Pneumoslide IgM

Product informative dossier

Index
Overview on Pneumonia (CAP) .………………………………………………... 2

Main variables to consider when diagnosing CAP ……………………......….. 3

Pneumoslide key features ………………….……………………………..…..… 4

Pneumoslide IgM product positioning …………………………………...…….. 5

Optimal product usage …………………………………………………...……… 5

Pneumoslide IgM assay procedure ……………………………………...…….. 6

Pneumoslide image gallery ………………………………….………………….. 7

Troubleshooting image gallery ……………………………….………......…… 10

Frequently Asked Questions ………………………………….………….….... 13

Troubleshooting guide …………………………………………………...…….. 22

Reference list ……………………………………………………………..….…. 23

Instructions for use …………………………………………………..…... annex 1

Demonstrative video ……………………………..………………...…….. annex 2

Pneumoslide IgM product dossier 1

Overview on Pneumonia (CAP)
Community-acquired pneumonia (CAP) is one of the most clinically important diseases in
adults, affecting 5 to 20 per 1,000 adults per year. Of these, at least 20 to 40% will require
hospitalization for the treatment of their pneumonia.

According to Bartlett, J. et al (Practice Guidelines for the management of Community- Acquired

Pneumonia in adults), pneumonia is the sixth most common cause of death in the United
States. From 1979 through 1994, the overall rates of death due to pneumonia and influenza
increased by 59% (on the basis of ICD-9 codes on death certificates) in the United States. Much
of this increase is due to a greater proportion of persons aged ≥ 65 years; however, age-
adjusted rates also increased by 22%, which suggests that other factors may have contributed
to a changing epidemiology of pneumonia, including a greater proportion of the population with
underlying medical conditions at increased risk of respiratory infection.
Annually, 2-3 million cases of CAP result in ~10 million physician visits, 500,000
hospitalizations, and 45,000 deaths in the United States. The incidence of CAP that requires
hospitalization is estimated to be 258 persons per 100,000 population and 962 per 100,000
persons aged ≥ 65 years. Although mortality has ranged from 2% to 30% among hospitalized
patients in a variety of studies, the average is ~14%. Mortality is estimated to be <1% for
patients not hospitalized. The incidence of CAP is heavily weighted toward the winter months.

Moreno, R. et al. Etiología de la neumonía adquirida en la comunidad en el adulto inmunocompetente.

2005. Rev Chil Infect 22 (supl 1): S18-S25.

English translation of headlines from table 2.

Table 2 - Etiology of adult community-acquired pneumonia depending on attending settings. Foreign studies.

CAP etiology Non-hospitalized Hospitalized patients Intensive Care Unit (UCI)

Patients (8 studies) (35 studies) (14 studies)
Moreno, R. et al. Etiology of immunocompetent adult community-acquired pneumonia. 2005. Rev Chil Infect 22 (supl 1):
S18-S25

The diagnosis of an infectious disease is always performed by the evaluation of a number of

parameters. Laboratory tests are another diagnostic option, but are not the only ones.
In order to achieve a diagnosis, the doctor must study and bear in mind clinical data of the
patient: symptomatology, clinical history, radiology tests, general analysis, etc. along with the
data from the microbiology laboratory.

Pneumoslide IgM product dossier 2

Depending on the patient’s age, disease evolution time, microorganism excretion rate, means
available at the laboratory, skills and experience of the technicians, etc., Pneumoslide IgM will
be a very useful diagnostic method for some patients and less useful for others.

When it comes to choosing the ideal method for the diagnosis of respiratory infections, the
following factors must be taken into account:

Depending on the geographic area where the assay is to be performed, some diseases may
have more incidence than others. Thus, the diagnosis of those most prevalent diseases will be
of value. It is therefore important to know the epidemiology in the area so that those
microorganisms to be studied can be precisely defined.

Moreover, in those seasonal geographic areas it is needed to know the prevalence of the
different microorganisms causing respiratory infections that are related to seasonal changes. In
those areas where no seasonal changes occur, the prevalence of the different microorganisms
is stable throughout the years.

