Clinical-Pathological Conference Series from the Medical University of Graz

The patient was admitted to the emergency room (ER) in a state of shock; his blood pressure was 95/45 mmHg, pulse rate 99 beats per minute, oxygen saturation 86% at room air, body temperature 36.8 °C. His skin was marbled, cyanotic and his back was livid; however, there was no rash or jaundice. Further clinical examination revealed tachycardia with rhythmic heart sounds and no murmurs, and vesicular breathing sounds in both lungs. The patient presented with abdominal guarding, diffuse abdominal pain most accentuated in the lower quadrants, livid limbs and blue acra. Electrocardiogram showed ST-elevation in leads V1–V3 and V6; echocardiography revealed significantly reduced left ventricular function and diffuse hypokinesia; there was no hemodynamically relevant valve abnormality. Coronary angiography was performed immediately and revealed a slow flow in all vessels, but no hemodynamically relevant stenosis. Since fulminant myocarditis and septic shock of unknown origin were suspected, blood cultures were taken three times and antibiotic treatment with meropenem was initiated. Computed tomography (CT) of the brain was unremarkable; CT of the abdomen showed fatty liver and one enlarged lymph node (1.4 × 0.9 cm) located above the celiac trunk.

Laboratory data on admission: Hemoglobin 13.2 g/dL (normal: 14.0–18.0 g/dL), leukocytes 2.45 G/L (normal: 4.0–9.0 G/L), 15% lymphocytes, 81% neutrophils, platelets 210 G/L (normal: 140–400 G/L), prothrombin time 48% (normal: 70–130%), creatine kinase (CK)-MB 28 U/L (normal: 1–24 U/L), high-sensitivity troponin I (hs-TnI) 95 ng/L (normal: <26 ng/L), creatinine 4.5 mg/dL (normal: 0.7–1.2 mg/dL), glomerular filtration rate (GFR) 14 mL/min/1,73m² (normal: 80–140 mL/min/1,73m²), C‑reactive protein (CRP) 32 mg/dL (normal: <0.5 mg/dL), D‑dimer 4491 µg/L (normal: < 500 µg/L), myoglobin 241 mg/mL (normal: 23–70 mg/mL), total bilirubin 2.3 mg/dL (normal: 0.1–1.2 mg/dL), alanine aminotransferase (ALT) 327 U/L (normal: 10–50 U/L), lactate dehydrogenase (LDH) 433 U/L (normal: 120–240 U/L), procalcitonin 90 ng/mL (normal: <0.5 ng/mL). Blood cultures were reported as negative. Polymerase chain reaction (PCR) in blood was negative for meningococci, pneumococci, Haemophilus influenzae, Staphylococcus aureus, streptococci group B and Listeria monocytogenes. Blood smear was negative for fragmentocytes, haptoglobin was 266/dL (normal: 30–200/dL). Despite intensive care including artificial ventilation at maximum oxygenation, administration of inotropes (dobutamine), vasopressors (norepinephrine and vasopressin), hydrocortisone (because of massive hemodynamic instability despite inotropes and vasopressor treatment) and antibiotics (additional levofloxacin and ampicillin), the patient did not become hemodynamically stable (pO₂ 59 mmHg, arterial pH 7.3, base excess –12, lactate > 10 mmol/L). Extracorporeal membrane oxygenation was not available. Less than 24 h after admission, the patient died of multiorgan failure.

An autopsy was performed and a diagnostic test result became available three days later.

Some bacterial infections are well-known to have the potential of causing fatal outcome (septic shock) even in previously healthy young persons. These include staphylococcal toxic shock syndrome, group A streptococcal toxic shock syndrome, group A streptococcal myocarditis, leptospirosis, gram-negative sepsis, typhoid fever, rickettsiosis, and infection with meningococci or Streptococcus pneumoniae [3].

