FOR IN VITRO DIAGNOSTIC USE ONLY

Field of application
The EuSepScreen Eurospital Kit is used to determine any presence - in biological samples such as blood, CSF, pleural and synovial fluid or any other normally sterile human biological fluid or material of bacterial DNA belonging to all Neisseria meningitidis serogroups (A, B, C, X, Y, W135, H, Z, 29 E, NT), all the 90 Streptococcus pneumoniae serotypes, serotypes b, c of Haemophilus influenzae and all Adenovirus serotypes (a-f).

Principle of the method
Meningitis and sepsis are extremely severe diseases; they cause high mortality (1,2) and require a rapid and precise determination for implementing both an appropriate therapy and an early prophylaxis whenever this is required (infections ascribable to Neisseria meningitidis or type-b Haemophilus influenzae) (3).
Furthermore, a rapid detection of these pathogenic agents contributes to toning down the news in the mass media, thereby preventing the spreading of panic associated with meningitis and similar diseases.
In theory, the germs capable of inducing meningitis or sepsis are innumerable, but in medical practice a small number of them is known to cause over 90% of meningitis cases (4).
In healthy children and adults, Meningococcus and Pneumococcus alone account for over 70% of meningitis/sepsis forms (4). Immunodepressed patients can be an exception, as they are prone to carrying other pathogens in addition to all the above-mentioned germs.
The cultural methods, to date regarded as the gold-standard for diagnosis, show a number of weak points: first of all, they require the presence of living and very vital germs, which means they can result to be falsely negative in subjects in whom the antibiotic treatment was introduced before the culture (5); furthermore they very much depend on the volume of the collected sample (6), as well as on the rapidity of sample transport and test execution after sample collection. A further and not less important problem is that these methods can require several days before a sample is found to be negative or, in the slow growth cases (5), many days may be necessary before concluding that the result is probably a false positive by contamination.
The methods based on molecular biology do not require living germs and, therefore, are not characterised by the sensitivity limitations that are typical of cultural methods (7,8). For this reason, such methods can result to be extremely effective in patients who have already received an antibiotic therapy, and are not affected by rapid transport (so much so that samples can be stored at room temperature even for a few days) and, finally, do not require any culture medium. The EuSepScreen kit is designed to obtain the highest sensitivity and specificity in the determination of most invasive bacterial diseases (meningitis, sepsis, pneumonia, etc.) affecting both children and adults by directly using biological samples such as blood, cerebrospinal fluid, pleural fluid, synovial fluid or any other normally sterile human biological fluid or material.
In the EuSepScreen kit, indicated for both paediatric and adult patients, the choice of included bacteria has been formulated on the basis of the current Italian epidemiological context, so as to include germs causing about 90% of the meningitis/sepsis cases among patients of these age groups (4).
The detection system can unmistakably identify the germ against which it is directed and without causing any cross-reaction with other germs or human DNA (7,9).
The inclusion of Adenovirus responds to a specific clinical need of differential diagnostics: it is well known that, from both the clinical and the laboratory point of view, Adenovirus can mimic a septic situation (10), with persistent high fever, marked neutrophilic leukocytosis and high indices of inflammation.
The results obtained with this method have demonstrated a 100% specificity (no false positive result) when based on both cerebrospinal fluid and whole blood, or other human biological fluids (7, 8).
The sensitivity of this method compared with the cultural method has turned out to be twice as high (200% sensitivity) in the case of meningitis and, with regard to pneumococcus, about 10 times higher (1000% sensitivity) in pneumonia (8).

