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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

Reverse-Transcription Loop-Mediated Isothermal Amplification

and alternative protocols for lower cost, large-scale COVID-19 testing:

lessons from an emerging economy

Ismael Segura-Ulate1; https://orcid.org/0000-0002-6069-6252

Alejandro Bolívar-González1; https://orcid.org/0000-0001-7305-2248

Germán Madrigal-Redondo1; https://orcid.org/0000-0002-9856-4044

Santiago Nuñez-Corrales2; https://orcid.org/0000-0003-4342-6223

Andrés Gatica-Arias3; https://orcid.org/0000-0002-3841-0238

1. Instituto de Investigaciones en Farmacia, Facultad de Farmacia, Universidad de Costa Rica, San Pedro, Costa Rica;

ismael.segura@ucr.ac.cr, abolivar1993@gmail.com, generacionlcr96@gmail.com

2. Illinois Informatics and National Center for Supercomputing Applications, University of Illinois Urbana-Champaign,

Champaign, Illinois, United States of America; nunezco2@illinois.edu

3. Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica;

andres.gatica@ucr.ac.cr

Received 08-VII-2021. Corrected 25-I-2022. Accepted 15-III-2022.

ABSTRACT

Introduction: Most successful cases of COVID-19 pandemic mitigation and handling have relied on extensive

reverse-transcription quantitative polymerase chain reaction (RT-qPCR). However, many emerging economies

have struggled with current molecular testing demands due to economic, technical and technological constraints.

Objective: To define a potential diagnostic protocol to increase testing capacity in current and post-pandemic

conditions.

Methods: We reviewed the literature, patents and commercial applications, for alternatives.

Results: We found a good potential in saliva samples, viral inactivation and quick RNA extraction by heating;

the use of an isothermal technology such as loop mediated isothermal amplification (LAMP) and naked eye

test-result visualization by in-tube colorimetry or turbidity.

Conclusions: Saliva samples with quick RNA extraction by heating and colorimetric LAMP are promising

options for countries with economic and infrastructure limitations.

Key words: emerging economies; molecular testing; SARS-CoV-2; saliva; loop mediated isothermal

amplification.

https://doi.org/10.15517/rev.biol.trop..v70i1.47407

BIOMEDICINE

The advent of SARS-CoV-2 and our col-

lective failure in pandemic handling: In late

2019 a SARS-like novel disease was discov-

ered in the city of Wuhan, China. By January

2020, the novel infection had been denomi-

nated COVID-19 and its causal pathogen was

identified as a new coronavirus named SARS-

CoV-2. The high infection rate of SARS-CoV-2

led to a rapid worldwide spread, forcing the

World Health Organization (WHO) to declare

COVID-19 a pandemic in April 2020. As of

today, early June 2021, there have been nearly

174 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

176 million official COVID-19 cases and more

than 3.8 million deaths worldwide (Johns Hop-

kins University & Medicine, 2020).

After many months of attempting to

achieve epidemiological management through

general containment measures (e.g. provisional

lockdowns, social distancing, mandatory use

of face masks) it has become apparent that

such measures alone do not suffice to keep the

virus in check and prevent its spread among

large swaths of the population. As it has been

the case with previous outbreaks, evidence

indicates that the most effective non-pharma-

cological strategies to contain the spread of

the virus include some combination of early

detection, contact tracing and isolation, like

the so called Find, Test, Trace, Isolate, and

Support (FTTIS) approach (Rajan et al., 2020).

This type of epidemiological management has

traditionally relied on a clear clinical diagnosis

that is confirmed by a reliable test. However,

due to the large proportion of asymptomatic

COVID-19 cases and the transmissibility of the

SARS-CoV-2 from those asymptomatic indi-

viduals, the only alternative to detect enough

cases to stop the spread of the virus is by using

large-scale or random testing (Gandhi et al.,

2020); doing so eliminates future pools of

newly exposed individuals that replicate and

amplify the contagion cycle. Countries that

have used large-scale testing, like South Korea,

appear to have been most successful at flatten-

ing the curve of cases, especially early during

the pandemic (Chang et al., 2020). Even under

the current situation of active mass COVID-19

immunizations or in a post-pandemic world, a

FTTIS approach will continue to be a necessary

tool in our epidemiological arsenal.

Unfortunately, any successful FTTIS

approach for COVID-19 epidemiological man-

agement is resource intensive and costly. As

evidence of this, scarcity or high cost of some

components necessary for a FTTIS approach

has prevented large-scale deployment of this

type of strategies across several countries.

Most prominently, scaling-up of testing capac-

ity using real-time quantitative polymerase

chain reaction (RT-qPCR), the current gold

standard technique for COVID-19 diagnosis,

stands as one of the most difficult bottlenecks

to overcome in order to implement the large-

scale random testing necessary for epidemio-

logical management. Due to the many intrinsic

technical difficulties and requirements of RT-

qPCR, it has been challenging if not impossible

for many countries around the world to scale up

the RT-qPCR testing capacity for COVID-19 to

meet the current needs. The struggle to ramp up

RT-qPCR testing capacity has been especially

obvious during periods with high numbers

of cases and in emerging economies with

more limited healthcare systems. Even when

pool-based sampling methods can increase the

population-level sensitivity of detection for at-

large strategic public health responses (Mutesa

et al., 2020), these remain constrained by the

same technical bottlenecks and do not exclude

intensive individual testing after a critical posi-

tivity rate has been reached.

Costa Rica is a middle-income emerging

economy with an internationally praised uni-

versal healthcare system which has nonetheless

struggled to meet several of the unexpected

demands imposed by the COVID-19 pandemic.