Indeed, there are two conspicuous facts in the patient’s history: (1) A sore throat and fatal myocarditis, and (2) a homemade sandwich (with ham and cheese) and diarrhea. Given the sore throat and myocarditis, infection with Corynebacterium diphtheriae should be considered, even though the incidence of diphtheria in developed countries has been declining following effective immunization programs since the 1920s [4‐6]. In the USA, only 55 cases of diphtheria were documented from 1980 through 2011, with only five cases being reported since 2000. Most cases occurred in nonimmunized or inadequately immunized persons; however, diphtheria continues to occur in other parts of the world. In the 1990s, a major epidemic was reported in all newly independent states of the former Soviet Union with more than 157,000 cases and more than 5000 deaths. In some of these countries, up to 80% of cases affected adults. Globally, reported cases of diphtheria have declined from 11,625 in 2000 to 4880 cases in 2011 [7]. The most common sites of diphtheria infections are the pharynx and the tonsils resulting in sore throat, anorexia and low-grade fever. Within 2–3 days, a bluish-white membrane forms and extends, which can vary in size from covering a small patch on the tonsils to covering most of the soft palate. This pseudomembrane firmly adheres to the tissue and attempts to remove it forcibly may cause bleeding. Extensive formation of pseudomembranes may result in airway obstruction. Patients with severe disease may further develop marked edema of the submandibular areas and the anterior neck along with lymphadenopathy, giving a characteristic “bull neck” appearance [7]. The most frequent complication in diphtheria and the most important predictor of mortality is myocarditis [8], which may occur early in the course of the disease or weeks later and can lead to heart failure [7].

Given the consumption of a homemade ham and cheese sandwich and subsequent diarrhea, various food-borne infections should be addressed as well. These include infection with salmonella, Toxoplasma gondii and Listeria monocytogenes, which are frequently associated with ham or cheese as a potential vector; however, taking into account that the incubation time of such diseases is longer than the onset of abdominal discomfort and diarrhea reported in this patient, and the fact that there were only two bouts of diarrhea, diarrhea in the context of sepsis is much more likely than diarrhea caused by a food-borne infection.

This leaves the following four infectious diseases in the differential diagnosis, which have the potential to cause death even in previously healthy young persons: (1) Rickettsial infection, (2) staphylococcal toxic shock syndrome, (3) group A streptococcal toxic shock syndrome and group A streptococcal myocarditis, and (4) leptospirosis.

Rickettsial organisms are a diverse group of gram-negative, obligately intracellular bacteria with variable pathogenicity. All species that are known to cause human disease depend on an arthropod vector (up to 24% of terrestrial arthropods carry rickettsial endosymbionts [9]) such as fleas, ticks, mites or lice [10]. According to serologic and genomic tests, the pathogens of human rickettsioses can be categorized as (1) spotted fever group rickettsiae (R. rickettsii: Rocky Mountain spotted fever, R. montanensis, R. amblyommii, R. parkeri), (2) translational group rickettsiae (rickettsiae belonging to the spotted fever group serologically, but genetically overlap with typhus group rickettsiae; these are rickettsiae with a much milder clinical course and no reported fatalities, such as R. felis and R. akari [11]) and (3) typhus group rickettsiae (R. typhi: murine typhus, also known as endemic or flea-borne typhus and R. prowazekii: endemic typhus, Brill-Zinsser disease) [10]. Moreover, diseases such as the Mediterranean spotted fever, and scalp eschar and neck lymphadenopathy (SENLAT), which is also known as Dermacentor-borne necrosis erythema lymphadenopathy (DEBONEL) or tick-borne lymphadenopathy (TIBOLA), are due to R. conorii [12] and R. raoultii [13]. Clinically significant rickettsial infections present with fever, headache and a rash with or without eschar; symptoms such as myalgia and arthralgia may occur. The clinical course of rickettsiosis is highly variable ranging from self-limiting in the case of R. akari [10] to myocarditis [14], fulminant organ failure and death due to infection with R. rickettsii [10]. The exact mechanisms by which rickettsia exerts the pathogenic effects on humans remain elusive. During infection, bacteria disseminate to endothelial cells which become dysfunctional and lose their integrity. Microvascular thrombosis and increased permeability of microvasculature with subsequent edema are common histologic findings in rickettsial infections [10]. Since the discussed patient did not present with a rash, the diagnosis of rickettsiosis is quite unlikely; however, another tick-borne disease presenting with fever (97% of cases), headache (81%), myalgia (68%), malaise (84%) as well as leukopenia and thrombopenia, but less frequently with gastrointestinal involvement (nausea, vomiting, diarrhea 25–68%) and only rarely with a rash (6%) is ehrlichiosis. Ehrlichiae are small, obligately intracellular bacteria with a gram-negative type cell wall that grow in cytoplasmic vacuoles to form clusters called morulae. Two distinct Ehrlichia species (E. chaffeensis and E. ewingii) and one Anaplasma species (A. phagocytophilia) can cause severe infection in humans. While infection with E. chaffeensis results in human monocytotropic ehrlichiosis, E. ewingii and A. phagocytophilia infect cells of the myeloid lineage, particularly neutrophils [15]. Ehrlichiosis may cause left ventricular dilatation and dysfunction [16] and in some cases may be fatal due to rapidly progressive myocarditis and multiorgan failure as reported for E. chaffeensis in a previously healthy adolescent [17]. Since the clinical course of ehrlichiosis is typically associated with fever, which was only reported for one day in the discussed patient, this diagnosis seems unlikely in this case.

