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Saliva-Controlled Sputum Culture (SSC) – A High Value

Diagnostic Tool For Deep Pulmonary Infections

Inna Gitelman

PII: S0167-7012(20)30131-7
DOI: https://doi.org/10.1016/j.mimet.2020.105986
Reference: MIMET 105986

To appear in: Journal of Microbiological Methods

Received date: 20 February 2020

Revised date: 27 May 2020
Accepted date: 15 June 2020

Please cite this article as: I. Gitelman, Saliva-Controlled Sputum Culture (SSC) – A
High Value Diagnostic Tool For Deep Pulmonary Infections, Journal of Microbiological
Methods (2020), https://doi.org/10.1016/j.mimet.2020.105986

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Saliva-Controlled Sputum Culture (SSC) – A High Value Diagnostic Tool For Deep
Pulmonary Infections

Inna Gitelman

email: inna.gitelman@mac.com
Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel

ABSTRACT

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A major obstacle to prompt diagnosis of fungal pulmonary infections is that deep sputum samples are
scarce, yet are frequently rejected if they contain saliva. We show that including saliva controls

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unfailingly distinguishes oropharyngeal flora from pulmonary fungi, thus preserving valuable samples
for analysis, expediting diagnoses and improving patient care.

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KEYWORDS: Saliva only::deep sputum conjoint cultures (SSC), saliva control protocol, aspergillosis,
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patient-assisted sputum collection, fungal pulmonary infections, rejected sputum, oropharyngeal flora
contamination, prompt diagnosis, optimization guidelines recommendations
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HIGHLIGHTS
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1. Saliva control of deep sputum cultures is invaluable for correct diagnosis

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2. SSC offsets the oropharyngeal flora growth in sputum cultures

3. In deep mycoses SSC prevents rejection of rare purulent sputum samples
4. SSC fosters the wellbeing of bedridden patients
5. Results strongly advocate inclusion of SSC in optimization guidelines of deep mycoses

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Respiratory fungal diseases bring high mortality, their incidence is on the rise worldwide, and they
increasingly strike the non-immunocompromized and immunocompetent patients (Baker et al., 1971
Borman et al. 2010l, Chrdle et al. 2012, Denning et al. 2014, Nosanchuk 2016, Pashley et al. 2012).
A key to successful treatment of deep respiratory fungal infections is prompt definitive diagnosis,
and a key 1st-line test is analysis of sputum samples. A vast amount of careful work has been done over
the years by clinical and research scientists, who have developed best practice recommendations for the
care of patients with invasive fungal diseases, including guidelines for rapid diagnosis of infectious
fungal pulmonary isolates (e.g., Center for Disease Control (CDC), USA, Infectious Diseases Society
of America and American Thoracic Society (IDSA/ATS), Schelenz et al. 2015 and refs. therein,
Freymuth et al., 2012: European guidelines, The UpToDate site and refs. therein). These
recommendations have been accepted widely and are being implemented, with certain modifications, in
medical centers across the world, and they include modern methods, which are fast and powerful.

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They also contain prescribed optimizations for both collection of sputum and of its processing
(Table 1, Recommendations column). The patients are carefully instructed as to when, where and how

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sputum can be collected. Then the labs are advised to accept those sputum samples, which meet certain
criteria, for example, if the volume is sufficient, if they were collected under supervision in a clinic, if
they contain no saliva, and so on (Table 1). If rejected, the test gets re-ordered and the patient resubmits
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another sputum sample; between 3-5 consecutive samples are recommended for a sound diagnosis.
Unexpected challenges arise, however, at this first, and seemingly undemanding, step of obtaining a
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quality sample (reviewed in Table 1). On one hand, there is the question of scheduling. In deep fungal
infections, the cough is mostly unproductive because the sputum comes from deep lying cavities. The
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expectorations are not only rare, but also sporadic and, as a rule, occur at night.
On the other hand, in addition to unpredictability of productive coughing, patients with deep
respiratory mycoses are often bed ridden. Altogether, these factors stand in the way of patients’ ability
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to provide sputum deemed acceptable by the current guidelines (Table 1, the Common Problems
column). In turn, multiple sputum samples get rejected leading to misdiagnosis and/or significant delay
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in correct diagnosis, with potentially disastrous outcomes.

We report on successfully resolving this problem by including an immediate saliva control culture
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for each expectorated sputum sample.

