Approach
Diagnosis requires a high index of suspicion, based on clinical presentation and epidemiological exposure, as leptospirosis can present with a wide range of clinical manifestations. Diagnosis can be confirmed by isolation of the organism from blood or cerebrospinal fluid (CSF), serology, histopathology with special staining, and polymerase chain reaction (PCR).
The standard approach for confirming the diagnosis is a two-test microscopic agglutination test (MAT) on paired acute and convalescent serum samples, although PCR is increasingly used, if available, to provide a more timely diagnosis during the acute phase.[50] The development of nucleic acid-based assays has increased, but one meta-analysis found significant heterogeneity among genetic markers in current tests; further studies are needed to identify the optimal test methods for leptospirosis.[51][52]
History
Identifying risk factors for acquisition of leptospirosis is of the utmost importance. Significant risk factors include direct or indirect contact with urine of infected animals, occupational exposure, water sports, outdoor recreational activities, and natural disasters.[1][2][3][4][6][24]
Review patient history, asking questions regarding any exposure to animals, water, and soil. Leptospires penetrate primarily through broken skin, mucous membranes, and inhalation of water, and less effectively through the conjunctivae.[34] Patients usually report a history of contact with animals, their urine, or contaminated water or soil.
Definition of a timeline between exposure and symptom development must be pursued. The incubation period is generally 7-14 days but ranges between 2 and 30 days.
Clinical presentation
Most infections (approximately 90%) are subclinical. Patients who develop symptoms usually experience an acute, self-limited febrile illness; however, 5% to 10% develop a more severe illness, which can include renal failure, hepatic failure, and pulmonary haemorrhage.[50] Severe illness can lead to multi-organ failure and death.
In patients progress to severe disease, the illness can be biphasic with a temporary decrease in fever between the two phases: an acute/initial phase, followed 5-7 days later by the immune phase.[50]
Acute septicaemic phase: patients present with high fevers, chills/rigors, headache, myalgia (often localised in the calves), abdominal pain, diarrhoea, nausea and vomiting, asthenia, anorexia, photophobia, conjunctival suffusion, and non-pruritic morbilliform rash lasting 1 or 2 days (morbilliform eruption is rare).
Immune phase: after 5-7 days, fever resolves and the patient develops symptoms related to the immune phase. Symptoms during this phase include severe eye pain, headache, photophobia, pulmonary symptoms (cough, dyspnoea, chest pain, haemoptysis), palpitations, conjunctival suffusion, muscle tenderness, mental status changes (delirium, coma), and focal neurological deficit.
Severe disease is associated with the immune phase and may manifest with renal failure, hepatic failure, and/or pulmonary haemorrhages.[53] Other presentations during this phase include myocarditis with arrhythmias, aseptic meningitis, and pancreatitis.[5] Patients who are immunocompromised may manifest with cardiovascular complications such as complete heart block, although this is a very rare presentation.[13]
In pregnant women, leptospirosis may lead to severe fetal and maternal morbidity and mortality. The presentation may mimic other viral, bacterial, and parasitic infections; acute fatty liver; pregnancy-induced hypertension; and haemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome.[54] Hence, leptospirosis in pregnancy is often misdiagnosed and under-reported.[55]
The clinical diagnosis of leptospirosis in tropical areas is often challenging, given that the clinical manifestations of leptospirosis are often indistinguishable from other endemic infections.[20] Co-infections can also occur and complicate the diagnosis, including dengue, malaria, rickettsia, scrub typhus, and HIV.[14][15][16][17][18]
Physical examination
Physical examination is extremely important in order to recognise particular manifestations of the disease, including signs that are pathognomonic.
Findings on physical examination differ among patients; they will be present according to the phase and consequent severity of disease. General examination findings during the acute/initial phase include high fevers up to 40°C (104°F), rigors, hypotension, tachycardia, lymphadenopathy, muscle tenderness localised in the calves (pathognomonic) and possibly in paraspinal muscles, and rarely a morbilliform rash.
