Keywords
influenza vaccination - COVID-19 - surveillance - influenza activity - Libya
Introduction
The World Health Organization (WHO) estimates that one billion influenza cases, three
to five million severe cases, and 290,000 to 650,000 influenza-related respiratory
deaths occur annually worldwide.[1] Experts in the Centers for Disease Control (CDC), United States, have predicted
a severe flu season for the 2021 to 2022 winter because of reduced population-level
immunity due to common influenza infections since March 2020 following the coronavirus
disease 2019 (COVID-19) pandemic.[2]
Significant declines in influenza activity have been observed worldwide since March
2020, except in certain tropical regions where small activity was identified. Reasons
for this were attributed to the emergence of severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) and the COVID-19 pandemic, where physical and social distancing, masking,
and travel restrictions played an important role.[1]
Between late 2019 and the end of 2020, only 19% of tested respiratory samples in global
surveillance were positive for the influenza virus.[3] In North Africa, influenza cases increased in late 2019 in Egypt and Morocco, while
influenza detection peaked in January to February 2020 in Algeria and Tunisia. Most
infections were influenza A (H1N1) pdm09 viruses representing 69% of all the viruses
identified between Nov 2019 and April 2020.[3]
In the northern hemisphere, the influenza season typically starts in October and commonly
peaks in January or February. In contrast, in the southern hemisphere, the season
starts in their winter months counterparts.[4] Most of Libya lies within the northern hemisphere and a small part within the southern
hemisphere, which suggests influenza can be all year round.
In Libya, COVID-19-positive cases reached 38.7% of tested samples during the omicron
surge in February 2022. Reverse-transcription polymerase chain reaction (RT-PCR) was
the primary diagnostic test employed in public laboratories in Libya, and rapid antigen
tests were also used in many point-of-care and filtration units. However, RT-PCR technology
was only recently introduced into the majority of these public laboratories during
the COVID-19 pandemic, and most other viral illnesses were diagnosed on a clinical
basis.
In addition, symptoms of influenza and other respiratory infection are similar to
that of COVID-19, making a diagnosis based on clinical presentation alone tricky.
Therefore, the causative virus must be tested to confirm the diagnosis.[2]
This preliminary report aims to estimate the frequency of common respiratory pathogens
in samples tested at the Libyan Biotechnology Center in Tripoli, Libya, referred for
COVID-19 testing for multiple reasons employing a multiplex RT-PCR molecular test.
Materials and Methods
Patient Population
This is a cross-sectional study. We analyzed nasopharyngeal swabs in viral transport
media from 2,186 samples that arrived at our laboratories at the Libyan Biotechnology
Research Center, Tripoli, Libya, between December 1, 2021, and January 31, 2022. The
inclusion criteria were all patients referred for SARS-COV-2 testing with clinical
features suggestive of an upper respiratory tract infection and/or pneumonia.
Influenza and SARS-COV-2 Detection
Collected nasopharyngeal swab samples were analyzed by Xpert Xpress Xpress SARS-CoV-2/Flu/RSV
(Cepheid, 904 Caribbean Drive Sunnyvale, CA 94089 USA) multiplexed real-time RT-PCR
test intended for the simultaneous qualitative detection of SARS-CoV-2, influenza
A, influenza B, and respiratory syncytial virus (RSV) viral RNA and the test was run
on the GeneXpert Xpress System (Cepheid, United States).
The manufacturer's instructions were followed. Briefly, using the pipette supplied,
approximately 300 μL of viral transport media of the swab was added to the assay cartridge.
No viral RNA extraction step was needed. The cartridge was loaded into our 16-module
GeneXpert Xpress System. The test was completed within approximately 37 minutes for
negative samples and depending on viral load for positive ones.
According to the manufacturer, the Xpert Xpress Flu/RSV test was evaluated against
multiple strains of influenza A H1N1 (seasonal pre-2009), influenza A H1N1 (pandemic
2009), influenza A H3N2 (seasonal), avian influenza A (H5N1, H5N2, H6N2, H7N2, H7N3,
H2N2, H7N9, and H9N2), influenza B (representing strains from both Victoria and Yamagata
lineages), and RSV subgroups A and B (RSV A and RSV B).[3]
Statistical Analysis
Frequency of cases, estimates of average cycle threshold (Ct), and scatter plot charts
were analyzed using the Cepheid GeneXpert system accompanying software.
Results
The majority of tested cases were negative for all viral panels. About 27% (589/2186)
of study patients tested positive for SARS-COV-2, 2.8% (61/2186) were positive for
influenza A virus, 0.18% (4/2186) influenza B virus, and 1.4% (31/2186) tested positive
for RSV ([Table 1]). Two patients had coinfection of influenza A and SARS-COV-2, and one had coinfection
of SARS-COV-2 and RSV. Ten cases were healthcare workers. All patients did not receive
influenza vaccination in 2021.
Table 1
Frequency of positive cases for the four viral targets (SARS-COV-2, influenza A and
B, and RSV) in the study sample (n = 2,186) from 01/12/21 to 31/01/22
|
Target analyte
|
Frequency of positive cases (%)
|
Average cycle threshold
|
|
SARS-COV-2
|
589 (26.9)
|
26.4
|
|
Influenza A
|
61 (2.8)
|
23.8
|
|
Influenza B
|
4 (0.18)
|
15.7
|
|
RRSV
|
31 (1.4)
|
29.5
|
Abbreviations: RSV, respiratory syncytial virus; SARS-COV-2, severe acute respiratory
syndrome coronavirus 2.