1. Sputum Collection and Culture. The essence of the procedure (Figure 1) is to collect a saliva
sample immediately after each expectorated pulmonary sputum sample. In the laboratory, parallel
processing and comparison of the paired samples allows interpretation of the culture results even in the
presence of the upper respiratory flora (Figure 1, red & blue plates diagram and Figure 2).
Commonly, deep purulent plugs (Figure 2A, red arrows) emerge intermixed with watery mucus.
Upon culturing, such sputum produces a mixed growth of filamentous fungi and Candida sp. (Figure
2C). The latter are ubiquitous in oro- and nasopharynx, and are only rarely found in the lower
respiratory system. Inclusion of the saliva control (Figure 2, B and D), and its comparison with
sputum culture allows, with good confidence, to exclude, in this case, Candida involvement in
pulmonary sites (Figure 2, C).
Thus, the saliva control is a simple, yet high value diagnostic tool because it renders mixed growth
pulmonary samples informative and feasible for further testing by cytology, rPCR-based and other
types of analyses.

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Saliva Controlled
Analysis Of Sputum
Culture

½ minute later
collect sputum collect saliva in
in ‘sputum’ cup ‘saliva’ cup

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+
sputum saliva

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in the lab: side-by-side culture

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Figure 1. Saliva Controlled Analysis Of Sputum Culture Enables Productive And Cost-effective
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Analysis Of Fungal Culture – the Protocol For Collection Of Paired Sputum-Saliva Specimens
1. For each event of sputum collection, the patient receives a pair of containers distinctly labeled as
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‘sputum’ and ‘saliva’. The containers are sterile, wide-mouth with tightly fitting screw top lids, marked
with the patient’s name. 2. In deep lung infections, productive cough is infrequent, sporadic and
happens more often at night. Therefore, patients must keep the containers handy - at all times, and
definitely next to bed at night - not to miss a rare sample. 3. The patient collects the expectorated
sputum in its container and closes it tightly 4. Then, right away, without drinking or swallowing, the
patient expresses saliva and collects it in the 2-nd, saliva container. Important - the patient must collect
a saliva sample after each sputum collection. 5. The patient must clearly record on both containers the
date, the time and any other pertinent information, like eating or drinking just prior to sputum
collection, preferably with a permanent marker.
In the lab: side-by-side culture of the paired sputum::saliva samples identifies the oropharyngeal
flora in the ‘saliva plate’ (little light blue circles for e.g., Candida sp. colonies). This, in turn, allows to
discern within the mixed growth, those fungal species that are specific to the deep sputum (coral-olive
circles represent filamentous fungi). A portion of the samples can be compared by cytology and/or PCR.
Thereby, the hard to get sputum specimens do not need to be rejected; this expedites accurate diagnosis
of the deepest fungal lung infections.

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A B

1 2 3 4 5 cm 1 2 3 4 5 cm
Deep Pulmonary Sputum -
purulent plugs (red arrows) Saliva control
intermixed with mucous secretions

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Mixed growth of filamentous Oropharyngeal Candida sp. in

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fungi and Candida colonies (blue saliva samples
arrows)
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Figure 2: Paired Saliva Controls Ensure That Pulmonary Samples Can Remain Informative
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Irrespective Of Intermixed Oral Flora.

A – a deep pulmonary sputum containing purulent plugs (red arrows) intermixed with mucous secretions; B -
the paired saliva control sample collected immediately after collection of the deep sputum; C & D - the fungal
growth produced by A & B, respectively.
Deep sputum is often expectorated in the form of purulent plugs in watery mucus (A), producing a mixed
growth (C) of filamentous fungi and Candida yeast (blue arrows). The immediate, cultured in parallel, saliva
control grows only Candida colonies, which represent the oropharyngeal flora (D). This allows subtraction of
the control results from the experimental, and reveals that the purulent plugs grow the filamentous fungi,
dominant here – Aspergillus fumigatus, and represent aspergilloma-associated pulmonary flora.
Procedure: The paired saliva control and the entire sputum sample, i.e., containing both the plugs and the
mucus, were cultured in parallel at identical conditions: they were plated, undiluted and unhomogenized, onto
Sabouraud dextrose agar (SDA) containing 50 μg/ml chloramphenicol (SC) and incubated at 37°C. Plates were
monitored and photographed unopened. As colonies appeared, their macroscopic and microscopic features were
examined. The identity of the fungi were confirmed: a) prior to culture a small portion was taken to prepare
sputum smears, which were stained with PAP, GMS, Gram and PAS for cytology; b) molecular identity of
grown single colonies was determined using PCR-based rRNA sequencing performed at HyLabs, Inc. In this
case, PET-CT demonstrated thick-walled cavitary lesions and aspergilloma within a bronchiectatic cavity.
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Table 1. The Saliva Control Culture Method Ensures the More Effective Use of Sputum Samples & Thus Averts
Misdiagnosis / Significant Under-detection of Deep Pulmonary Infections - An Overview.
Current guidelines for optimization of (left column) include recommendations for both the how and the when of sputum
collection (lines 1-6) and for sputum acceptance protocol by the laboratory (lines 7-12). They are implemented widely in
North American and European medical establishments (e.g., the http://www.uptodate.com and multiple references
therein); they work well for bacterial or viral disease - but not for deep pulmonary mycoses. The problems with obtaining
adequate sputum (center column) arise at multiple levels. The saliva control cultures (SSC) avert such problems (right
column) and we show why and how this is so.