Main variables to consider when diagnosing CAP

ƒ Serological response depending on infection’s ubiquity
In primoinfections, type IgM and IgG response appear. In reinfections, IgM is sometimes absent
and some others it is of low intensity, although IgG seroconversion does occur. If the infection is
very common (frequent in aerial transmission infections), primoinfection appear in the
childhood. For those infections whose incidence is lower, primoinfection may occur during the
whole lifetime.

ƒ Age range of the population to be analyzed

It is important to know whether the population to be diagnosed is adult or child. Generally, child
populations excrete a higher quantity of microorganisms when they get infected by viruses
causing a respiratory infection, and they produce IgM antibodies against the agents responsible
for that infection. In adult populations the excretion rate is usually lower; also on many
occasions IgM are not produced, since the adult has been previously in contact with the
microorganism many times.

ƒ Qualification of the laboratory

The diagnostic method to be used will depend on the equipment of the laboratory where the
diagnosis is to be performed, and on the staff’s skills and training.

ƒ Clinical evolution of the patient

The advisable techniques to be used will depend on when the patient actually goes to the
doctor. In initial phases of the disease, some options are microorganism culture, molecular
biology or antigen detection techniques. In more advanced phases of the disease where
excretion rates of the microorganism are minor, it is advisable to perform serological methods.

ƒ Sample type
The diagnostic technique to be used will depend on the type of patient, skills of the staff in
charge of collecting the sample and on the means available for the shipment of the sample to
the laboratory where it is to be analyzed. Sometimes it is not possible to obtain a respiratory
sample in some patients, due to his/her age, symptomatology or any special circumstance; in
these cases, blood samples are needed for serological analyses. Most health workers are well
trained to obtain blood samples but not all are properly trained for obtaining high quality
respiratory samples. When a quick and secure transport condition shipment of the respiratory
samples cannot be guaranteed, it may be advisable to obtain blood samples rather than
respiratory ones.

Pneumoslide IgM product dossier 3

Pneumoslide key features
Pneumoslide IgM allows presumptive diagnosis by measuring IgM responses. It allows to
perform 10 tests (10 slides of 10 wells)

ƒ This test is especially designed for the study of those microorganisms likely to cause
respiratory infections and that can be diagnosed by serological methods. There are other
microorganisms causing respiratory infections that cannot be diagnosed by serological
methods.
ƒ In comparison with other commercial brands in the market, Vircell’s test is easier to perform.
It is also more user-friendly when seeing it on the fluorescence microscope (there are less
parameters and they are easier to be found than the biochips).
ƒ Vircell has a well containing Hep-2 cells. This allows the identification of those sera with
anticellular or antinuclear antibodies likely to cause unspecific reactions leading to the test
to be invalid.
ƒ Pneumoslide presence in the world is shown in the following chart:

Historical Sales Contribution by region (NSLIDEM)

Africa Russia
Asia Middle East
1% 6%
6% 6%
Australia
0%

LATAM
19%

EU
62%

Pneumoslide IgM product dossier 4

Pneumoslide IgM product positioning

Summing up, it is necessary to deeply evaluate a number of factors in order to decide which
diagnostic techniques are the most appropriate to achieve the best diagnosis, bearing in mind
circumstances and available means.

Optimal product usage

Pneumoslide IgM product dossier 5

Pneumoslide IgM assay procedure

Pneumoslide IgM product dossier 6

Pneumoslide images gallery
Microscope visual reading results

Legionella pneumophila serogroup 1 (positive and weak positive)

Mycoplasma pneumoniae (positive and weak positive)

Coxiella burnetii (positive and weak positive)

Pneumoslide IgM product dossier 7

Chamydophila pneumoniae (positive and weak positive)

Adenovirus (positive and weak positive)

Respiratory syncytial virus (positive and weak positive)

Pneumoslide IgM product dossier 8

Influenza A (positive and weak positive)

Influenza B (positive and weak positive)

Parainfluenza 1, 2 and 3 (positive and weak positive)

Cell control

Pneumoslide IgM product dossier 9

Troubleshooting image gallery
Contaminated sample

Inappropriate washing

Pneumoslide IgM product dossier 10

Legionella and Coxiella edges

Clots

Pneumoslide IgM product dossier 11

Frequently Asked Questions
1- What is the most common methodology for respiratory tract pathogens within the
international market? Who are the most representative manufacturers?
Taking into account the general considerations above, the main etiological agents responsible
for respiratory infections could be detected with different methodologies depending on the type
of hospital.

