Possible intraindividual evolution of SARS-CoV-2 in nasopharyngeal and anal swab in an octogenarian: a case report
Jamal Zaini1,*, Andika Chandra Putra2, Asep Muhamad Ridwanuloh3, Zahrah Saniyyah4, Budi Haryanto5, Ahmad Rusdan Handoyo Utomo6, Anik Budhi Dharmayanthi7, Anggia Prasetyoputri8, Ade Andriani9, Hariyatun
Hariyatun10, Isa Nuryana11, Syam Budi Iryanto12, Sugiyono Saputra13, Andri Wardiana14,Ratih Asmana Ningrum15 Abstract
Introduction COVID-19 is an emerging infectious disease that remains to be further investigated.
Case report Here, we describe a case of COVID-19 in an octogenarian woman with comorbidities who slowly recovered during hospitalization, but died due to sudden cardiac death after 2 weeks of hospitalization. Her nasopharyngeal and anal swabs returned positive for SARS-CoV-2 by RT-PCR on day 7 of hospitalization. The NGS showed possible intraindividual evolution of virus. The sample from the nasopharyngeal swab yielded a B.1470 variant classified as clade GH. This variant showed mutation in the spike gene D614G; N gene; NS3 gene; NSP2 gene and NSP12 gene. The sample from the anal swab showed similar mutation but with additional point mutation in spike gene S12F and was classified as B.1.465 variant.
Conclusions The possibility of the gastrointestinal tract that served as reservoir for virus mutation accumulation should also be considered and the potential impact of viral fecal transmission in the environment should be further investigated.
Keywords SARS-CoV-2, evolution, anal swab, octogenarian.
Introduction
COVID-19 in geriatric patients poses significant challenges as they have weakened immune responses mostly accompanied by comorbidities. The course of disease progression1
Received: 11 November 2021; revised: 30 April 2022;
accepted: 20 May 2022.
1MD, PhD, Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, Universitas Indonesia, Persahabatan National Respiratory Referral Hospital, 13230, Jakarta Timur, Indonesia; 2MD, PhD, Faculty of Medicine, Universitas Yarsi, 10510, Jakarta Pusat, Indonesia, Persahabatan National Respiratory Referral Hospital, 13230, Jakarta Timur, Indonesia; 3MSc, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia;
4MD, Faculty of Medicine, Universitas Yarsi, 10510, Jakarta Pusat, Indonesia; 5MD, Department of Microbiology, Persahabatan National Respiratory Referral Hospital, 13230, Jakarta Timur, Indonesia; 6PhD, Graduate School of Biomedical Science Master Program, Universitas Yarsi, 10510, Jakarta Pusat, Indonesia And Dharmais Hospital National Cancer Center, 11480, Jakarta, Indonesia; 7PhD, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia; 8PhD, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia;
9PhD, National Research and Innovation Agency Republic
is sometimes unpredictable.1,2 The immunocompromised host in geriatrics has reduced capabilities to fight viral infection in COVID-19, therefore there is a possibility of viral persistence with the potential to become a
of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia; 10MSc, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia;
11MSc, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia; 12MSc, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia;
13PhD, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia; 14PhD, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia;
15PhD, National Research and Innovation Agency Republic of Indonesia, Jalan Raya Bogor KM 46 Cibinong, Bogor 16911, Jawa Barat, Indonesia.
*Corresponding author: Jamal Zaini, [email protected] Article downloaded from www.germs.ro
Published June 2022
© GERMS 2022 ISSN 2248 – 2997 ISSN – L = 2248 – 2997
reservoir for viral evolution in the targeted organ.3 COVID-19 is a systemic disease that not only affects the respiratory system but other systems as well. The gastrointestinal (GI) tract is one of the common targets. The anal swab is considered as a potential specimen for detecting SARS-CoV-2 but its routine clinical use for diagnosis is uncommon.4 On the molecular level, whole-genome sequence of SARS-CoV-2 could be obtained from an anal swab, highlighting a potential risk of fecal-oral transmission.5
Here, we report an octogenarian patient with COVID-19 with comorbidity who died suddenly despite clinical improvement during hospitalization. The matched nasopharyngeal and anal swab returned positive results for SARS- CoV-2 viral gene (using RT-PCR), from which we proceeded to perform whole genome sequencing.
We discuss its clinical profile and genomic sequencing results along with potential implications.