After a substantial investment in consumables,

facilities, training, personnel and equipment the

official capacity for RT-qPCR COVID-19 tests

has plateaued at 4 500 tests per day. However,

this number does not reflect the real number of

daily tests –a fraction of daily tests corresponds

to certification of known cases for nosocomial

purposes– and the level at which COVID-19

testing is performed in Costa Rica yields very

high positivity rates (i.e. percentage of positive

cases over total tested individuals), around 36

%. This positivity rate, however, may increase

during epidemiological peaks (Barquero, 2020)

or decrease when public policy measures have

a significant effect. The current testing strategy

mainly targets either symptomatic individuals

or those with a known immediate epidemiolog-

ic linkage, excluding most of the asymptom-

atic cases and leaving plenty of loose ends to

maintain effective epidemiological tracing and

management (Ministerio de Salud, 2020). In

point of fact, the high positivity rate correlates

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with complete contact tracing being infeasible

since July 2020. Furthermore, it is unlikely

that Costa Rica will be able to further increase

RT-qPCR-based COVID-19 testing in the short

term to reach a positivity rate of less than 5 %,

which is the threshold initially recommended

by the WHO for proper epidemiological sur-

veillance (World Health Organization, 2020).

This concern led our interdisciplinary research

group to look for effective and efficient alter-

natives that may help Costa Rica to drastically

improve the handling of the ongoing health

crisis. The current review and opinion article

describes our conclusion that alternatives to the

traditional RT-qPCR-based COVID-19 testing

are necessary for Costa Rica to improve the

outcome of this crisis, and that this lesson can

be generalized to other developing nations and

emerging economies. This idea is beginning

to materialize in a clinical validation of some

of these testing alternatives in an effort coor-

dinated by several researchers and healthcare

officials from different Costa Rican institu-

tions. We also recount here some of the lessons

learned that may prove to be valuable for other

countries and healthcare systems battling with

similar conditions.

The bottlenecks and limitations of the

traditional RT-qPCR-based COVID-19 test-

ing: Our current dependence on RT-qPCR

originates from the well-known flexibility and

reliability of this technology. The basic form

of PCR was developed 37 years ago. Since

then, many different forms of PCR have been

developed to detect all sorts of pathogens and

remains especially useful for diagnosis of

emerging infectious diseases. Theoretically,

PCR could be used to detect any pathogen

present in a biological sample and, to this

day, RT-qPCR remains the primary diagnostic

option for several viral and bacterial infections.

Due to its intrinsic amplifying properties, all

forms of PCR have very high sensitivity and its

targeting of pre-defined and carefully selected

genetic sequences secures a very high –and

sometimes nearly perfect– specificity. How-

ever, nucleic acid amplification technologies,

including RT-qPCR, tend to be more expensive

and technically more complex than other types

of diagnostic testing options. In the case of

COVID-19 testing, it is unfortunate that simple

and inexpensive alternatives (e.g. antibody and

antigen testing) either miss the early infectiv-

ity window or lack the high sensitivity levels

required to become an effective tool for epide-

miological surveillance (Benzigar et al., 2021;

Nagura-Ikeda et al., 2020).

As mentioned previously, RT-qPCR is a

complicated test to perform. Most of the han-

dling steps of this test must be carried out by

either a highly trained laboratory technician or

“pipetting” robots (Fig. 1).

Under normal circumstances, total pro-

cessing time in the laboratory (not including

sample collection, transportation and storage)

for this type of RT-qPCR test is 3 to 6 h

depending on whether steps have been auto-

mated by the use of robots or carried out by

technicians. Furthermore, the most prevalent

workflow with RT-qPCR must be carried-out

in a biosafety level 2 (BSL-2) laboratory in

order to protect the personnel from contagion

due to aerosols arising from the samples, which

means that every COVID-19 testing labora-

tory must have at least one BSL-2 cabinet.

Additionally, RT-qPCR requires a real-time

thermal cycler to be performed, which lim-

its the rate of sample processing in a facil-

ity to the combined number of sample spots

or “wells” available among all the real-time

thermal cyclers in that facility. It should be

noted that the most common configuration of

a real-time thermal cycler is 96 wells and the

turnaround time is typically 2 h for a 2-in-1

reaction encompassing both retrotranscription

and amplification. Thus, the total sample pro-

cessing rate using the most prevalent RT-qPCR

protocol is limited by a composition of differ-

ent bottlenecks that are difficult to overcome

individually in any healthcare system, let alone

all of them together. These bottlenecks include

i) the number of BSL-2 laboratories available,

ii) the processing or “pipetting” capacity of the

technicians or robots and iii) the availability of

“wells” per real-time thermal cycler to carry

176 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

out the amplification and detection. In order to

scale-up testing capacity by RT-qPCR, all of

these bottlenecks must be overcome together

in every single testing facility since a weak link

in the chain may hamper the investments and

improvements in other steps of the workflow

or even render them useless.

MATERIALS AND METHODS

We reviewed the corresponding literature,

patents and commercial applications for alter-

native technologies and protocols to detect

SARS-CoV-2 in human samples that do not

rely on RT-qPCR. Likewise, we reviewed the

corresponding literature, patents and commer-

cial applications for evidence of alternative

sample collection and genetic material extrac-

tion that does not rely on nasopharyngeal

swabs and traditional RNA purification. From

this information we elaborated an argument for

the use of alternative technologies that could

effectively overcome the current bottlenecks of

RT-qPCR-based diagnosis.