USEFULNESS OF THE TECHNIQUE

LARGE HOSPITAL
ANTIGEN DFA CULTURE SEROLOGY IgM SEROLOGY IgG MOLECULAR
LEGIONELLA
PNEUMOPHILA OK OK OK OK OK
MYCOPLASMA
PNEUMONIAE OK OK OK
COXIELLA BURNETII OK OK
CHLAMYDIA
PNEUMONIAE OK OK OK
ADENOVIRUS OK OK OK OK
RESPIRATORY SYNCITIAL
VIRUS OK OK OK OK OK OK
INFLUENZA OK OK OK OK OK OK
PARAINFLUENZA OK OK OK OK

SMALL HOSPITAL
ANTIGEN DFA CULTURE SEROLOGY IgM SEROLOGY IgG MOLECULAR
LEGIONELLA
PNEUMOPHILA OK OK OK
MYCOPLASMA
PNEUMONIAE OK OK
COXIELLA BURNETII OK OK
CHLAMYDIA
PNEUMONIAE OK OK
ADENOVIRUS OK OK OK
RESPIRATORY SYNCITIAL
VIRUS OK OK OK OK
INFLUENZA OK OK OK OK
PARAINFLUENZA OK OK OK

The sensitivity of the test would also vary depending on the technology used.

SENSITIVITY OF THE TEST

SEROLOGY
ANTIGEN DFA CULTURE SEROLOGY IgM IgG MOLECULAR
LEGIONELLA
PNEUMOPHILA HIGH LOW HIGH HIGH HIGH
MYCOPLASMA
PNEUMONIAE HIGH HIGH HIGH
COXIELLA BURNETII HIGH HIGH
CHLAMYDIA
PNEUMONIAE HIGH HIGH HIGH

Pneumoslide IgM product dossier 12

ADENOVIRUS LOW HIGH MODERATE HIGH
RESPIRATORY SYNCITIAL
VIRUS HIGH HIGH HIGH MODERATE HIGH HIGH
INFLUENZA LOW MODERATE HIGH MODERATE HIGH HIGH
PARAINFLUENZA LOW HIGH MODERATE HIGH

The moment in which the diagnosis takes place will also determine the most appropriate
technique to be used.

DIAGNOSIS TIME

ANTIGEN DFA CULTURE SEROLOGY IgM SEROLOGY IgG MOLECULAR

LEGIONELLA
PNEUMOPHILA EARLY EARLY INTERMEDIATE LATE EARLY
MYCOPLASMA
PNEUMONIAE INTERMEDIATE LATE EARLY
COXIELLA BURNETII INTERMEDIATE LATE
CHLAMYDIA
PNEUMONIAE EARLY INTERMEDIATE LATE
ADENOVIRUS EARLY EARLY INTERMEDIATE LATE
RESPIRATORY SYNCITIAL
VIRUS EARLY EARLY EARLY INTERMEDIATE LATE EARLY
INFLUENZA EARLY EARLY EARLY INTERMEDIATE LATE EARLY
PARAINFLUENZA EARLY EARLY INTERMEDIATE LATE

2- What is the deficiency of Pneumoslide IgM in the clinical application? What are the
solutions?
A fluorescence microscope must be available at the laboratory. Otherwise, it would have to be
provided.
The laboratory must have technicians with training in visualization of immunofluorescence
techniques. Otherwise, training must be given to the technicians.
It may happen that the patient goes to the doctor at a very early phase of the disease and
antibodies may not have appeared yet. In this case, the doctor will have to ask for another blood
sample 7-10 days after the initial visit.