Supplied materials
(quantity sufficient for 8 tests)

• Water for control reconstitution 1x0.5 ml
• Reagent A 1x1.5 ml
• Positive control (lyophilized) 1x0.4 ml
• Mix 1 (red test tube) 1 strip x 8 test tubes
• Mix 2 (green test tube) 1 strip x 8 test tubes
• Mix 3 (clear test tube) 1 strip x 8 test tubes

Composition of the supplied materials/reagents

• Water for control reconstitution
  1 Eppendorf vial containing 0.5 ml of water for PCR to be used for positive control reconstitution.
• Reagent A
  1 1,5 ml vial, green cap, containing master mix diluent. Ready for use.
• Positive control
  1 vial, red cap, containing reactive DNA for all Mixes. Lyophilised, to be resuspended on first use.
• Mix 1
  1 strip with 8 red test-tubes sealed by aluminium foil. Each test tube contains, in dried form, the detection system for the determination of Neisseria meningitidis and Streptococcus pneumoniae. Warning: avoid direct exposure to light.
• Mix 2
  1 strip with 8 green test-tubes sealed by aluminium foil. Each test tube contains, in dried form, the Adenovirus and Haemophilus influenzae detection system.
  Warning: avoid direct exposure to light.
• Mix 3
  1 strip with 8 clear test-tubes sealed by aluminium foil. Each test tube contains, in dried form, the human genomic DNA detection system. Warning: avoid direct exposure to light.

Required, but not supplied, instruments and materials

• 5, 20, 100, 200, 500 μl pipettes
• Disposable sterile tips with filter, 5, 20, 100, 200, 500 μl pipettes.
• Vortex.
• Microcentrifuge for test tubes
• Rotor centrifuge for microplates.
• Amplifier with 520 and 550 nm reading filters.
• 0.2 ml test tubes with optical grade caps.
• Optical grade microplates.
• 0.2 ml test tube holder
• Universal PCR Master Mix

Performance criteria
This method allows the identification of bacterial (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) or viral (Adenovirus) DNA in a biological sample after a nucleic acid extraction process.

Sample collection
The EuSepScreen kit requires the use of biological samples such as blood, CSF, pleural and synovial fluid or any other normally sterile human biological fluid or material.

Sample preparation
The sample is composed of DNA extracted from biological samples such as blood, CSF, pleural and synovial fluid or any other normally sterile human biological fluid or material. The DNA can be extracted with the most common DNA extraction methods, but for top performance the use of the Eu-Gen Estrazione kit (code 9132) is recommended.

Sensitivity and specificity
40 DNA samples extracted from biological samples, previously characterised at a reference centre, have been tested by this method. The results obtained with EuSepScreen have shown 100% sensitivity and specificity.

Storage
All kit components shall be stored at 2 €“ 8°C.

Stability after the first opening
All components, if used according to the instructions given in the General advices section, are stable until the date shown in the label.

Stability in transport
An accelerated stability study has shown that all the device components are stable after being stored at 37°C for 96 hours.

Sample storage
If not immediately submitted to amplification, the purified DNA can be stored at 2-8°C for 3 months, or at -20°C for longer periods: in this case, it is advisable to divide it into aliquots so as to avoid freezing and unfreezing it repeatedly, which could deteriorate the DNA. A new use of the DNA stored at -20°C has been checked by Eurospital only by using the Eu-Gen Estrazione kit reagent (code 9132). The use of other extraction kits requires that the relevant procedures of use be checked by the laboratory to assess the stability and preservability of the extracted DNA.

Positive control resuspension
Upon the first use, briefly centrifuge the Positive Control vial. By using sterile tips only, add 0.4 ml of water for control reconstitution. Shake with a mechanical agitator (vortex) for at least 15 seconds to achieve an appropriate resuspension of the material contained in the control test tube.
Briefly centrifuge the vial to remove the drops on the walls and under the cap. The positive control thus resuspended is ready for use. The stability remains as indicated in the label.

DNA amplification procedure
From each of the three mixes contained in the kit, with a pair of scissors separate one test tube for each patient to be analysed from the 8 test-tube strips. Place the test tubes in a vertical position in an appropriate holder, which is not supplied;
After piercing the aluminium foil sealing the test tube with the tip, add 37.5 μl of Reagent A to each Mix; pipette a couple of times to resuspend the pellet adhering to the bottom;
Add to each mix 62.5 μl of Universal PCR Master Mix (reagent required but not supplied); pipette a couple of times to obtain a homogeneous solution;
By using test tubes or microplates dedicated for the thermal-cycler, prepare, for each sample, 4 test tubes per Mix (a total of 12 test tubes);
Dispense 20 μl of each Mix into each of the four test tubes, according to the following table:

For each Mix, carry out the following steps, according to the table below; Do not add anything to test tube nr.1 (negative reaction control, White, non template control or NTC); Add 5 μl of extracted sample to test tubes nr. 2 and 3 and use the pipette a couple of times to mix the solution (S1); Add 5 μl of positive control to test tube nr. 4 and use the pipette a couple of times to blend the solution (positive control, C+);

Analyse the signals obtained in each test tube.