RESULTS AND DISCUSSION

Testing technology requirements for

effective public health responses: In terms

of public policy making, the role of test-

ing technologies is to inform strategic and

tactical recommendations. These are devised

using test outcomes obtained from a limited

number of COVID-19 samples that must be

both sufficient and representative (Hilborne et

al., 2020). Most generally, recommendations

aim to address the most relevant and current

stage of an epidemiological crisis. At the onset

and early stages, FTTIS aims to contain and

remove infective individuals to prevent further

spread, particularly of asymptomatic cases, as

a way to delay or even prevent reaching a rapid

growth phase; simulation of COVID-19 spread

under various systematic testing regimes sug-

gests that remotion of asymptomatic individu-

als is one of the main mechanisms behind the

effectiveness of scalable testing technologies

(Núñez-Corrales & Jakobsson, 2020). If a rapid

growth phase is reached, testing must intensify

proactively as a way to understand how the

Fig. 1. Schematic workflow of COVID-19 testing using the standard RT-qPCR protocol. Two separate steps require sample

handling and pipetting by a technician or robot. Red biohazard symbols illustrate steps with potentially bio-contagious

samples, while gray biohazard symbols represent steps that no longer have contagious potential. Created with BioRender.

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underlying population structure fuels the rise

in cases, and then systematically craft measures

capable of preventing the overload of health-

care systems and irreversible economic dam-

age. When mitigation efforts are successful and

the situation is stable, testing can be optimized

to detect and control new outbreaks and drive

the epidemic process to a manageable level

until pharmacological alternatives are applied

and herd immunity is reached. The ability to

use testing as an anticipatory tool depends on

the existence of testing technology capable

of scaling rapidly and reliably across several

orders of magnitude depending on the stage

of the epidemic process. As evidenced by the

reasoning above, RT-qPCR cannot particularly

provide such flexibility.

COVID-19 testing technologies must

strive to minimize false negatives by increas-

ing their sensitivity (West et al., 2020) since

these represent individuals that can restart

and amplify even more the contagion cycle.

In addition, testing must also provide the

flexibility to address intrinsic uncertainties in

the process (Gray et al., 2020). Both can be

resolved by re-testing individuals. Essentially,

an adequate testing technology not only should

scale to accommodate sudden increases in new

cases but also allow re-testing of known cases

whose outcome was uncertain or whose epide-

miological link is updated. In a situation such

as the Costa Rican one where RT-qPCR testing

is extremely limited, this forces a compromise

between discovering new cases and certifying

the recovery of existing ones; testing people

before they are released from hospitalization

takes priority. Statistically, we start by looking

at the disjoint sensitivity between two con-

secutive tests and σA∨B given by

and since the same test is applied twice (i.e.

A=B)

which is a monotonically increasing con-

vex function in the domain σA ∈ [0,1] with

range σ2A ∈ [0,1]. Succinctly, applying two

consecutive disjoint tests reduces the prob-

ability of false negatives. Conversely, specific-

ity τA∨B decreases with each test quadratically

for A=B as

Arguably, lower specificity of two disjoint

tests is not a significant concern as long as the

specificity of a single test is high enough. For

instance, τA = 0.95 for a single test produces a

combined disjoint specificity of τ2A ≈ 0.9. False

positives are in this case benevolent since the

protocol for a new positive case entails manda-

tory isolation for two weeks, a time beyond the

12 days required for more than 97.5 % of indi-

viduals to have ended their incubation period

(Lauer et al., 2020). Worst case scenario, excess

false positives lead to a limited number of indi-

viduals being isolated, decreasing the pool of

available susceptible individuals slightly. In

consequence, any scalable testing technology

for COVID-19 must ensure high specificity

geared towards repeated testing of individuals

for rapid disambiguation. Saliva-based tech-

nologies such as the alternative reported in

this article overcomes this limitation thanks to

having a specificity at least comparable to that

of traditional nasopharyngeal swabs samples

(Chen et al., 2020; Takeuchi et al., 2020; Wil-

liams et al., 2020; Wyllie et al., 2020).

Finally, time-to-outcome is a critical vari-

able for COVID-19 testing technologies. The

ability to perform contact tracing to stop conta-

gion depends on rapidly acquiring and process-

ing samples from suspected individuals or from

proactive measures. The complexity RT-qPCR

entails prevents having certified test outcomes

within the first 24-48 h in most cases. This

time is critical for contact tracing to identify

potentially exposed individuals and extend the

search promptly; simulation results suggest that

performing the testing-contact tracing cycle

within the same day can significantly reduce

the effective reproductive number regardless of

contact tracing technology (Kretzschmar et al.,

2020). Since performing the test depends on

specialized equipment and trained personnel, it

cannot usually be geographically decentralized.

178 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

Hence, a scalable technology should also aim

to be time and geographically scalable, particu-

larly in entry-level health attention centers.

Simplified sample collection by using

saliva: Nasopharyngeal and oropharyngeal

swabs are the main method of sample collec-

tion for COVID-19 testing (Fig. 1). However,

the invasive nature of this procedure can be

uncomfortable for many patients and may

even cause aversion to COVID-19 testing,

potentially reducing public compliance with

health authorities. Nonetheless, the presence

of SARS-CoV-2 in saliva is indisputable and

saliva samples for COVID-19 testing have

been proven to be effective in the detection

of the virus in a non-invasive manner using a

RT-qPCR-based protocol (Chen et al., 2020;

Takeuchi et al., 2020; Williams et al., 2020;

Wyllie et al., 2020).

When sensitivity for COVID-19 testing

has been compared between nasopharyngeal

swabs and saliva samples using traditional viral

RNA extraction methods and amplification by

RT-qPCR, the saliva sample has shown only a

slightly lower sensitivity than its comparison

standard, thus, both types of samples should

be regarded as equally useful from a clini-

cal standpoint. In the case of specificity, both

technologies have shown near perfect scores

(Jamal et al., 2021; Pasomsub et al., 2020;

Procop et al., 2020; Teo et al.,2021; Uwamino

et al., 2020).