3- What kinds of sera are required for Pneumoslide IgM test? What is the influence in the
test result if it is Hyperlipidemia, pollution, hemolysis or jaundice sera?
As indicated on the Instructions for use, Specimen Collection and Handling:
Blood should be collected aseptically using venipuncture techniques by qualified personnel. Use
of sterile or aseptic techniques will preserve the integrity of the specimen. Serum samples are to
be refrigerated (2-8ºC) upon collection or frozen (-20ºC) if the test cannot be performed within 7
days. Samples should not be repeatedly frozen and thawed, to avoid immunoglobulin titer
decrease, especially IgM. Do not use hyperlipemic or contaminated sera. Samples containing
particles should be clarified by centrifugation.

4- What is the worldwide authorized and standard test method for the virus at present?

Serological methods Methods of microorganism detection

Legionella IFA Antigen detection in urine

Mycoplasma ELISA, Complement PCR

Pneumoslide IgM product dossier 13

 Fixation, Microaglutination

Coxiella IFA None

Chlamydia MIF PCR

Adenovirus IFA PCR

RSV IFA Antigen Detection (only children)- PCR/Cell Culture (adults)

Influenza Complement Fixation PCR/Cell Culture

Parainfluenza IFA PCR/Cell Culture

5- - What is the incidence of the nine pathogens in clinical population? Please let us
know the prevalence of each pathogen.
Multiple infections are not common. In some cases, a polyclonal reactivation may happen due
to an acute infection caused by a microorganism. This polyclonal reactivation would then cause
IgM antibodies against other infectious diseases suffered by the patient during his life. This
would be a strange fact and if two or three wells were found to be positive, the doctor would
have to evaluate the prevalence of the microorganisms found depending on the geographical
area and season of the year. The doctor will also have to ask for another serum sample in 15-
21 days for further analysis.
In general terms the main causes of pneumonia requiring hospitalization are Mycoplasma,
Legionella and Chlamydia

Data from an active surveillance study performed in Ohio in 1991, showing age-specific rates of community-acquired
pneumonia due to the major bacterial pathogens. M. pneumoniae infections were diagnosed by seroconversion, using
CF tests. These data demonstrate that M. pneumoniae causes a relatively large proportion of pneumonias of sufficient
severity to warrant hospitalisation among persons younger than 50 years but that it is also an important cause of
pneumonia in older age groups. Sp, S. pneumoniae; Mp, M. pneumoniae; Lp, L. pneumophila; Cp, C. pneumoniae

M. pneumoniae causes up to 40% of cases of community-acquired pneumonias and as many

as 18% of cases requiring hospitalisation in children (maximum incidence between 5 to 15
years of age). The most typical syndrome in children is tracheobronchitis.
M. pneumoniae infection is ordinarily mild, and many adult cases may be asymptomatic. 3 to
10% of infected persons develop pneumonia. It occurs endemically and epidemically (3-5 years)
worldwide in children and adults. Climate and geography are not thought to be of major
significance.
Outbreaks of M. pneumoniae infections also tend to occur in summer or early fall. The long
incubation period and relatively low transmission rate have been involved in the prolonged
duration of epidemics of M. pneumoniae infections.

Pneumoslide IgM product dossier 14

The distribution of organisms varied from year to year, with M. pneumoniae ranking second to
influenza A virus as the most frequent pathogen encountered during the surveillance period. It
can persist for variable periods in the respiratory tract following infections that have resolved
clinically. Numerous outbreaks of M. pneumoniae infections have been documented in the
community or in closed or semiclosed settings such as military bases, hospitals, religious
communities, and institutions for the disabled.
Legionella pneumophila causes Legionnaires disease, a severe multidisease involving
pneumonia, and Pontiac fever (a self-limited flu-like illness). Legionellosis is a disease that has
emerged in the last half of the 20th century because of human alteration of the environment.
Water is the major reservoir for Legionella, and the bacteria are found in fresh water
environments worldwide.
Coxiella burnetii infection (Q fever) could be asymptomatic in 60% of the cases. In the acute
phase it causes febrile illness, atypical pneumonia, or a granulomatous hepatitis. Evolved forms
cause endocarditis. The history of Q fever in China began with the recognition of cases of
primary “atypical pneumonia” back in 1951. The distribution of Q fever in China is countrywide.

