Introduction

Crimean-Congo haemorrhagic fever (CCHF) is a devastating viral tick-borne disease which causes sudden fever, headaches, muscle aches, nausea and haemorrhagic symptoms (bleeding). The Crimean-Congo hemorrhagic fever virus (CCHFV) is classed as a priority pathogen for which research and development are urgently needed by the World Health Organisation (WHO) due to its high case fatality rate, potential for nosocomial (hospital) outbreaks and challenges in prevention and treatment.¹ CCHFV, a Nairovirus of the Bunyaviridae family, is spread mainly through tick bites or direct contact with infected tissue or bodily fluids from livestock. Those in the livestock industry (e.g., slaughterhouse workers, farm employees and veterinarians) are most at risk.² Person-to-person transmission, whether through contact with bodily fluid or air droplets, is less common.³   

CCHF, originally called Crimean haemorrhagic fever, was first identified as a disease when Soviet soldiers stationed in Crimea developed haemorrhagic fever symptoms following tick bites during World War II.³ Congo was added to its name in 1969 after being discovered to be the cause of the same disease in the Congo basin.³ The disease is now endemic in parts of Africa, Asia and Eastern Europe, but climate change and the migration of livestock and wild animals and to a lesser extent, travelling with pets, has led to CCHF spreading to new areas such as Western Europe.⁴ With a case fatality rate of 10-40%, early and accurate diagnosis is crucial to managing outbreaks and providing timely medical intervention.⁵ In this article, we will explore current diagnostic methods for detecting CCHF, examining laboratory and molecular approaches and recent advances that enhance diagnostic accuracy and speed.

Overview of diagnostic needs for crimean-congo hemorrhagic fever

The diagnosis of CCHF presents unique challenges. Given its high fatality rate and the overlapping symptoms with other haemorrhagic diseases (e.g. Ebola, Dengue or Marburg virus disease) early identification is essential for appropriate management and containment.⁶ Effective diagnostic methods for CCHF need to:

• Detect the virus in the acute phase of infection when symptoms are most pronounced and the virus is most detectable⁷
• Differentiate CCHF from other diseases with similar presentations
• Ensure safety for laboratory personnel due to the highly infectious nature of CCHFV

Traditional diagnostic approaches

Use of medical history,  symptom presentation and clinical assessment of those suspected to have CCHF is useful in locations where laboratory facilities may not be available. 

Clinical assessment based on symptoms and history

A CCHV diagnosis is made if at least two of the following are present:

• Fever,  i.e. oral  temperature  >38°C
• Headache  
• Generalised pain or joint pain 
• Fatigue or weakness
• Diarrhoea 
• Bleeding  

AND

• Either tick exposure history and/or resident in, or recent travel to a CCHF endemic region within the previous two weeks⁷ 

Using symptoms and stages for initial clinical assessment

• Incubation period (no obvious symptoms after exposure):
  • Usually 3-7 days, but can be as long as 13 days
    • 1-5 days after a tick bite 
    • 5-13 days after infected bodily fluid 
• Pre-haemorrhagic phase (1-5 days):
  • Sudden fever, redness of the upper body, muscle aches, headache, nausea, vomiting, and sensitivity to light and sounds, similar to a migraine episode
  • In some cases, inflammation of the eyes (conjunctivitis and congested sclera)
  • Because of the range of symptoms, suspicion of CCHF based on potential exposure and travel history is often enough for a diagnosis at this stage⁸ 

• Haemorrhagic Phase (3-5 days after onset of symptoms lasting 1-3 days):
  • Starts with a rash, then progresses to bleeding from multiple sites
    • This includes bruising, nosebleeds, vomiting or coughing up blood, blood in urine or stool, bleeding from genitals and even internal bleeding, such as in the brain or abdomen⁸ 
    • At this stage, there might also be confusion, delusion, headaches, neck stiffness, or seizure-like symptoms, but the way CCHF affects the brain needs further study
    • Multiple organs (e.g. the heart and pancreas) may also be affected, potentially leading to multiple organ failure⁸

• Convalescent phase (starts 10-20 days after onset of symptoms and  can last up to 1 year):
  • Most recover without complications
  • Some experience fatigue, weakness, memory loss, visual loss, hearing loss, hair loss, weight loss, damage to nerves, heart and breathing complications and even post-traumatic stress disorder⁸
  • Fortunately, reinfection is rare even in endemic areas⁸    

It is necessary for healthcare professionals to know differential diagnoses for CCHF. These are other haemorrhagic fevers with similar symptoms to CCHF (e.g., dengue and Ebola). Laboratory testing can help rule these out.