From a practical standpoint, using saliva

samples for COVID-19 testing offers several

advantages over traditional swabs that could

be very beneficial during a large-scale testing

initiative. Such advantages include better pub-

lic acceptance and compliance, shorter sample

collection times, reductions in frontline person-

nel in charge of sample collection and even the

development of sample self-collection to be

shipped to or dropped-off at a testing facility.

Another advantage of using saliva samples

is the availability of many types of low-cost

sterilized screw-capped containers that can be

used to collect liquid samples, including small

urine and stool sample cups, falcon tubes and

an assortment of other laboratory contain-

ers such as cryogenic tubes. The use of this

readily available variety of containers would

make the saliva sample collection process less

susceptible to the common market shortages

experienced during the last few months for

most COVID-19 testing consumables, includ-

ing medical swabs.

Quick viral RNA extraction and virions

inactivation by heating: Traditional RNA

extraction is a lengthy and costly process

requiring highly trained technicians or spe-

cialized equipment like “pipetting” robots in

order to isolate RNA from other biochemi-

cal components like DNA, proteins and lip-

ids. Even though purified RNA is the ideal

genetic material to perform retrotranscription

and subsequent amplification in RT-qPCR or

any isothermal amplification technology, it is

not necessary to have this material completely

isolated from other biochemical components

present in some biological samples. In fact,

most forms of PCR and almost any nucleic

acid amplification technology allow for some

level of flexibility regarding DNA, protein and

lipid carryover. In most samples containing

trace amounts of these types of biochemical

components such as saliva and swabs, it is

perfectly possible to perform a direct sample-

to-amplification protocol bypassing traditional

RNA extraction (Esbin et al., 2020). Quick

viral RNA extraction by means of a heating

step has been developed as an alternative meth-

od of viral RNA extraction. This quick heating

step allows viral RNA to be accessible for the

retrotranscriptases and polymerases used in

PCR or isothermal amplification. Alternatively,

some protocols may either replace or comple-

ment the rapid high temperature heating step

with a long mid-temperature heating step, such

as 65 °C for 30 min after mixing the sample

with a lysis buffer that contains detergents

and a highly active protease like Proteinase K

(Esbin et al., 2020; L’Helgouach et al., 2020).

This long mid temperature heating step with

a lysis buffer seems to achieve similar results

to the quick high temperature heating step in

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regard to facilitating the release of the viral

RNA and the two methods might work inter-

changeably. However, the fact that the rapid

high temperature heating step achieves good

results without the need for a lysis buffer makes

it more attractive to our goals of simplifying

test development. Furthermore, one more point

must be taken into account when it comes to

this simplified method of viral RNA extraction,

and that is RNA degradation. Because the viral

RNA is released into a biological sample with

other components, it immediately becomes a

target for endogenous RNAses in that sample.

This problem can be disregarded if the sample

is tested immediately, but sample storage for

any length of time might have an effect on

the detectable viral RNA in it. For that reason,

some protocols may also include in the sample

buffer or the reaction mix RNAses inhibitors or

carrier RNA as protectors of the integrity of the

viral RNA (Wei et al., 2021).

Quick viral RNA extraction using a heat-

ing step has one more important advantage

over the traditional RNA extraction. During

a heating step at 95-98 °C for 10 to 15 min,

any virions potentially contained in a saliva

sample are inactivated and all samples can be

regarded as of low biosecurity risk after that

point (Fig. 2). If sample processing protocols

are devised in which sample containers are

properly disinfected at the point of collec-

tion and never opened inside the laboratory

before the heat inactivation step, processing of

all these samples could be carried out in any

BSL-1 facility.

This reduces the biosecurity equipment

necessary to process samples, most notably the

biosafety cabinet. It also opens the possibility

of establishing temporary testing facilities in

entry level healthcare attention centers that

normally don’t have BSL-2 laboratories and

other places of interest that could not support

a traditional clinical laboratory (e.g. schools,

airports, factories, etc.). It must be mentioned

that the protocol developed by Wei et al (2021)

uses no heating step and takes the saliva sample

directly into a single step RT-LAMP reaction.

This alternative protocol seems advantageous

for currently functioning testing laboratories as

it greatly simplifies most of the testing work-

flow; however, we consider that this method

still does not fit well with our interests of

Fig. 2. Schematic workflow of simplified COVID-19 testing combining saliva sample, quick viral RNA extraction,

RT-LAMP and results visualization by in-tube colorimetry. Red biohazard symbols illustrate steps with potentially bio

contagious samples, while gray biohazard symbols represent steps that no longer have contagious potential. Since the first

step inside the laboratory is the sample heating to release the viral RNA extraction and inactivate the virions, and this step

is performed without opening the sample container, the entire process can be carried out in a BSL-1 laboratory. Created

with BioRender.

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completely simplifying COVID-19 testing

since bypassing of all heating steps would

mean that the sample must still be handled in a

BSL-2 laboratory.

The advantages of isothermal ampli-

fication technologies: Different nucleic acid

isothermal amplification technologies have

been developed over the last few decades.

These include Nicking Endonuclease Sequence

Amplification Reaction (NESA), Ligase Chain

Reaction (LCR), Recombinase Polymerase

Amplification (RPA), Strand Displacement

Amplification (SDA) and Loop Mediated Iso-

thermal Amplification (LAMP), among others

(Fakruddin et al., 2013; Kiesling et al., 2007;

Zhao et al., 2015). All of these technologies

have the capacity to detect specific genetic

sequences in the same way that RT-qPCR does;

however, all forms of isothermal amplification

have one important advantage over the dif-

ferent forms of PCR; as the term isothermal

implies, these technologies perform the entire

amplification reaction at a single temperature

and therefore, there is no need for a thermal

cycler. Thus, the reaction for the different

forms of isothermal amplification can be car-

ried out using any stable heating source such as

a water bath, a heating block, a hoven or even

a water container on top of a heating plate. This

particularity allows the development of isother-

mal amplification protocols using only basic

laboratory tools like pipettes and opens the pos-

sibility of creating equipment-free diagnostic

tests. Furthermore, the number of reactions or

samples that can be performed with isothermal

amplification does not rely on the number of

spots or “wells” available in a device but rather

the number of tubes with samples that can be

fitted into a heat source like a water bath.