Pneumoslide IgM product dossier 15

Q FEVER: The Biology of Coxiella burnetii. Ed. CRC Press- October 1991. Chapter 12: COXIELLA BURNETII IN
CHINA. Yu Shurong- Department of Microbiology Third Military Medical Collage. Chongqing, Sichuán. People’s
Republic of China.

The increasing number of recognized cases of Q fever, especially cases of chronic disease, is a
cause of concern in China. Early diagnosis and treatment of patients may play a very important
role in preventing recurrence of Q fever o chronic disease.

Chlamydia pneumoniae courses asymptomatic in 90% of the cases. Pneumonia, asthma and
conornary artery disease are its main manifestations.
If it is assumed in adults, that the annual incidence of community acquired pneumonia is 0.1%,
that C. pneumoniae is responsible for 10% of these and that the annual seroconversion rate is
1.5%, then only 1 in 150 C. pneumoniae infections will result in pneumonia.

Adenovirus is responsible for 7% of all respiratory diseases in children, 20% of acute

conjunctivitis in children, 3-10% of gastroenteritis cases in children, myocarditis and pericarditis
and infections in immunocompromised patients (it is one of the most significant fatal infections
in bone marrow transplants)
The heterotypic anamnestic rise that occurs early in the infection may obscure a fourfold rise in
the titer of antibody to the infecting serotype when the acute phase specimens are not collected
in the first few days. Detection of IgM is one of the earliest laboratory indicators.

The public health problem caused by Respiratory Syncytial virus is exemplified by the fact
that approximately two-thirds of infants are infected with RSV in the first year of life; one-third of
those infected develop lower respiratory tract disease, 2.5% are hospitalised (more than
90,000 children in the United States every year), and 0.1% die
It accounts for approximately 50% of all pneumonia and up to 90% of the reported cases of
bronchiolitis in infancy. Outbreaks of RSV disease are abrupt in onset and can last up to
5 months. These epidemics occur annually at regular, predictable intervals.

Influenza is an acute self-limiting viral disease of the upper respiratory tract. Influenza has a
considerable public health impact worldwide each winter because of its ability to spread rapidly
through populations by coughs and sneezes from infected people. During seasonal influenza
epidemics 5-15% of the population is affected with upper respiratory tract infections. Seasonal
epidemics are associated with substantial demands on healthcare resources and considerable
costs due to increases in general practice consultation rates, clinical complications,
hospitalisations, drug treatment and absence from work. Although difficult to assess, it is

Pneumoslide IgM product dossier 16

estimated that worldwide between 250,000 and 500,000 people die from severe illness as a
result of an influenza virus infection every year.
Influenza is an enveloped RNA virus which matrix protein and nucleoprotein allows
differentiation between A, B and C. Influenza A has subtypes according hemagglutinin and
neuraminidase: H1N1, H3N1, H3N2 and H5N1.
Influenza causes flu, vomiting and diarrhoea in children, bronchitis, otitis, croup and pneumonia.
On April 2009, a novel H1N1 (nH1N1) reassortant causing human infections in Mexico and
USA was identified by the CDC. Continued identification of new cases indicates sustained
human-to-human transmission of this novel influenza A virus. Most confirmed cases have been
characterized by self-limited, uncomplicated febrile respiratory illness and symptoms similar to
those of seasonal influenza; however, vomiting or diarrhea was common. Some patients
required hospitalization due to more severe disease.
This new H1N1 virus may be detected either in direct samples or cultures by direct
immunoassays targeting common epitopes of influenza A viruses. Yet, these assays can not
identify the novel virus from the current circulating seasonal strains. This identification can be
only achieved by sequencing (only available in a limited number of laboratories) or by RT-PCR
targeting specific sequences of the new virus.