Validation of obtained results.
Make sure that the signals, read by a 520 nm filters, obtained with Mix 3 in both test tubes containing the sample to test (S1) are positive;
Make sure that, for each mix, the signals obtained for the NTC control are negative for both reading filters (520 and 550 nm);
Make sure that, for each mix, the signals obtained with the positive control (C+) are positive for both reading filters (520 and 550 nm);
Check that, for Mixes 1 and 2, the signals obtained in the two test tubes containing the sample to test (S1) are concordant: both positive and both negative for each reading filter used (520 and 550 nm).In the absence of at least one of the above-mentioned conditions, the test shall be repeated.

Interpretation of results.
The positiveness obtained with the sample in Mix 3 indicates that the extraction of nucleic acids has been carried out correctly.
For Mixes 1 and 2, the presence of a signal for a certain filter used (520 and 550 nm) indicates the presence, in that sample, of genetic material belonging to the pathogenic agent according to the following pattern:

Mix 1:
Positive with 520 nm = Neisseria meningitidis
Positive with 550 nm = Streptococcus pneumoniae

Mix 2:
Positive with 520 nm = Adenovirus
Positive with 550 nm = Haemophilus influenzae

Precautions
Always wait for the reagents to reach room temperature before starting the test.
Scrupulously comply with the procedural instructions. In the event of an accident or sickness, call a doctor immediately and show him/her the container, the label and, if possible, the safety data sheet.
Warning: it is very important not to mix components belonging to different lots.
If the procedure is not carried out under a hood, always use a mask to avoid test tube contamination.
It is advisable to carry out the various stages of sample preparation (extraction), reagent preparation and amplification in separate and well delimited areas.
Mixes 1, 2 and 3 contain specific DNA sequences, substances the dangerousness of which has not been fully tested.

Limits of the test
The EuSepScreen kit has been optimised by means of the extraction reagents of the Eu-Gen Estrazione kit (code 9132). The use of other extraction kits requires that the relevant procedures of use be checked by the laboratory, with regard to the stability and preservability of the extracted DNA.
The DNA should be used immediately after being extracted from the blood. If not immediately submitted to amplification, purified DNA can be stored at 2-8°C for 3 months, or at -20°C for longer periods: in this case, it is advisable to divide it into aliquots so as to avoid freezing and unfreezing it repeatedly, which could deteriorate the DNA. There are no available data on EuSepScreen performance with DNA samples stored for longer periods and extracted with different systems.

General advices
Always use sterile tips with filters.
Adopt all precautions to avoid reagent contamination.
Do not leave the mixes contained in the aluminium minigrip bags exposed to light.
Once the required number of test tubes have been taken, immediately put the rest of the strip back into the aluminium minigrip bag. Make sure that the desiccant is inside.
Do not touch the tips with your hands.
Always wear protection gloves to avoid contaminating the reagents. Use only test tubes, caps or adhesive foils of optical grade.
Do not mix components belonging to different lots.
The EuSepScreen kit is not to be used for purposes different from those stated in the Field of application and repeated in the Performance criteria.

Troubleshooting

• No signal
  Wrong setting of reading filters.
  Errors in reagent dispensation or reagent omission.
  Inhibitory effects of the sample: insufficient DNA extraction and purification processes.
  Kit stored incorrectly.
  Check the amplifier performance.
• The signal intensity is too low.
  Deterioration of the detection system owing to incorrect reagent handling and/or storage.
  Very low DNA concentration in the sample to be analyzed.
  Inappropriate mix resuspension.
  Air bubbles trapped inside the amplification test tubes.