In addition, isothermal amplification tech-

nologies perform the entire reaction faster than

PCR since it is not necessary to change the

temperature of the reaction. Likewise, the reac-

tion master mix for isothermal amplification

can be developed to contain both a retrotrans-

criptase and a polymerase that work in parallel

and at the same temperature, thus performing

both reactions at the same time and reducing

the total time required.

For example, this combination of two steps

into one reaction creates what is known as

retrotranscription-LAMP (RT-LAMP). These

key differences reduce the total reaction time

of isothermal technologies when compared to

standard RT-qPCR protocols. As an example,

most RT-LAMP protocols for SARS-CoV-2

detection take 30 min to complete a single step

reaction while the standard RT-qPCR diagnos-

tic protocol usually takes 1 to 2 h to complete

both retrotranscription and amplification.

Despite all of these advantages, isother-

mal amplification technologies in general still

suffer from limitations compared to the more

traditional PCR. In our experience, probably

the greatest drawback of developing tests based

on isothermal technologies is the lack of a

well-developed manufacturing network with

ready-to-use consumables such as recombi-

nant enzymes, master mixes and additives.

In our search for this type of products to use

in RT-LAMP, we found a limited number of

manufacturing companies that commercialize

them but the number of providers around the

world and the variety of products is but a small

fraction of what can be found in the market for

PCR consumables. Another limitation com-

pared to PCR is the lack of options to perform

tests using primers with differential affinity by

temperature such as touchdown, gradient or

nested PCR. In the particular case of LAMP,

it also suffers of other drawbacks. It requires a

complex system of 6 different primers, which

is far more than the 2 primers used for PCR

(Fakruddin et al., 2013; Li et al., 2017; Notomi

et al., 2015). Furthermore, at least 2 of these

primers are hybrid sequences are not found in

the targeted DNA. Therefore, this complex set

of primers must be designed and validated in

silico using bespoken programs. However, a

few different tools are available for free (such

as Primer Explorer https://www.primerexplor-

er.jp/e/) and some others are offered under a

commercial license. Because of this complex

design, the primers sets required for RT-LAMP

may not be suitable for some genomic areas,

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especially repetition-rich loci. However, those

particular obstacles are unlikely to affect the

capacity of RT-LAMP to detect most pathogens

since these types of repetitions are not common

in the genome of prokaryotes and viruses.

In this article we are not describing in

detail the complex interaction of RT-LAMP

primers or its mechanism of amplification.

However, Eiken Chemical Co., the original

inventors of LAMP, offer a detail description

of its principle in their website both in the form

of an illustration (http://www.loopamp.eiken.

co.jp/e/lamp/principle.html), as well as an ani-

mation (http://www.loopamp.eiken.co.jp/e/

lamp/anim.html).

In this balance between pros and cons,

isothermal amplification technologies have not

been considered as an alternative that could

challenge the predominance of traditional

forms of PCR either for clinical or research

use until recently. In the case of LAMP, it

has mainly remained in the fringes of the

clinical field as a simple and low-cost alterna-

tive for the development of field deployable

diagnostic tests for animal and agricultural

diseases for which RT-qPCR-based testing in

a laboratory is not cost-effective. RT-LAMP

also seems to have gathered momentum as an

inexpensive alternative diagnostic technology

for pathologies that occur in areas that lack the

healthcare infrastructure to provide an expen-

sive option like RT-qPCR. This latter case is

clearly illustrated by the different triple testing

protocols that have been independently devel-

oped to differentially diagnose dengue, zika

and chinkungunya; three mosquito transmitted

viral diseases with similar clinical presenta-

tions and commonly found in the same tropical

areas of emerging economies (Ganguli et al.,

2020; Priye et al., 2017; Yaren et al., 2017;

Yaren et al., 2018). However, the particular

needs created by COVID-19 could change the

vision of RT-LAMP from fringe alternative to

a secure position within the mainstream of the

technological spectrum for clinical diagnosis

(Khan et al., 2020). In our case, we chose RT-

LAMP over other isothermal amplification

technologies because of its several advantages,

including the fact that it is the most maturely

developed and most used of these alternatives.

Another reason to choose LAMP as the iso-

thermal amplification technology for massive

testing is the current lack of intellectual prop-

erty protections in most countries. The LAMP

technology was one of the first isothermal

amplification technologies to be developed. As

such, its initial intellectual property protections

have recently expired or are close to. LAMP

was first patented in Japan by Eiken Chemi-

cal Co. in November 1998 (Japan patent No.

JP2000283862) and suffered an anticipated

expiration in 2019; however, at least the pro-

tection granted for this invention in the United

States is anticipated to expire in November

2021 (US patent No. US7494790B2). In the

case of Costa Rica, we have found no appli-

cations or patents granted that protect the

original or any subsequent inventions by Eiken

Chemical Co., or by other inventors regarding

LAMP. We believe this lack of intellectual

property protections includes most emerging

economies in Latin America, Africa and most

of Asia and Eastern Europe. This lack of legal

protections allows the development of testing

protocols faster and at a lower cost compared

to technologies that must be acquired from a

licensed manufacturer or licensed directly from

a patent’s owner.

