HMPV RT-PCR test in Hyderabad, India

Diagnosis of HMPV

Human Metapneumovirus (HMPV) is a respiratory virus belonging to the Paramyxoviridae family. It causes respiratory infections that range from mild cold-like symptoms to more severe respiratory illnesses, particularly in young children, the elderly, and immunocompromised individuals.

Clinical features of HMPV

Human Metapneumovirus (HMPV) typically presents with respiratory symptoms, from mild or moderate upper respiratory tract infections to more severe lower respiratory tract involvement. Common clinical features include cough, fever, wheezing, shortness of breath, and in some cases, pneumonia or bronchiolitis.

• Upper respiratory symptoms: Cough, nasal congestion, rhinorrhea, sore throat.
• Lower Respiratory symptoms: Wheezing, shortness of breath, tachypnea, chest retractions.
• Systemic symptoms: Fever, malaise, fatigue, myalgia.
• Severe cases: Pneumonia, bronchiolitis, respiratory failure. 
  

Given below is a detailed step-by-step outline of the process:

1. Sample collection

• The process begins with sample collection, where respiratory samples such as nasopharyngeal or oropharyngeal swabs, nasal aspirates, or bronchoalveolar lavage (if clinically indicated) are obtained.
• These samples are placed in a viral transport medium (VTM) to preserve the RNA and transported to the laboratory promptly under cold conditions (2–8°C). 

2. RNA extraction

• RNA extraction is a significant step in the RT-PCR test for detecting Human Metapneumovirus (HMPV), aimed at isolating viral RNA from the sample. The process begins with lysing the sample to release RNA using a lysis buffer.
• The RNA is then purified through methods such as silica-based spin columns, magnetic beads, or automated RNA extraction systems. 

3. Reverse transcription

• The transformation of Viral RNA into complementary DNA (cDNA) is done by reverse transcription, which is necessary for amplification in the RT-PCR procedure. The reverse transcriptase enzyme, primers, and other required materials are combined with the isolated RNA in this stage.
• The mixture is then incubated for 15–30 minutes at a temperature of 42–50°C, allowing the synthesis of cDNA from the viral RNA template. 

4. PCR amplification

• PCR amplification is conducted to amplify the HMPV-specific cDNA to detectable levels. The process begins by adding the cDNA to forward and reverse primers containing reaction mixture specific to the HMPV genome (targeting conserved regions like the N, L, or F gene), a probe for real-time detection (labelled with a fluorophore and quencher), DNA polymerase, dNTPs, and a reaction buffer. 
• The amplification is carried out using a thermal cycler, by starting with an initial denaturation step at 95°C for 2–3 minutes. This is followed by 40–45 denaturation cycles for 15 seconds at 95°C and annealing at 55–60°C for 30–60 seconds, depending on the primers and probe used.

5. Detection and analysis

• Detection and analysis involve identifying the amplified products to confirm the presence of HMPV. This detection can be achieved through two main methods.
• In real-time RT-PCR, fluorescence emitted by the probe during each amplification cycle is measured, with Ct (cycle threshold) values used to determine the presence or absence of the virus. A lower Ct value indicates a higher viral load. 
• On the other hand, because it relies on gel electrophoresis to visualize the amplified DNA fragments, classical PCR—which is relatively rare—requires extra steps and tools for interpretation.

6. Quality control

• To guarantee the precision and dependability of the RT-PCR test for HMPV, quality control is essential. It entails including internal, external, and positive controls into the testing procedure.
• A positive control, such as synthetic HMPV RNA or a known HMPV-positive sample, confirms that the assay can detect the virus.
• A negative control, such as RNase-free water or a non-template control, ensures there is no contamination or false-positive result.
• Additionally, an internal control, such as an endogenous gene like human RNase P, verifies the integrity of the sample and the efficiency of the RNA extraction and amplification processes. 

7. Interpretation of results

• The interpretation of results in an RT-PCR test for HMPV is based on the Ct (cycle threshold) value and control validation. 
• A positive result is indicated by a Ct value below the threshold, confirming the presence of the virus. 
• A negative result is concluded when there is no amplification or when the Ct value exceeds the threshold, suggesting the absence of the virus. 
• Results are considered inconclusive if controls are invalid or if Ct values are borderline, necessitating a repeat of the test to ensure accuracy.

8. Reporting

The results of the RT-PCR test for HMPV should be documented in a detailed laboratory report. The report must include the following information: 

• The sample type (e.g., nasopharyngeal swab, nasal aspirate)
• The target gene (e.g., N, L, or F gene of HMPV) 
• The Ct value (indicating the cycle threshold and viral load), and 
• The test interpretation (whether the result is positive, negative, or inconclusive). 
• This ensures that the findings are clearly communicated for clinical decision-making.
  

Precautions

• Ensure the use of RNase-free equipment and consumables to avoid RNA degradation.
• Adhere to biosafety protocols to prevent contamination and ensure a safe working environment.
• Confirm the accuracy and integrity of reagents and controls before initiating the test.

This RT-PCR workflow offers high sensitivity and specificity for detecting HMPV in clinical samples. 

Other Major diagnostic procedures of HMPV virus are as given below:

• Antigen detection: Direct antigen detection methods like immunofluorescence (DFA) or enzyme immunoassays (EIA) can identify the virus, but these are less sensitive than PCR.
• Serology: Serological tests for HMPV-specific antibodies (IgM, IgG) can support the diagnosis, but they are not commonly used for acute diagnosis due to delayed antibody response.
• Imaging: Chest X-ray may show signs of pneumonia, interstitial lung infiltrates, or other findings indicative of a viral respiratory infection. 
  

Differential Diagnosis

Other respiratory pathogens, such as Influenza, Respiratory Syncytial Virus (RSV), Parainfluenza virus, Adenovirus, and SARS-CoV-2, need to be excluded, as they share overlapping clinical features.

Duration of RT- PCR for HMPV

RT-PCR is considered the most sensitive and specific method for detecting HMPV. It's performed in special laboratories and uses target-specific probes to detect amplified products. A real-time RT-PCR test for human metapneumovirus (HMPV) typically involves multiple cycles of thermal cycling, with the total time to complete the test taking up to 8 hours.