There are more than 5 x 106 lower respiratory infections (LRI) each year in the United States in
children younger than 5 years. Parainfluenza virus (HPIV-1 to HPIV-3) has been found in as
many as one-third of these infections. In addition, Parainfluenza virus causes upper respiratory
infections (URI) in infants, children, and adults and, to a lesser extent, LRI in the
immunocompromised, those with chronic diseases and the elderly.

AGE EPIDEMIOLOGY URI LRI REMARKS

PAR-1 7-36 months fall/biennial croup 50% bronchiolitis 10%

PAR-2 12-24 months fall/biennial croup 10%

PAR-3 <6 months summer/yearly croup 10% bronchiolitis 10%, pneumonia, more serious illness
tracheobronchitis

5 - Which factors can lead to false positive for Pneumoslide IgM test?
• Presence of anticellular or antinuclear antibodies.
These antibodies may appear in several autoimmune diseases. In these cases, the
Pneumoslide’s well #10 may present a fluorescence staining and all wells containing cells
(wells 2, 5, 6, 7, 8, 9 and 10) will present fluorescence. In these cases, immunofluorescence
must not be used. Other techniques have to be used instead.
• Inappropriated washing step:
1. Instructions for use must be strictly followed. Otherwise, false positive results
may appear.
2. A washing solution not prepared or stored according to the working instructions
could lead to false positive results.
• Inappropriated incubation step:
1. Incubation for longer than specified in the working instructions can cause
backgrounds.

Pneumoslide IgM product dossier 17

 2. Backgrounds can be produced due to the fact that the wells dried up during
incubation. It is important to use for incubation steps a humid chamber as we
mention in the working instructions.
3. Incubation at a temperature higher than 40 ºC
• Sorbent treatment:
Pneumoslide IgM has a step of treatment of the sera with sorbent and further centrifugation in
order to remove the precipitate. If this step is not carried out and if the precipitates are
resuspended when collecting the centrifuged serum, these may lead to background.

6- Which factors can lead to false negative for Pneumoslide IgM test?
• If the sample has been obtained at a very early phase of the disease, when IgM
antibodies have not appeared yet.
• In reinfections by virus causing respiratory diseases in adult populations, IgM antibodies
do not always appear. Therefore, Pneumoslide may give negative results even if the
disease does exist.
• The following factors must be taken into account in order to avoid false negative results
o Check the microscope and UV lamp
o Check the absence of contamination of fluorescent conjugate
o Check the mounting medium covers the whole well
o Check that the components of the correct kit have been used.
o Check that the technique has been carried out following the working
instructions.
o Do not assay at a temperature lower than 30ºC.
o Do not use any product other than the PBS included in the kit as a wash buffer
o Avoid washing excessively prolonged or too vigorous

7- EUROIMMUN company also has an IFA kit with a reactive time of 30+30 minutes, while
Vircell’s reactive time is 90+30 minutes, which can be too long. Could the reactive time
be shortened?
Immunoglobulins IgM are bigger than Immunoglobulins IgG and reaction time with antigenic
epitopes is longer than the one for the Immunoglobulins IgG. It is scientifically agreed worldwide
that longer incubation times must be used, usually 90 minutes. Shorter incubation times could
reduce the technique’s sensitivity.

8- During the assay procedure, could we incubate straight in boiled water instead of
using a humid chamber?
We do not have any experience in that. In any case, preparing a humid chamber is easy: a
container with a tap (Petri dishes, a box…) that assures a secure closing (hermetic closing is
not needed). On the bottom of the box or dish, a filter paper or cotton damped with water must
be placed.

Pneumoslide IgM product dossier 18

9- Which details should we pay more attention to while using Pneumoslide IgM?
From the technical point of view, the most important aspects are as follows:
Procedure of centrifuging the sample after treatment with sorbent: It is important to remove the
treated sample carefully avoiding the precipitates not to resuspend, since they may interfere
with the visualization of the sample leading to false positives or invalid readings.
Do not scratch the wells when placing the samples and during other handling procedures. This
could make the antigens come off leading to readings hard to interpret.
To have experience in visualizing fluorescence on microscope.
To have a fluorescence microscope available with an appropriate maintenance that guarantees
high quality images.