Abdominal examination findings of hepatosplenomegaly and abdominal tenderness, and findings on eye examination of photophobia, jaundice, and bilateral conjunctival suffusion (pathognomonic), may be elicited in both the acute/initial and immune phases of the disease.
The immune phase may reveal signs of consolidation secondary to pulmonary haemorrhage, and rales and wheezes associated with pulmonary oedema on respiratory examination. Cardiac examination may reveal arrhythmias, such as atrial fibrillation, atrial flutter, heart block, and premature ventricular contractions, while the neurological examination reveals nuchal rigidity, delirium, weakness, and paralysis.
Investigations during acute/initial phase
Routine laboratory tests during the initial phase include the following:
FBC and differential: leukocytosis and thrombocytopenia in the absence of disseminated intravascular coagulation.
Urinalysis: mild proteinuria, pyuria, haematuria, and hyaline or granular casts.
Microscopy: spirochetes can be visualised by darkfield microscopy on urine and blood specimens.
Cultures: Leptospira can be isolated from blood and CSF during acute/initial phase for the first 7-10 days of the disease. Culture is insensitive and slow and is therefore not recommended as the sole diagnostic method.[50]
Serology: Leptospira anti-IgM antibodies will be positive during acute/initial phase. Positive IgM-based screening tests such as enzyme-linked immunosorbent assay (ELISA) IgM should be confirmed with MAT on paired acute and convalescent samples, if available.[50][56]
PCR: if available, can provide a timely confirmation of the diagnosis during the acute phase, through detection of Leptospira DNA in blood or urine, or in CSF (for patients with meningitis).[50][56]
Investigations during immune phase
Laboratory findings in the immune phase include the following:
FBC and differential: anaemia secondary to haemorrhage
LFTs: show elevated aminotransferases (<200 mg/dL), elevated conjugated bilirubin (≤1368 micromol/L [80 mg/dL]), and elevated alkaline phosphatase
Metabolic panel: indicates elevated urea and creatinine and hypokalaemia
Pancreatic enzymes: amylase and lipase may be elevated in the presence of pancreatic involvement
CSF analysis: cell counts below 500 cells/mm³, lymphocytic pleocytosis, elevated protein, and normal glucose
Serology: rising titre levels between the acute and convalescent phase will confirm diagnosis
CXR: indicated in patients with clinical signs of pulmonary involvement and shows small nodular densities and patchy alveolar infiltrates, and air-space nodules
ECG and cardiac monitoring: should be performed in all patients, owing to associated arrhythmias secondary to myocarditis
Abdominal ultrasound: should be done in patients with abdominal pain and hepatomegaly or splenomegaly
Renal biopsy: usually performed in the immune phase, owing to renal involvement.
Direct methods of confirmation
Darkfield microscopy:
This is an illumination technique used to enhance the contrast in unstained samples. This test provides direct visualisation of spirochetes from blood or urine specimens, or visualisation of agglutination in the MAT. It is positive during acute/initial phase. Darkfield examination has poor sensitivity and specificity.
Isolation and culture of leptospires:
Leptospires can be isolated and cultured from blood, CSF, and peritoneal dialysate fluids during the first 10 days of disease. Urine can be cultured after the first week of illness. Fletcher's media is the selective culture media for leptospirosis. However, culture is insensitive and slow and is therefore not recommended as the sole diagnostic method.[50]
Staining methods:
Techniques such as silver staining, immunoperoxidase staining, and immunofluorescent staining may show the presence of the organism in tissue samples. These tests are not widely used due to a lack of commercially available reagents and low sensitivity rates. The three staining techniques have low sensitivity.
PCR:
Can provide a more timely diagnosis during the acute phase, if available. Genetic sequencing can be amplified from serum, urine, aqueous humour, and other tissues. The best or preferred specimen is serum collected in the first week of illness (in the first 4 days is ideal). PCR can also be performed on CSF fluid from a patient with signs of meningitis or on urine collected at least 1 week after symptom onset. However, the transience of leptospires in body fluids means that a negative PCR test does not exclude leptospirosis.[57]
There is substantial variability between studies assessing the accuracy of PCR and real-time PCR, and it is uncertain whether PCR or real-time PCR is better in detecting leptospirosis. There is preliminary evidence that PCR is more sensitive on blood samples collected early in the disease course.[58] [
]
Choice of methods of confirmation will depend on availability at local laboratories, and physicians should consult local testing protocols.