Exclusion of rickettsial infections in this case reduces potential candidates in the differential diagnosis to staphylococcal or group A streptococcal toxic shock syndrome, group A streptococcal myocarditis and leptospirosis. Epidemiologic investigations demonstrated that 50–70% of cases of staphylococcal toxic shock syndrome are associated with menstruation and the use of highly absorbent tampons. Evidence of Staphylococcus aureus infection is not a prerequisite for the development of the disease. The staphylococcal toxic shock syndrome results from the elaboration of an enterotoxin or the structurally related enterotoxin-like toxic shock syndrome toxin-1 (TSST‑1), which causes more than 90% of menstrual cases. Nonmenstrual cases are predominantly caused by enterotoxins [15]. TSST‑1 is a superantigen produced by 5–25% of Staphylococcus aureus isolates [18] that binds primarily to the alpha-chain of the class II major histocompatibility complex exclusively through a low-affinity (or generic) binding site on the surface antigen N‑terminal domain. This stimulates human T cells that express VB 2, which results in the expansion of both CD4 and CD8 subsets of T lymphocytes releasing large amounts of interleukins 1 and 2, and tumor necrosis factor [19]. A staphylococcal toxic shock syndrome typically begins with nonspecific flu-like symptoms including fever, hypotension and erythroderma of variable intensity. Mucosal involvement such as conjunctival hyperemia is also common. The clinical course may rapidly progress to symptoms such as vomiting, diarrhea, confusion, myalgia and abdominal pain, reflecting the multisystemic nature of the disease. Desquamation of the skin can be observed one or two weeks after the onset of the disease. Since the discussed patient did not present with the described symptoms, the diagnosis of staphylococcal toxic shock syndrome seems unlikely in this case [20].

Lancefields’s group A streptococci consist of a single species, Streptococcus pyogenes, which is an organism associated with a variety of suppurative infections. Group A streptococci produce a large number of extracellular products such as streptolysins S and O, streptokinase, DNases, protease and pyrogenic exotoxins A, B and C, that may be important in local and systemic toxicity and in the spread of infection through tissue [15]. Pyrogenic exotoxins cause the rash of scarlet fever, and have been linked to severe invasive infections, including necrotizing fasciitis and a systemic syndrome termed the streptococcal toxic shock syndrome (mortality rate up to 60%) [21, 22]. After bacterial inoculation, which frequently occurs via a small wound, the incubation period is 1–4 days. Intake of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to worsen the course of the disease by increasing the bacterial virulence and production of pyrogenic exotoxins [22]. Among a number of cell surface components expressed by group A streptococci, the major cell surface protein is M protein. The presence of this protein on a group A streptococcus correlates with its capacity to resist phagocytic killing. This is because M protein molecules bind to plasma fibrinogen and so inhibit complement activation and deposition of opsonic complement fragments on the bacterial cell [15]. Indeed, both a small wound and the intake of NSAIDs that might have triggered the clinical course of a potential streptococcal infection were present in the discussed patient; however, typical clinical symptoms of streptococcal toxic shock syndrome include sudden diffuse severe pain with fever, flu-like symptoms and a scarlet-like rash (in up to 10% of cases) [23]. Initial manifestation of abdominal pain and cholecystitis, followed by acute respiratory distress syndrome, renal and hepatic failure and disseminated intravascular coagulation is rare but has been reported [24]. Furthermore, group A streptococcus can induce acute, nonrheumatic myocarditis resembling acute myocardial infarction with ST-elevation [25]. Although no epidemiologic data are available that estimate the incidence of group A streptococcus-induced myocarditis, it may be responsible for more cases than previously thought.