Current laboratory diagnostic methods for detecting CCHF

There are two main types of laboratory CCHFV diagnostic methods: 

• Direct tests (molecular techniques):
  • Can be performed during the early stages of the disease
  • The virus is isolated either through cell culture, viral antigen detection or detecting viral genetic material⁹
• Indirect tests (serological techniques):
  • Performed from one week to four months and sometimes up to 5 years following infection^9,11 
  • Viral antibodies (protective proteins which the body produces to fight infection) produced in response to CCHFV infection are detected⁹ 
  • Can confirm recent or ongoing infection
  • Prognostic indicator: low antibody production is linked to a low chance of survival¹¹

Blood is the preferred sample for diagnostic tests, however, saliva, urine and other bodily fluids can be used in cases where a blood sample cannot be taken.¹¹   

Molecular techniques

Viral isolation and culture

Early diagnosis of CCHF is key to treating people with the virus and to preventing of the spread of the CCHFV. The ideal test is therefore one which detects the virus early.⁹ This can be done by isolating and culturing the virus. Though diagnosis is definitive with viral isolation and culture, add to that the benefit of being able to closely observe the virus, and this comes with challenges: 

• Viral isolation of CCHFV is only possible in high-containment (biosafety level 4) facilities with the highest level of protection for their workers, the environment and the public. This greatly affects the number of laboratories able to use it
• Detection of the virus is highest 1-5 days after infection, leading to potential errors if blood samples are collected after this window and safety issues for healthcare workers collecting and examining samples, as the virus is easy to pass on during this window
• Results after 3-7 days
• Not the best test for routine diagnosis, given how resource-intensive and time-consuming it is⁹

Laboratories tend to focus on the most effective, manageable techniques to diagnose CCHF in their locations, with several techniques enabling the detection of the virus's genetic material directly. These are explored below.

Nucleic acid amplification tests (NAAT)

Reverse transcription polymerase chain reaction (RT-PCR)

Due to CCHFV’s diversity of strains, nucleic acid amplification tests (NAAT), where the virus’s genetic material is copied repeatedly, are recommended. RT-PCR (a type of NAAT) has become the gold standard for CCHFV diagnosis due to its high sensitivity and specificity, especially in the early stages of infection. It is also more accessible than viral isolation and culture.¹⁰ RT-PCR involves the conversion of viral RNA into complementary DNA (cDNA) through reverse transcription, followed by amplification of specific viral gene sequences– in simple terms, making multiple copies of information about the virus, allowing easier detection.¹¹ Real-time RT-PCR also estimates viral load (the amount of virus in an infected person’s blood).

Advantages of RT-PCR include:

• Early detection: Detects viral RNA within days of symptom onset¹¹
• Estimation of viral load: Useful for monitoring disease progression
• Can detect a wide range of CCHFV strains¹⁰

Limitations include:

• High costs
• Need for specialised equipment and highly trained personnel
• Strict (level 4) biosafety protocols are required, as the virus cannot be inactivated¹⁰

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a more sensitive test to quantify (determine the amount of) RNA in a sample. It is a more reliable, faster, safer approach to RT-PCR with lower biosecurity requirements. The test’s major challenge is its abilityto detect every single strain of CCHFV. Scientists have developed a method to detect all strains by either:

• Using multiple primers and probes to cover all strains⁹
• Using primers and probes targeting a part of the RNA that is common in all strains⁹

Synthetic RNAs from deactivated CCHFV strains can be used as a template to eliminate potentially contaminated samples or as calibrators during measurements.⁹  

Nested reverse transcription polymerase chain reaction (nested RT-PCR)

Nested reverse transcription polymerase chain reaction (nested RT-PCR), which is conducted during most RT-PCR tests, is when a second round of amplification takes place in order to increase the sensitivity of the test and to confirm the result of the initial amplified PCR product.⁹ Disadvantages due to repeated tests on a single sample include:

• Higher chance of errors
• Time consuming
• Prone to cross-contamination⁹

Reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a faster, cheaper diagnostic method for CCHFV, involving one-step amplification of viral RNA at a constant temperature. RT-LAMP, which is also an NAAT, has demonstrated high sensitivity and specificity in detecting CCHFV, even in locations where level 4 biosafety is not possible, and is becoming an increasingly popular diagnostic method.⁹

The advantages of LAMP include:

• Speed: Results can be obtained in less than an hour
• Simplicity: Complex training is not required
• Minimal equipment requirements: Can be performed without complex laboratory infrastructure, is transportable and has been beneficial in low-resource settings¹³
  • Level 3 biosafety protocols are sometimes used if level 4 is not possible, and depending on national guidelines¹¹  

Serological techniques

Serological tests detect antibodies produced by the immune system in response to CCHFV infection. Most tests target the CCHFV N protein, which causes a fast, strong, lasting immune response; however, severe cases of CCHF do not usually have a detectable antibody response.¹ Immunoglobulin M (IgM) and immunoglobulin G (IgG) are the target antibodies for most serological tests. 