Indirect detection methods of confirmation
Microscopic agglutination test (MAT):
Live antigens of leptospires react with serum samples and then they are examined for agglutination using darkfield microscopy. MAT is the reference standard for confirming leptospirosis but is a complex test to perform and interpret. This method will provide delayed confirmation because it involves pairing of acute and convalescent sera collected 2 weeks apart. A serologically confirmed case of leptospirosis involves a fourfold increase in MAT titre to 1 or more serovars between acute and convalescent phases. A single MAT titre of 1:800 on any sera or identification of spirochetes on darkfield microscopy, when accompanied by the appropriate clinical scenario, is strongly suggestive of recent or current infection.[5][59] A single titre of 1:200 following symptom onset is suggestive evidence of infection, especially if the case is from a non-endemic region.[60]
Indirect haemagglutination assay:
This technique has low sensitivity on acute-phase specimens. Higher sensitivity is reached when both acute-phase and convalescent-phase specimens are tested.[61]
ELISA for IgM antibody detection:
IgM antibodies can be detected after the fifth day of illness. The detection of IgM antibodies can be used as a screening test to increase the diagnostic capacity of laboratories, especially in developing countries.[61][62] Results should be confirmed, ideally with MAT or PCR if available.[57]
Latex agglutination and western blot:
Possible alternatives to IgM ELISA or MAT where these are not available. In one study both latex agglutination and western blot were shown to be sensitive and specific for the confirmation of leptospirosis.[63]
Quantitative PCR (qPCR):
Has been evaluated as an aid to provide an accurate and fast diagnosis in patients presenting with clinical manifestations suggestive of leptospirosis in endemic areas.[64] Some studies have found that leptospiraemia levels measured by qPCR are associated with disease severity when obtained early in infection; this may be a valuable method to predict clinical course.[44]
Emerging tests:
While not yet widely employed, lateral flow assays are a promising diagnostic modality, although there is considerable variability in published data regarding their sensitivity (55% to 93%) and specificity (57% to 99%).[65] Several factors may account for this variability (including variations in reference tests and the stage of infection when the testing was performed, with a lower sensitivity described in the first week of infection).[65]
Current evidence suggests that interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-8, IL-10, and tumour necrosis factor (TNF)-alpha levels are higher in severe cases of leptospirosis in comparison with mild cases.[66] The day of infection appears to be an important determinant of cytokine level; further studies are needed before cytokine levels can be used to monitor infection.[66][67]
Imaging
CXR may show bilateral small nodular densities and patchy alveolar densities in patients with pulmonary involvement during the immune phase. Cases in which chest radiographs detect air-space nodules have been associated with severe leptospirosis.[68]
Abdominal ultrasound may show acalculous cholecystitis, hepatomegaly, and splenomegaly.
ECG and cardiac monitoring
An extensive variety of cardiac arrhythmias can be identified, such as atrial flutter, atrial fibrillation, tachycardia, premature ventricular contractions, and heart block.
Renal and lung biopsy
Renal biopsy is usually performed during the immune phase. It is recommended in patients with renal involvement in order to document the type of kidney injury. The feasibility of performing a renal biopsy is dependent on the clinical status of the patient and available resources. It can reveal acute interstitial nephritis or immune-complex glomerulonephritis. Leptospira can also be found in the renal parenchyma. Histological findings in fatal cases include mixed tubulointerstitial inflammatory cell infiltrate of lymphocytes, plasma cells, and polymorphonuclear leukocytes with areas of tubular necrosis.
Lung biopsy reveals pulmonary congestion and focal areas of haemorrhage indicative of severe haemorrhagic pneumonitis in the immune phase.[69][70][71] At autopsy, histological examination of the lung tissue shows damage to the capillary endothelium, interstitial and intra-alveolar haemorrhage, diffuse alveolar haemorrhage, and severe airspace disorganisation.
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