The Ct trend for influenza-positive patients showed that most cases presented during
early disease were reflected by lower Ct (mean Ct 23.8 for influenza A and 15.7 for
influenza B; [Fig. 1]). Ct trend for SARS-COV-2 positive patients showed that most cases were between
January 14 and January 29, while RSV cases were scattered throughout the month ([Fig. 2]).
Fig. 1 Cycle threshold (Ct) trend for influenza A and influenza B positive cases. Green
squares are positive samples and blue circles are negative samples (above 40 Ct for
positivity set by the test manufacturer).
Fig. 2 Cycle threshold (Ct) trend for severe acute respiratory syndrome coronavirus 2 and
respiratory syncytial virus positive cases. Green squares are positive samples and
blue circles are negative samples (above the 40 Ct for positivity set by the test
manufacturer).
Discussion
We observed for the first time since the COVID-19 pandemic the emergence of new positive
influenza cases in samples from suspected COVID-19 patients in our laboratory using
a multiplex assay for SARS-COV-2, influenza, and RSV detection.
We identified 65 cases positive for influenza (∼3%). Four cases were influenza B,
and all other patients were influenza A. The assay we used only indicates if it is
influenza A or B. We estimate a higher proportion because many cases were not tested
and probably had a milder illness.
The US most circulating influenza strain is A (H3N1). It would be advantageous to
type influenza strains to assess the epidemiological status and track variants. Surveillance
performed at the end of 2019 and early 2020 showed that most circulating A(H1N1)pdm09
viruses were the same phylogenetic cluster.[1]
In our sample, few patients were healthcare workers. This subgroup should have received
their vaccination for influenza since the beginning of the fall season.
We emphasize the importance of making RT-PCR influenza detection assays more widely
available in public and private laboratories. This detection method could be efficiently
utilized in most public COVID-19 diagnostic laboratories because they were recently
established and equipped with RT-PCR machines and trained personnel. RT-PCR method
has a sensitivity of approximately 80% and a specificity of 90%.[4]
There are potential consequences in discovering patients diagnosed with the influenza
virus during this time when COVID-19 cases are expected to peak, allowing new highly
transmissible Omicron subvariants to emerge. The first issue is the difficulty in
making the correct diagnosis without specific viral testing like RT-PCR. Another critical
concern is that the same health resources needed for COVID-19 control will also be
utilized to fight any influenza activity surge, leading to an extra burden on the
health system.
Also, because of the COVID-19 pandemic, all suspected cases of pneumonia are routinely
managed as SARS-COV-2 infections risking patients with influenza not receiving the
appropriate treatment. The use of corticosteroids in influenza is not recommended
because it is associated with higher mortality and nosocomial infections.[5] When influenza patients are admitted to COVID-19 isolation units based on their
high-resolution computed tomography findings only without viral testing, there is
a risk of coinfection with SARS-COV-2, potentially increasing morbidity and mortality
of the patient.
The gold standard for confirming influenza virus infection is RT-PCR or viral culture
of samples taken from nasopharyngeal or throat swabs. Rapid diagnostic tests for influenza
are available and are becoming more widely used. These tests have high specificity
but only moderate sensitivity.[6]
A Libyan national advisory committee on influenza control was established in 2014,
and an official recommendation for influenza vaccination was made in 2013.[7] Influenza vaccination in Libya was unavailable in 2021 and the winter of 2022 for
the first time since the official recommendation.
The flu season activity most commonly peaks in February and can last into May. Also,
during most flu seasons, different flu viruses spread throughout the season. Current
US flu vaccines are designed to protect against four different flu viruses: A(H1N1),
A(H3N2), and two flu B viruses. The American CDC recommends influenza vaccination
despite evidence showing reduced vaccine effectiveness against the current predominant
H3N2. However, some protection against those H3N2 viruses was found.[8]
The COVID-19 pandemic hugely reduced the number of influenza detections and adversely
affected the generation and reporting of virus characterization because of reduced
human and laboratory resources and increased workload due to the response to the pandemic.[9] However, new genetic strains were reported, and some had spread globally.[1]
[9]
This research, however, is subject to several limitations. Unfortunately, we did not
have enough RT-PCR reagents in our laboratory to screen more patients for an extended
period and cover hospitals.
As the COVID-19 pandemic continues, the most significant concern is the development
of an influenza outbreak in the upcoming months. As mentioned above, population immunity
to influenza has decreased because fewer people were exposed to the virus. Therefore,
continuing annual influenza vaccination is still critical to increasing population
immunity.
In a monthly update by the WHO Eastern Mediterranean Region, the October 2022 statistics
of 17 countries that reported influenza data to FluNet and/or EMFLU showed that the
number of influenza cases increased in October compared with the previous 4 months.[10]
Due to the circulation of both influenza and SARS-COV-2 viruses during this season,
coinfection is possible and should be considered. Also, it is crucial to develop updated
guidelines for managing patients with suspected viral pneumonia during this time and
offer education programs for healthcare providers to ensure proper diagnosis and treatment
of such cases.
Conclusions
The results of the present study revealed that influenza infections were also rising
along with COVID-19. As the COVID-19 pandemic continues, the most significant concern
is the development of an influenza outbreak in the upcoming months. Therefore, continuing
annual influenza vaccination is critical to increasing population immunity. There
is a need for continuous monitoring in national laboratories to detect any zoonotic
events and important viral evolution. National influenza surveillance and testing
should be conducted. Furthermore, sequencing and antigenic characterization should
be performed regularly. Providing molecular testing for respiratory viruses including
influenza and respiratory syncytial virus in addition to SARS-COV-2 could not be more
emphasized to prevent a potential tripledemic.
