >65 years of age saw a 92% decrease in invasive pneumococcal disease caused by PCV7 serotypes, despite not being vaccinated themselves, because of reduced transmission of S. pneumoniae from children to adults. Due to the selective pressure of the vaccine, non-vaccine serotypes of S. pneumoniae such as 19A subsequently became the predominant causes of invasive streptococcal disease. In 2010, a 13-valent pneumococcal conjugate vaccine (PCV13) was FDA approved. It includes all seven S. pneumoniae serotypes contained in PCV7, plus six additional serotypes (1, 3, 5, 6A, 7F, and 19A). PCV13 provides coverage against 2/3 of all serotypes responsible for invasive pneumococcal disease in children under 5 years of age.

Follow up

The patient had an uneventful hospital stay. All subsequent blood cultures were negative and susceptibility testing found the patient’s S. pneumoniae isolate to be susceptible to penicillin, cefotaxime, and clindamycin. The patient and was discharged home after 24 hours of observation with a 7 day course of amoxicillin.



  • Manual of Clinical Microbiology, 11th edition
  • Pediatric Red Book, 2015 Report of the Committee on Infectious Diseases, 30th edition


-Erin McElvania TeKippe, Ph.D., D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

Procalcitonin: Sepsis Marker Extraordinaire?

Sepsis is one of the most common causes of significant morbidity and mortality in hospitalized patients as well as the most common cause of death in ICU patients.  In addition, the earlier sepsis is identified and treated, the better the prognosis for the patient. We actually do not have a biochemical marker which can be used to effectively diagnose sepsis. Sepsis diagnosis depends on finding microbial infection by culture, and while PCR methods do exist to quickly identify bacteremia, in most institutions cultures take at least 24 hours to grow.  To aid in the diagnosis, clinicians can check three biomarkers commonly considered “sepsis” markers: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and procalcitonin (PCT).

Despite being very different tests, these three assays are ultimately indicators of inflammation or the inflammatory response. ESR is a simple manual test that measures how far red cells sediment out of a blood sample in one hour. It is used as a marker of inflammation but is quite unspecific; several conditions can cause inflammation. The ESR can tell a clinician that inflammation exists but not the cause of that inflammation CRP is an acute phase reactant protein. Its production by the liver increases in acute inflammation. However, its levels will be affected by liver dysfunction. PCT is a pro-hormone produced by extra-thyroidal immune cells within 2-4 hours of a bacterial insult or an inflammatory response.

Deciding whether a biomarker is a good indicator of sepsis is made difficult by its complex pathology. Studies that show one marker performs better are contradicted by other studies that show it does not. The utility of PCT for predicting sepsis remains controversial for this reason. However PCT has shown to be useful for predicting prognosis in sepsis. Increasing PCT concentrations correlate with increasing severity and a poor prognosis. Decreasing or low concentrations indicate a good prognosis. PCT is also being used to guide antibiotic therapy, although this use should be limited to non-surgical/trauma ICU patients, which is where the studies have been done. Thus although PCT proponents consider it to be the best available biomarker indicator of sepsis, none of the three tests have been shown to be good at diagnosing sepsis. Unfortunately, all three of these biomarkers are indicative of an inflammatory response and not specific for sepsis itself. However, once sepsis is known, all three biomarkers can be used to monitor its progression and response to therapy.

If you’d like to read more about PCT and sepsis, you can do so here:



-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.