10- Due to Pneumoslide IgM being an IgM detection technique for the first infected, for
the repeatedly infected, does this technique have any limitations? What sales arguments
we could use for this product?

Bacterial infections causing pneumonia that can be diagnosed by serological methods

(Legionella, Mycoplasma, Chlamydia, Coxiella) have a low prevalence and the cases of
reinfections throughout the lifetime are not high. That is why the diagnosis in adult population
can be made by the determination of IgM immunoglobulins against Legionella, Mycoplasma,
Chlamydia and Coxiella, making Pneumoslide a very useful diagnostic technique.

For viruses causing respiratory symptoms, it is possible to find adult populations with IgM
antibodies. However, in many reinfections IgM antibodies do not appear anymore, making
Pneumoslide of no use in these cases. In child population, where most of the respiratory
infections are primoinfections, the determination of IgM by Pneumoslide is a very useful
technique.

11- For the kits in the same batch, can we just do one negative and positive control?

Yes, it is possible to perform only one negative and positive control when using several kits from
the same batch.

12- Compared with gelatin particle method, what is the advantage of a Pneumoslide IgM
kit?

Gelatine particle methods measure total antibodies, therefore differentiating IgM (indicating an
acute process) from IgG (that may correspond to a past infection) is not possible.

Pneumoslide IgM product dossier 19

13- There is sometimes a small amount of fluorescence in well # 10, does it mean the test
is invalidated?

A faint immunofluorescence signal in well # 10 does not mean an invalid test. To consider the
test as invalid for containing antinuclear or anticellular antibodies, well # 10 should present an
intense stain. Besides, fluorescence would also appear in those wells containing cells other
than well # 10: 2, 5, 6, 7, 8 and 9.

14- Sometimes fluorescence is not totally visible in the whole well; also some cells show
fluorescence and some others do not. In such a case, how to interpret negative or
positive results?

Negative and positive controls included in the kit offer very clear results. Until gaining some
experience with the tests, it is important to pay special attention to positive and negative
controls before the microscopic visualization of the samples.

According to the working instructions, the reaction is positive when apple green nuclear,
cytoplasmatic and/or peripheral fluorescence is visible, in 1-15% of the cells for positive sera to
adenovirus, influenza, RSV or parainfluenza (peripheral pattern is most frequent with weak
sera; in parainfluenza and RSV dyed together with the previous pattern can be observed); apple
green fluorescence in all the bacteria in the case of Legionella, Chlamydophila or Coxiella;
apple green fluorescence in the periphery of the cell for positive sera to Mycoplasma can be
observed.

15- In spite of appearing fluorescence in cell membrane, cytoplasm or cell nucleus, how
to interpret positive? Must it form a fluorescence circle or an almost fluorescence circle
when we interpret positive?

In the case of weak positive sera a peripheral pattern in 1-15% of the cells to adenovirus,
influenza, RSV or parainfluenza can be observed.

16- If drying with hot air or a 37 degree incubator, will it affect the fluorescence
observation?

Following the working instructions, after washing procedures you have to allow the slide to air
dry. When we want to speed up the slide dry, we use a dryer, as it allows sweeping away the
washing remains. Additionally, the slide has to be inclined while getting dried. Concerning the
use of an incubator at 37 ºC, we do not recommend it.

17- What are the requirements in washing time and washing intensity? Would there be
any difference depending on the type of shaker, the speed of shaker or the shaking time
used in the washing? What is the influence like?

As mentioned in the instructions for use, after the incubation step briefly rinse the slide with a
gentle stream of PBS (avoid directing PBS at wells) and immerse it in PBS while shaking gently
on a shaker (orbital shaker or waving shaker) for ten minutes. Dip wash slide briefly in distilled
water.