Indeed, the history of a sore throat, fever and myocarditis strongly hints at group A streptococcal infection in the discussed patient. According to the literature, the mean latency of pharyngitis or tonsillitis and the onset of chest pain in patients with group A streptococcus-induced myocarditis ranges from 3 to 5 days [26‐28]. Such latency would basically be compatible with the clinical course of our patient; however, he never complained of chest pain, which finally makes the diagnosis of group A streptococcus-induced myocarditis unlikely.

This finally leads to the discussion of leptospirosis, which is a zoonosis caused by spirochetes of the genus Leptospira [29]. Leptospirosis is endemic in tropical and temperate regions [30], but the disease has been recognized as an emerging global public health problem because of its endemic proportions and increasing incidence in both developing and developed countries [31, 32]. Leptospirosis presents with protean and nonspecific manifestations along with nonspecific findings in routine laboratory investigations, such as increases in CRP, CK, creatinine and transaminases, and thrombopenia [33]. Symptoms are usually a flu-like syndrome with fever, headache and myalgia that may resolve spontaneously; however, in some cases, the disease runs a severe course, leading to multiorgan failure with hemorrhage, hepatic, renal and pulmonary injury and septic shock [33, 34]. The mortality rate in leptospirosis ranges from 3% to 5% in severe cases and is primarily due to acute hepatic or renal failure, myocarditis, pulmonary hemorrhage and multiorgan failure [35, 36]. Leptospirosis is a biphasic disease characterized by an early phase (anicteric) lasting about one week and a delayed immune phase (icterohemorrhagic) in which most complications occur [29, 30]. This disease should be highly suspected whenever febrile patients who were previously healthy present with septic shock, acute renal and hepatic dysfunction and respiratory failure requiring intensive care [37] as presented in this case. Conjunctival suffusion was not reported in the discussed patient but would be an important clinical feature supporting the diagnosis of leptospirosis. Cardiac involvement with occurrence of acute myocarditis may be found in leptospirosis [38‐40]; however, the frequency and extent of cardiac involvement in this disease are underreported and the pathophysiology is poorly understood. A variety of electrocardiographic changes may occur with atrial fibrillation, atrioventricular conduction blocks and nonspecific ventricular repolarization abnormalities being the most common. Electrolyte abnormalities are likely to contribute to electrocardiographic changes; direct effects on Na⁺, K⁺ and Cl⁻ transporters in the renal tubules have been postulated. Furthermore, histopathological changes from postmortem studies have shown myocardial inflammation and vasculitis in patients with cardiac involvement of leptospirosis [38]. Myocarditis usually occurs during the 5th and 7th day of leptospiral infections; significant left ventricular dysfunction is rare [35]. Fulminant leptospiral myocarditis requires early aggressive management including extracorporeal membrane oxygenation support if needed. Management of hemodynamic instability associated with leptospiral myocarditis is primarily supportive. Although yet unvalidated, the additional use of high-dose pulsed steroids may be beneficial [41].

There were many candidates on the differential diagnosis list in this patient, but in view of the entire constellation of findings, leptospirosis (Weil’s disease) seems to be the most likely final diagnosis. This should be confirmed by a microscopic agglutination test (MAT) revealing antibodies against leptospires.

The peritoneal cavity contained 150 mL of turbid fluid. The liver was enlarged with a coarse border and multiple irregularly distributed bright yellow areas in the liver capsule (suspicious of ischemic areas). It weighed 2.5 kg. Histology showed massive steatosis (grade 3) with moderate inflammatory infiltration of the portal fields, and portal and focal septal fibrosis (stage 1), but no evidence of viral hepatitis (Fig. 1). The serosa of the bowel was covered and clotted with fibrin; histology showed massive infiltration with neutrophils and multiple bacteria, mainly of coccoid structure, which is typically found in fibrinous-putrid peritonitis (Fig. 2). The kidneys appeared normal on gross examination and displayed focal interstitial lymphocytic infiltration and mild arteriosclerosis on histology. The spleen, pancreas and stomach were macroscopically and histologically unremarkable.

Microbiological investigation revealed Streptococcus anginosus on the peritoneum. Stool cultures and tests for parasites including Cryptosporidium parvum, Giardia lamblia and Entamoeba histolytica were all negative.