Guidelines/ Common Problems with Guidelines - Saliva Control Culture (SSC)

Recommendations *
Collect samples with little or no
1.
saliva or postnatal discharge
at Multiple Levels. An Essential Solution
1. Sputum entering the oral cavity stimulates Samples with secretions are retained
saliva secretion within the time it takes to - SSC subtracts any oropharyngeal

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get and open a screw-cap container. contamination of fungal origin.
Rinse mouth with saline or 2. Does not avert presence of saliva nor of Scheduling deep sputum is impossible
2.

water before collecting sputum
Collect sputum 1-2hr before
3.
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postnatal discharge – but is also unneeded thanks to SSC.
3. The food bulk is removed by swallowing. In sputum cytology, saliva-only is an
Residual particles may interfere with sputum invaluable control, for any ‘foreign’

4.
eating

Collect sputum 1st thing in the -p

cytology (but do not raise saliva levels).
4. It is impossible to expectorate deep
matter as well
With the Saliva Control Culture (SSC)
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morning cavitary sputum voluntarily on schedule patients are free of schedules ->
Collect sputum at the same 5. ‘Collect-on-a-schedule’ yields samples that -> free to collect true deep samples,
5.
time for 3-5 consecutive days are wrong, that are not true sputum thus avoid testing of false-negatives->
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Collect early morning specimen

6. Supervision can neither help remove saliva -> SSC allows testing saliva-containing
6. under the direct supervision of
from samples nor induce sputum production. samples, ∴ no worry of false-positives
a nurse or a physician
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Reject sputum if not collected 7. As #6 above. Also - not only deep sputum SSC averts unnecessary re-ordering
7.
in the ordering client’s clinic is rare, it is produced, as a rule, at night. and testing of extra samples
Reject samples with saliva or 8. As #1 above. Also, deep sputum is often SSC enables subtraction of oral flora,
8.
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other mucus mucopurulent to begin with. thus prevents unnecessary rejection->

Reject samples with too much 9. ‘a lot’ vs. ‘a little” may fluctuate with and preserves sputum samples
9.
saliva different techs and/or on different days. informative for diagnosis
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Reject samples if it ‘is not quite
10. possible’ [quote] to separate
sputum plugs from saliva
Reject samples of less than 2 –
11.
15 ml in volume.
12. Accept only 1 sputum per 24hr
10. This is also a judgment call; some techs
are more successful in such separations.
11. In deep cavities, plugs get compacted and
dense, they are rarely >2 ml/sputum volume.
12. Purulent sputa are expectorated as a rule
in spells of 3-4/ day, every several days.
SSC ensures that technician time is
not wasted on removing saliva to get
‘clean’ purulent plugs
SSC saves from sick patients with rare
sputum to keep resubmitting samples
The multiple benefits of SSC, e.g.,
expedited diagnosis & cost efficiency -
cannot be overstated.

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2. Patient Instructions. Critical to the success of sputum tests is knowledgeable participation of
patients. While sputum collection is seemingly undemanding, many patients even if well educated,
surprisingly, cannot distinguish regular saliva from postnasal drip, or coughing up pharyngeal
secretions from the deep pulmonary cough. The reason, it seems, this is not something people normally
reflect on. But even the most basic explanation enables patients to distinguish among various types of
secretions, which in turn gives them the confidence to recognize and collect the correct samples.
Therefore – in addition to instructions on patient-assisted sputum collection as given in the Legend to
Figure 1, the patient must be made aware:
i) that orally cleared secretions are not all the same, but differ according to their origin: e.g., normal
saliva, postnasal drip, oropharyngeal, tracheo-bronchial and the deep lungs sputum;
ii) that the patient can identify the different secretion types based on the differences in their textures
and sensations they produce. The patient’s own description of their coughing patterns helps them
incredibly in this task.
iii) that the correct collection of samples is crucial for the correct diagnosis and for the patient’s well

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being.