It is important to do the wash step shaking the slides in order to avoid background. If it is
impossible to have a shaker to do the washing step without shaking, you should do the washing
step for 15 minutes.

18- Could you explain the origin of the antigens coated in the wells?

Pneumoslide IgM product dossier 20

The wells containing viruses (5: adenovirus, 6: RSV, 7: influenza A, 8: influenza B, 9:
Parainfluenza 1, 2 and 3) are a mixture of cell culture infected by the specific viruses and non
infected cells following this scheme:

- Adenovirus: virus grown in Hep-2 cell line. The well contains infected and non infected cells.
- RSV: virus grown in Hep-2 cell line. The well contains infected and non infected cells.
- Influenza A and B: viruses grown in LLC-MK2 cell line. The wells contain infected and non
infected cells.
- Parainfluenza 1, 2, 3: viruses grown in LLC-MK2 cell line. The wells contain infected and non
infected cells.

Each viral well contains 1-15% of infected cells inactivated with formaldehyde. Slides are fixed
with acetone.

19- Does it need to be performed in sterilized conditions?

Sterile conditions are not required.

20- How about the stability of the kit?

As you can see in the working instructions:

Storage requirements:
Store at 2-4ºC. Do not use the kit regents beyond the expiration date printed on the label. Kits
are stable through the end of the month indicated in the expiration date when stored closed and
at 2-8ºC.

Storage of reagents once opened:

Reconstituted PBS: 4 months at 2-8ºC, never beyond its expiration date. Rest of the
components: Refer to package label for expiration date (at 2-8ºC)

Stability and handling of reagents:

Handle reagents in aseptic conditions to avoid microbial contaminations. Use only the amount
of sorbent, PBS, control serum and conjugate solutions required for the test. Do not return the
excess solution into the bottles. After reconstitution store PBS at 2-8ºC and do not use if
turbidity appears.

Troubleshooting guide
ABSENCE OF FLUORESCENCE IN POSITIVE CONTROL
• Check UV Lamp
• Check absence of contamination in positive control
• Check absence of contamination in conjugate
• Check that the mounting medium covers the whole well
• Check that the components of the correct kit have been used

BACKGROUND FLUORESCENCE IN CONTROLS

• Check the washing procedure
• Check the washing solution
• Check incubation time (longer incubation times may lead to background)
• Check that wells did not become dry during incubation: a humid chamber should be
used for incubation

Pneumoslide IgM product dossier 21

FLUORESCENCE IN OVER 50% OF THE CELLS IN SERUM SAMPLES
• Unspecific fluorescence, probably due to autoimmune disease

ANTIGEN DETACHES FROM WELL

• Check that the washing solution does not contain detergent.
• Check that slides do not scratch one another during the washing and drying steps.

BLURRED FOCUS
• Check the microscope
• Check that the objective is not stained with glycerol

Pneumoslide IgM product dossier 22

Reference list

1. El-Sahrigy, S. Abdel-Rahman, Abou Shady, Attia and Goma. 2006. Pneumoslide- M

Technique for Rapid Detection of Atypical Pathogens in Critically ILL Children with
Lower Respiratory Tract Infections. J. Med. Sci. 6

2. Grdanoska, T., Petrovska, M., Cvetkovic, D., Kotevska, V., Dokic-Trajkovska, E.,
Kondova, I. and Panovski, N. Pneumo-slide: serological investigation of human
respiratory infections. 2nd Balkan Conference of Microbiology. Thessaloniki. 2001.

3. Kondova, I., Anastasovska, A., Kondov, G., Petrusevska, S. and Grdanovska, T.

The Incidence of Atypical Agents as Cause of CAP in Hospitalized Adults. Posters
Presentation. 12th ICID - Lisbon, Portugal - June 15-18, 2006.

4. Sayan, M., Kilinc, O., Yuce, A., Ucan, E. S. and Genc, S. 2003. Seropositivity against
atypical pneumonia agents demonstrated in patients with community-acquired
pneumonia. Mikrobiyol Bul.37 (4): 247-53.

Pneumoslide IgM product dossier 23