Clinical parameters of RT-LAMP vs

RT-qPCR for COVID-19 diagnosis: Several

recent studies have published data on the detec-

tion of SARS-CoV-2 using RT-LAMP from the

2 main types of samples (nasopharyngeal swabs

and saliva). These publications also describe a

wide variety of RNA extraction methods and

targeting primers. While an exhaustive review

of all of these variables is beyond the scope

of this article, Table 1 summarizes the articles

published or pre-printed studies where SARS-

CoV-2 is detected in clinical saliva samples or

virions spiked saliva using RT-LAMP. Table 1

also summarizes the type or RNA extraction

used (traditional RNA extraction vs heating

step) and the gene or genes targeted by the

RT-LAMP primers sets. It must be noted that

182 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

while some detection methods use a single set

of RT-LAMP primers, it is possible to use up to

2 sets of primers in some cases. The RT-LAMP

kit developed by New England Biolabs to detect

SARS-CoV-2 uses 2 different sets of RT-LAMP

primers targeting the N and E genes, effectively

multiplexing the detection reaction. However,

mixing different RT-LAMP primers sets should

always be evaluated on a case-by-case basis.

In the case of RNA extraction methods, it

should be noted that traditional RNA extrac-

tion from saliva samples secures a sensitivity

that closely matches the current gold standard

with RT-qPCR. However, the quick viral RNA

extraction from saliva by a heating step may not

yield the same level of detection, but it still pro-

duces a sensitivity that is clinically useful for

most cases. The highest clinical sensitivity (97

% compared to the gold standard) of a saliva

RT-LAMP test for SARS-CoV-2 detection was

achieved by Wei et al. (2021) in a direct saliva-

to-reaction solution without any form of RNA

extraction. This LAMP based test also has the

lowest Limit of Detection (LOD), with 2 copies

of viral RNA per µL, which is on par with the

most sensitive RT-qPCR tests designed to date.

However, the formulation developed by Wei

et al. (2021) requires some specialized materi-

als such as a buffer containing carrier RNA to

protect the viral RNA in the sample. Whether

this method could be mass produced at a cost

that still makes it competitive to RT-qPCR has

not been established. Nonetheless, the protocol

created by Wei et al. (2021) is currently used at

Columbia University to test students and facul-

ty and the technology was licensed to Sorrento

Therapeutics, in order to obtain FDA approval

and commercialize it in the United States under

the name COVI-TRACE.

With all this in mind, it is clear that RT-

LAMP can achieve clinical values of sensitiv-

ity, sensibility and LOD that are close to those

of the current gold standard. Nonetheless, these

clinical parameters must be evaluated during

the development of any RT-LAMP-based test,

especially if this technology is coupled with

detection from a saliva sample or using quick

viral RNA extraction by a heating step.

TABLE 1

RT-LAMP performance in the detection of SARS-CoV-2 presence in saliva sample

Reference Type of RNA

extraction Genes targeted* Specificity** Sensitivity** LOD

Ben-Assa et al., 2020 heating step N High High Medium to high viral

loads

Bhadra et al., 2021 heating step Orf1ab, N, E Not

determined

Not

determined

3.3 x 106 copies/ml

Howson et al., 2021 traditional RNA

extraction

E High High Viral loads high in

dilutions (1:40 to 1:640)

L’Helgouach et al., 2020 heating step Not described 95.70 % 72.70 % NA

Lalli et al., 2020 heating step Orf1ab, N High High 102 viral particles per

reaction

Lamb et al., 2020 traditional RNA

extraction

Orf1ab Not

determined

Not

determined

3.802 ×10^10 to 228

copies of virus

Nagura-Ikeda et al., 2020 traditional RNA

extraction

Orf1ab, N High High Viral loads vary with

time

Wei et al., 2021 direct

saliva-to-reaction

Orf1ab 100 % 97 % 2 copies of viral RNA

per μL

*For simplicity, all primers targeting any of the different genes within the Orf1a or Orf1b loci are simply referred to as

targeting Orf1ab.

**Sensitivity and specificity are relative to a RT-qPCR test.

183

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

Simplified options to detect amplifica-

tion of genetic material by RT-LAMP and

other isothermal amplification methods:

Theoretically, RT-LAMP and all other forms

of isothermal amplification can be quantified

and detected in real time using fluorescence.

For example, RT-LAMP can be performed

and quantified in real time using any tradi-

tional double-strand DNA binding dye such as

SYBR-Green in a real time thermal cycler (Da

Silva et al., 2020; Fakruddin et al., 2013; Khan

et al., 2020; Notomi et al., 2015). However,

using an isothermal amplification technology

in a thermal cycler defeat most of its advan-

tages and thus, simpler methods of detection

have been devised in order to visualize and

even quantify RT-LAMP amplification results.

These simplified methods of detection include:

UV detection shows in-tube presence of

amplicons by naked eye using an UV light

source and a marker that fluoresces under such

light if nucleic acids amplification has occurred.

These UV sensitive markers include traditional

double-strand DNA binding dyes such as ethid-

ium bromide, SYBR-Green, SYBR-Safe and

ionic detection markers such as calcein. UV

detection can also be semi-automated using

devices that emit and detect light at the correct

spectra, such as a fluorescence plate reader.

Colorimetric detection uses colorimetric

dyes that change the color of the solution if

amplification has occurred. These include pH

sensitive dyes such as phenol red or metal ion

sensitive dyes such as hydroxynaphthol blue

(HNB) (Goto et al., 2009). These color changes

can be determined in-tube by naked eye or

using a light absorbance device equipped to

measure the correct spectra (e.g. 650 nm for

HNB). It should be noted that pH sensitive dyes

such as phenol red may not be well suited as a

detection method for samples with high or low

pH as enough of these conditions may be car-

ried over from the sample to the final reaction

and cause interference with the results detec-

tion. This type of carryover can potentially

happen with the saliva of some individuals, as

well as in swab samples that were preserved

in universal or viral transport media (UTM

and VTM, respectively). In such cases, metal

ion sensitive dyes such as HNB might be the

preferred option.