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BOX 1: Mul ple Benefits of Saliva Controlled Sputum Tes ng
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1. Cost / work efficiency 2. Pa ent advancement 3. Disease management

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- price reduc on - no need for permits long-term monitoring

- tests are noninvasive
hospitaliza on or ambulatory of treatment effec veness
visits – facilitates collec on of cri cal in pulmonary mycoses
- price reduc on - no need for samples according to sputum
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facilitates personalized medic-

high cost diagnos c tests
- price reduc on - sputum
culture & cytology: are
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produc on, rather than a ine approach to treatment
standard on-for-all schedule
- facilitates the supply of hard
- pa ent treatment more

to get deep sputum samples:

inexpensive diagnos c tests
- price reduc on - no need to
separate sputum plugs from
saliva
independent of their
economic situa on, i.e, from
availability of transporta on,
personal accompaniment to
the clinic
- stops rejec on of sputum if
intermixed with saliva
- cancels restric ons on the
me of the day for sample
collec on

Expedites Correct Diagnosis

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3. CONCLUSIONS.
The results strongly advocate inclusion of saliva controls culture (SSC) in the optimization
guidelines of deep mycoses. This is critical to rectify the current situation of preventable under
detection of often-fatal fungal infections.
The problem is three-pronged: On one hand, there are the false positives commonly produced by the
oropharyngeal flora. On the other hand, samples collected in compliance with current guidelines, e.g.,
“first thing in the morning for 3-5 consecutive days”, will most likely not be true deep sputum samples
and therefore are often scored as false negative.
Thus, at the same time, a sputum culture may be both: false negative for deep respiratory fungi,
AND false positive for the upper fungi. N.B., this is precisely in patients suffering from deep fungal
infections, who are required to provide at least three sputum samples, while their cough is usually
unproductive. Then, numerous samples contain saliva; these are actively rejected even prior to culture,

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and additional ones are ordered (Table 1).
Thus, the delay in or misdiagnoses are not uncommon even in obvious candidates - the

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immunocompromized patients, where pathogenic fungi are expected and are looked for. The situation
can be considerably worse for those with apparently healthy immune systems, in whom deep fungal

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infections are not usually suspected yet can lead to serious and fatal disease (Denning and Chakrabarti
2017).
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With the incidence of high mortality fungal diseases rising worldwide, the role of effective testing
becomes ever more significant.
The saliva control culture protocol presented here is a high value tool for better diagnosis of
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pulmonary fungal infections because:

i) SSC preserves the hard to obtain but informative sputum samples, which are otherwise commonly
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discarded in large numbers due to difficulty to comply with sputum collection guidelines. This waste is
particularly indefensible in cases of sick bedridden patients with largely unproductive cough.
ii) SSC enables subtraction of any oropharyngeal growth and thus drastically reduces the concern
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for false positive results.

iii) The use of saliva controls releases sputum collection from timetables, bringing down the number
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of false negatives because it yields significantly high rates of true sputum samples.
iv) For cytological analysis of sputum, saliva-only slides serve as valuable controls, among others,
for spotting the false negatives and true sputum confirmation.
v) For diagnosis, SSC increases the ratios of true sputum samples and thus offers significant savings
for both the patients and the medical establishment.

To summarize: SSC is de facto a new addition to the current procedures; it does not interfere with
existing guidelines, yet allows for non-standard, patient- and disease-oriented testing for deep
pulmonary infections in the context of unproductive cough. It is simple, cost-efficient for prevention of
misdiagnoses and expediting correct diagnoses.
Thus, the use of saliva controls (SSC) along with sputum cultures is clearly beneficial for everyone
involved and is highly recommended for inclusion in the sputum testing optimization guidelines.

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Pashley CH, Fairs A, Morley JP, Tailor S, Agbetile J, Bafadhel M, Brightling CE, Wardlaw AJ.
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The UpToDate site:

http://www.uptodate.com/contents/sputum-cultures-for-the-evaluation-of-bacterial-pneumonia

CONFLICT OF INTERESTS
The author declares that there are no conflicts of interests.
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Conflict of interests: The author declares that there are no conflict of interests.

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