Even though the fulminant clinical course would let one expect more pronounced findings on autopsy we finally diagnosed multiorgan failure due to sepsis caused by leptospirosis.

The symptoms of acute-phase leptospirosis include sudden onset of fever, myalgia and conjunctival suffusion; nausea, diarrhea, vomiting and chills may also be present [58]. The great majority of infections are either subclinical or mild and run a self-limiting anicteric course [30]. In about 10% of affected patients, infection leads to severe and possibly fatal Weil’s disease, which is characterized by hemorrhage, renal failure and jaundice caused by intrahepatic cholestasis and direct hepatocyte damage [29]. Hemorrhage can affect different organs; hyphema may occur in rare cases [59] due to autoimmune mechanisms [60]. More than 10³ leptospires per mL of blood prior to the introduction of antibiotics is associated with a higher risk of developing severe leptospirosis [61]. Leptospirosis has protean manifestations and mimics the clinical presentations of many other diseases. Since this disease can also cause severe acute respiratory distress syndrome (SARS), its diagnosis may be even more difficult at times of the pandemic caused by SARS coronavirus 2 (SARS-CoV-2) [62]. The broad clinical spectrum seen in leptospirosis is presumably the result of infection with different Leptospira spp. inducing different cellular and molecular mechanisms consequently leading to different disease expression; however, the details of these mechanisms remain to be elucidated.

Leptospirosis is more frequently found in tropical areas, but also occurs in temperate areas including Europe. In recent years, the disease has gained increasing attention as an emerging infectious disease of global importance [29, 63] with an increasing incidence in both developing and developed countries [31]. According to the estimates of the Leptospirosis Epidemiology Reference Group of the World Health Organization, approximately 1.03 million cases occur globally each year resulting in some 58,900 deaths [64]. In Austria, the annual epidemiological reports on communicable disease showed 8–11 cases of leptospirosis per year until 2010 [65]. With 24 cases, this number has more than doubled in 2019 [66]; however, data provide evidence of a high prevalence (29%) of antibodies against Leptospira spp. in Austrian male adults [67]. Most frequently serovar Canicola (16.5%), which is linked to dogs, and serovar Hardjo (12%), which is excreted by cattle, are found [51, 67]. Furthermore, data from southeast Austria and Upper Austria show that the vast majority of leptospirosis cases are autochthonously acquired and are associated with being exposed during activities in the woods and wet areas, as well as after contact with rodents [32, 68].

Different tests are available to diagnose leptospirosis. Serology is the most frequently used diagnostic tool. The microscopic agglutination test (MAT), which detects agglutinating antibodies in serum, is the reference standard test for serological diagnosis of leptospirosis with high sensitivity and specificity [69, 70]; however, MAT is limited by its subjectivity, and it requires the maintenance of live leptospires and a convalescent sample for conclusive results [71]. The standard criterion for a positive MAT is a titer of 1:400 or a fourfold increase in antibody titer in endemic countries, or a titer of 1:100 or above in nonendemic countries [72]. Besides MAT, rapid point-of-care IgM assays are available. Since IgM antibodies against leptospires become detectable during the first week of infection [29], these assays often become positive before MAT [69]. Molecular-based diagnostic testing is increasingly used for diagnosis of leptospirosis. Although quantitative PCR-based assays offer the ability to quantify the bacterial load in clinical specimens, the sensitivity (18–50%) is limited [71].

Leptospires can be isolated from blood or cerebrospinal fluid during the first 7–10 days of infection, and from urine during the 2nd and 3rd week of disease [73, 74]; however, culturing is difficult, insensitive and requires prolonged incubation (up to 3 months); the specific culture media are only available in few specialized laboratories [29]. Dark-field microscopy to see organisms in blood or urine is fraught with false positives and false negatives and is therefore not recommended [75].

Treatment of severe leptospirosis includes supportive care and use of appropriate antibiotics. Recommended regimens and doses are based on the severity of the disease. For mild disease, doxycycline, ampicillin or amoxicillin are preferred [76, 77], while for the treatment of severe leptospirosis, penicillin G, ampicillin or ceftriaxone are indicated [78]. Considering increasing antibiotic resistance and patients with penicillin allergy, the broad-spectrum third generation cephalosporins ceftriaxone and cefotaxime have been shown to be as effective as penicillin G in the treatment of severe leptospirosis [78, 79].

Leptospirosis