Turbidity detection measures the change in

turbidity of the LAMP solution due to the natu-

ral precipitation of magnesium pyrophosphate

as byproduct of DNA synthesis (Mori et al.,

2001). Turbidity change can be determined in-

tube by naked eye or using a turbidometer. The

use of some turbidometers such as the Loo-

pamp Realtime Turbidimeter (LA-500, Eiken

Chemical Co., Japan) also allows for real-time

quantification of the RT-LAMP amplification.

CRISPR Cas12a/Cas13a detection relies

on genetic material that has been pre-amplified

by isothermal amplification, including LAMP

or RPA, to detect specific genetic sequences

using targeted digestion with a CRISPR sys-

tem using either Cas12a, Cas12b or Cas13a

enzymes. The genetic sequences specifically

cleaved by the CRISPR Cas12/Cas13 enzymes

can be detected using a lateral flow chromatog-

raphy strip and visualized directly by naked eye

(Khan et al., 2020).

We observe that none of these meth-

ods of amplification detection are exclusively

designed or used for RT-LAMP. In fact, all of

them can be used to detect amplicons produced

by PCR or other forms of isothermal amplifica-

tions. However, these alternative methods for

amplification detection will probably play a

crucial role in the development of easily scal-

able diagnostic tools for COVID-19, especially

those methods that do not require costly and

specialized amplification or detection equip-

ment such as thermal cyclers or plate readers.

More specifically, our attention should be

focused on detection methods that allow the

visualization of the final result directly in the

reaction tube, bypassing time-consuming extra

steps. In general, the coupling of an isothermal

technique such as RT-LAMP with a form of

amplicon detection by the naked eye such as

in-tube colorimetry promises to produce diag-

nostic tools for COVID-19 that are effective,

simple, easily scalable and low-cost.

184 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

Public attitudes toward COVID-19 mas-

sive testing: While no official information has

been gathered so far regarding public attitudes

towards COVID-19 testing among the Costa

Rican population, a preliminary statistical sur-

vey was performed by the School of Statistics

at the University of Costa Rica to determine

self-reported public knowledge and attitudes

towards COVID-19 testing technologies, both

available and potential (Ramírez-Hernández

& Madrigal Pana, 2020). The survey was

applied to a sample of 1 287 residents within

the national territory in October 2020 with

ages 18 years and over, who own a cell phone.

An assessment of both the willingness of the

population to undergo COVID-19 testing and

of using a new test based on a saliva sample

was included in the questionnaire.

Results suggest a mostly positive response

of the general public to COVID-19 testing.

In general, 84.1 % of the population would

accept being tested were a massive diagnosis

program implemented. A significant portion

of the population (78.6 %) claims to have

information about the type of sample required

for COVID-19 testing, with most responders

referring to the nasopharyngeal swab protocol.

Belief in the effectiveness of testing as a tool

to gain control over this pandemic, however,

is much lower (66.8 %). Even when further

statistical probing and analysis must occur,

current disaggregate data from this survey

appear to indicate that individual health and

financial concerns drive public perception of

COVID-19 despite perceived uncertainty about

its effectiveness as a strategy to mitigate and

control the spread of the virus. This latter point

highlights the need to provide the general pub-

lic with a clear picture of the mechanism by

which massive testing operates at population

levels (i.e. removing a large portion of asymp-

tomatic carriers and exhausting large shares

of the infective pool) in an effort to further

improve compliance and public trust as part of

official public health communication initiatives

(Lazarus et al., 2020).

Most significantly, the situation appears

to be even better for saliva-based COVID-19

testing. Willingness to receive a saliva-based

tests ranks high (92.3 %), with a similar

response for the prospect of a round of testing

once per week (89.5 %). Disaggregate respons-

es by sex, educational attainment, nationality

or subjective income did not exhibit signifi-

cant differences with respect to the sample

mean. Willingness to receive the test, however,

appears to decrease for individuals above 50

years or more (77.6 %), and increases in pro-

portion to one’s perception of being at risk (low

risk: 75.8 %; medium risk: 85.2 %; high risk:

86.8 %). These data suggest that information

about saliva-based testing technologies must be

specifically tailored to age groups via different

media platforms to ensure maximum coverage.

Another determinant factor that must be studied

at depth is trust in public health infrastructures

regarding data privacy, in which perceived

consequences of testing data mismanagement

for individuals may vary per country.

Finally, out-of-pocket costs per test appears

to be a major factor for public acceptance of

saliva-based testing technologies in Costa Rica.

The largest fraction of the sample in the study

was that of individuals who would accept the

test only if it is free for the population (37.1 %).

As out-of-pocket cost increases, a smaller share

of the population reports willingness to receive

the test. A price point equal to or below US $10

appears to be required in order to ensure at least

a substantial proportion of the population (62.9

%) are likely to perform testing at a scale suf-

ficient to have a reasonable impact individually

and collectively.

Succinctly, the prospect of saliva-based

testing in Costa Rica –an emerging econo-

my with significant infrastructure and socio-

economic challenges– is overall positive and

encouraging. Even when the mechanism by

which massive testing helps mitigate and con-

tain the pandemic appears not to be well

understood, individual health and financial

concerns seem to drive the perceived need for

massive testing. Saliva-based testing appears

to attain better public perception, possibly

due to perceptions of being less intrusive than

nasopharyngeal swabs, while still in need of

185

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

better public health campaigns if deployed.

As expected, out-of-pocket costs drive public

acceptance in the midst of a public health crisis

with strong reverberations into the economic

and social fabric of a mid-income country.