Advantages of serological testing:

• Reliable results, even in the later stages of the disease.
• IgG detection aids in understanding disease prevalence and exposure history in a population.

Limitations of serological testing:

• IgM and IgG antibodies take time to develop, making serological methods less effective in the early phase of infection compared to direct methods like PCR
• There is a risk of false positives due to antibodies from similar viral infections; therefore, tests to confirm a result are sometimes needed⁹
• The need for laboratory infrastructure and trained personnel can be a challenge in resource-limited settings⁹

Below are examples of commonly used serological techniques.

Enzyme-linked immunosorbent assays (ELISA)

Enzyme-linked immunosorbent assays (ELISA) are the most widely used serological method to diagnose CCHF. ELISA detects and measures immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies in the blood.

• IgM antibodies: Detectable within the first week of infection, serving as an indicator of recent exposure
• IgG antibodies indicate past infection and can be detected for up to five years
• Reliable and accurate.
• Fast results⁹

Immunofluorescence assays (IFA)

Immunofluorescence assays (IFA), which are faster than ELISA, involve labelling antibodies with fluorescent dyes that can then bind to viral antigens, which are visualised under a fluorescence microscope. IFAs are best at determining those without viral infection but not as good at diagnosing those with the virus (high specificity, lower sensitivity).¹⁴ IFAs are labour-intensive, often requiring specialised equipment and trained personnel.¹⁴  

Historical serological techniques

Serological testing methods for CCHFV diagnosis, such as complement fixation, agar gel diffusion precipitation, haemagglutination and neutralisation tests, were widely used in the past. They have since been replaced by ELISA and IFAs due to their reliability and increased sensitivity.¹⁰

Innovations in CCHF diagnosis

Lateral flow assays (LFAs)

Lateral flow assays (LFAs) are portable diagnostic tests that work similarly to pregnancy or COVID-19 tests, providing results within minutes. LFAs offer rapid, cheap, easy-to-interpret results, making them valuable for initial screening in remote areas or during outbreaks. The development of lateral flow assays to diagnose CCHF has been attempted once, but was found to be an inadequate diagnostic tool due to low sensitivity due to a too small sample size.^9,10

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) methods use RNA-guided enzymes to identify specific CCHFV sequences, offering high sensitivity and specificity similar to RT-PCR but at a potentially lower cost. CRISPR-based diagnostics are still in the experimental phase for CCHF but show promise for use in low-resource settings due to their portability and ease of use.⁹

Biosensors

Point-of-care (POC) tests, laboratory tests conducted near the site of patient care, for CCHFV are not yet commercially available.⁹ Modern diagnostic approaches require specialised laboratory equipment or trained personnel to operate, but CCHFV-endemic regions are typically rural with limited resources, with answers lying in POC tests.⁹ Biosensors, a type of POC test, detect the presence or amount of virus in a sample by detecting biomolecules. Further research is needed, however, before this technology is available commercially.^9,10

Potential advantages:

• Speed
  • Results in minutes
• Specificity, reliability and safety
• Ease of use
  • No professional training required⁹

Novel biomarkers

Micro RNA (miRNA) is a naturally occurring short non-coding RNA that is involved in the function of human cells, including immune response, growth, and death. miRNA technology may potentially fill gaps that current diagnostic tools cannot, by serving as biomarkers or biosensors.⁹ Further study is needed.

Conclusion

Accurate and timely diagnosis of CCHF remains a challenge, especially in low-resource settings and during outbreaks. Non-laboratory methods provide important retrospective information, while molecular and serological approaches like RT-PCR and ELISA allow precise diagnosis. Emerging diagnostics, including biosensors, POC and CRISPR-based tools, bring hope for rapid, accessible testing to areas with limited laboratory infrastructure. The continued development of these tools will be crucial in improving response times and reducing mortality rates associated with CCHF. As technology advances, the goal remains to create diagnostic methods that are fast, reliable and scalable, addressing both the clinical and biosafety challenges that CCHF currently poses.