Given the dominant magnitude of negative

economic impacts of COVID-19 in comparison

to testing costs and other forms of mitigation in

Organization for Economic Co-operation and

Development (OECD) member states (López-

Valcárcel & Vallejo-Torres, 2021), RT-LAMP

may become an ideal low-cost alternative for

developing nations and emerging economies

since its overall cost may be absorbed by

public health organizations or international

relief efforts as a means to approximate a

situation of zero out-of-pocket expenses for the

general public.

Could RT-LAMP replace RT-qPCR as

the main option for large-scale COVID-19

testing?: Given the current situation in which

it is infeasible for many healthcare systems to

reach the necessary levels of COVID-19 test-

ing to adequately manage the health crisis, it is

unlikely that such a goal will be met unless we

develop practical alternatives to the traditional

RT-qPCR-based testing. Simplified alternative

protocols for different steps of the process all

promise to expand our diagnostic capacity and

help bridge the testing gaps. These simplified

alternatives include isothermal amplification

technologies like RT-LAMP, use of saliva

samples, quick viral RNA extraction by heating

and results visualization by naked eye using

in-tube colorimetry. While these alternative

protocols have been proven to work together,

they do not necessarily constitute an “all or

none” package. In fact, some of these processes

were originally designed to operate as part of

a simplified testing protocol using RT-qPCR;

for example, viral RNA directly extracted

from saliva by a quick heating step has been

successfully used as the start material for RT-

qPCR, a substitution that dramatically reduces

the cost and turnaround time for COVID-19

testing (Esbin et al., 2020). Conversely, others

have developed a direct-swab-to-amplification

protocols in which a traditional nasopharyngeal

swab is used but the commonly used RNA

extraction is replaced by a quick heating step,

and the amplification is performed either by

RT-qPCR or RT-LAMP (Bruce et al., 2020;

Dao-Thi et al., 2020). Regardless of the pleth-

ora of potential protocol variations that can be

developed by exchanging alternative options

in each step, the coupling of all or most of the

simplified alternative technologies into a single

uncomplicated and low-cost testing protocol

might be the only feasible option for many

developing countries where equipment such as

real-time thermal cyclers, pipetting robots and

biosecurity cabinets might be difficult to obtain

or economically burdensome. For that reason,

we are not surprised that research groups from

other emerging economies have arrived at the

exact same conclusions (Ohilebo et al., 2020).

On the other hand, given the very high

sensitivity and specificity of RT-qPCR, it is

unlikely that RT-LAMP or other alternative

technologies will replace the gold-standard

for COVID-19 diagnosis in the short term.

Also, the already installed RT-qPCR-based

testing capacity will continue to be abso-

lutely necessary during the pandemic. Further-

more, expansion of the RT-qPCR-based testing

capacity should continue until the end of this

crisis whenever and wherever it is feasible and

cost-effective. Even so, it is likely that alterna-

tive COVID-19 diagnostic options will emerge

in many parts of the world using RT-LAMP

as well as parts of the simplified testing pro-

tocols for sample collection, RNA extraction

and result detection. Some of these alternative

protocols may also be tailored to function as

part of a modified RT-qPCR diagnostic test in

an attempt to increase sample processing rates

in traditional laboratory settings. Nonetheless,

healthcare systems that are already struggling

to bridge the testing gaps are likely to continue

suffering from some level of deficit during the

rest of the pandemic, and the only sensible

decision will be to make use of all available

options. With this in mind it is unlikely that

RT-LAMP and other alternative COVID-19

diagnostic protocols will replace the traditional

186 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 173-189, January-December 2022 (Published Mar. 18, 2022)

RT-qPCR test but rather that they will become

complements that could strengthen each other’s

weaknesses and thus reciprocally fill their

gaps. In summary, COVID-19 has unveiled

the need for an ecosystem of affordable and

effective testing technologies capable of scal-

ing to different intensities, geographies and

development possibilities of countries during

a pandemic. RT-LAMP exemplifies such an

alternative alongside RT-qPCR, with the poten-

tial of enabling the discovery of new methods

capable of solving the hurdles of today as a

way to more effectively anticipate and respond

to the public health crises of tomorrow.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

RESUMEN

Amplificación isotérmica mediada por bucle de

transcripción inversa y protocolos alternativos para

pruebas COVID-19 a gran escala y de bajo costo:

lecciones de una economía emergente.

Introducción: la mayoría de los casos exitosos de miti-

gación y manejo de la pandemia de COVID-19 se han

dado mediante pruebas basadas en la reacción en cadena

de la polimerasa cuantitativa (RT-qPCR por sus siglas

en inglés). Sin embargo, muchas economías emergen-

tes han tenido problemas con las demandas actuales de

pruebas moleculares debido a limitaciones económicas,

técnicas y tecnológicas.

Objetivo: Definir un protocolo de diagnóstico potencial

para aumentar la capacidad de prueba en las condiciones

actuales y posteriores a la pandemia.

Métodos: Revisamos la literatura, las patentes y las aplica-

ciones comerciales, en busca de alternativas.

Resultados: Encontramos un buen potencial en muestras

de saliva, inactivación viral y extracción rápida de ARN

por calentamiento; el uso de una tecnología isotérmica

como la amplificación isotérmica mediada por horquillas

(LAMP, por sus siglas en inglés) y la visualización del

resultado de la prueba a simple vista mediante colorimetría

o turbidez en el tubo.

Conclusiones: Las muestras de saliva con extracción

rápida de ARN por calentamiento y LAMP colorimétrico

son opciones prometedoras para países con limitaciones

económicas y de infraestructura.

Palabras clave: economías emergentes; pruebas mole-

culares; SARS-CoV-2; saliva; amplificación isotérmica

mediada por bucle.

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