Case report
An 82-year-old female was referred to our center with a COVID-19 confirmed case with diabetic gangrene on her left toes. She had initially been hospitalized in a nearby hospital due to diabetic left toe gangrene and had been planned for surgical management. The symptoms before hospital admission were painful swollen left toe and low-grade fever for 2 weeks. No cough or difficult breathing was reported. She had not been vaccinated against COVID-19 since no COVID-19 vaccine was available for the public at that time. She had never been diagnosed with COVID-19 before. She lost her appetite, with difficult oral intake, weakness, dyspepsia, and enuresis during 3 days of hospitalization in a nearby hospital. Screening for COVID-19 through naso-oropharyngeal SARS- CoV-2 antigen test and IgG for SARS-CoV-2 were found to be positive, therefore she was then transfered to a COVID-19 referral hospital. All cases positive for SARS-CoV-2 and with comorbidity had to be referred to referral hospital for multidisciplinary COVID-19 treatment at that time.
Upon arrival in the Emergency Unit, she looked pale and weak, but compos mentis with
blood pressure 130/90 mmHg, heart rate 90 beats/minute, respiratory rate 20 breaths/minute with oxygen saturation in the room air of 94%
but increased to 98%-99% with 2-3 L/m on nasal cannula. Physical examination showed rales on both sides of the lung. Swollen left toes were noted. An initial SARS-CoV-2 RT PCR from nasopharyngeal-oropharyngeal swab was positive (RdRp gene Ct value of 25.01 and Gene E Ct value of 24.39).
Laboratory findings showed leukocytosis, neutrophil to lymphocyte ratio (NLR) 10.9, normal procalcitonin, normal C-reactive protein, high blood glucose, and normal troponin I. ECG was also normal. Chest X-ray was consistent with mild pneumonia. The patient was diagnosed as COVID-19 confirmed, gangrene digiti II, III, and IV pedis sinistra, with uncontrolled diabetes mellitus. The patient was given oseltamivir 75 mg BID for 7 days, multivitamin once a day, anticoagulant (subcutaneous heparin 5000 IU BID) for 7 days, subcutaneous insulin treatment (Novorapid 16 units TID), and 7 days intravenous antibiotics (ampicillin-sulbactam 1.5 g TID plus ciprofloxacin 500 mg BID) plus supportive care. Oseltamivir and all medications were given based on the local Indonesian COVID-19 guideline at that time. Her clinical condition gradually improved, she was able to communicate and was weaned off of supplemental oxygen on day 3 of hospitalization.
She underwent debridement, amputation of digiti II, III, and IV pedis sinistra on day five without any complication.
On day seven of hospitalization, she complained of gastrointestinal discomfort but the symptom was relieved after administration of proton pump inhibitor. An anal swab and nasopharyngeal swab were also taken in the same day and sent to the laboratory for SARS-CoV-2 testing. The patient was planned for homecare.
On day 14, the patient suddenly reported chest pain and dyspnea, and resuscitation was initiated, but the patient died after cardiopulmonary resuscitation. Troponin I taken just before her cardiac arrest showed significantly high concentration (640 pg/dL) compared to the initial admission (3.8 pg/dL) with a normal D dimer level. Sudden cardiac death was suspected.
The nasopharyngeal swab was positive for SARS-CoV-2 RdRp and E genes and surprisingly the anal swab also returned positive for SARS- CoV-2 genes with RdRp 27.06 and E 26.37 by RT-PCR. The samples were then prepared for whole genome sequencing using Oxford Nanopore Technology (ONT, USA) platform.
The whole genome sequencing from the nasopharyngeal swab showed 0% NN percentage of SARS-CoV-2 overall sequence with mutation in spike protein located in D614G. Based on clade and Pango lineage, the virus was classified as GH and B.1.470 (Pango v.3.1.11 2021-08-09).
Interestingly, the whole genome sequencing from the anal swab sample also showed readable sequences, with 3.84% NN percentage of SARS- CoV-2 overall sequence. There were new mutations located on the spike protein located in S12F and non-structural protein 6 (NSP6) in D159G in the anal swab sample as shown in Figure 1.
Figure 1. The 3D structural visualization of the spike glycoprotein with amino acid changes
identified. The mutation was shown in red (S12F and D614G). Amino acid substitution and protein modelled by I-TASSERtm, followed
by polishing the generated protein structure using 3Drefine. The protein structure was
visualized using PyMOLtm.
The virus sample from the anal swab was classified as clade GH and B.1.465 lineage. The details of the mutation are shown in Table 1.
Discussion
Elderly patients have more unusual clinical features compared to younger ones. Clinical signs and symptoms of COVID-19 in the elderly vary greatly depending on immune status and comorbidities.1,2 They rarely show high-grade fever because of the lower baseline temperature in elderly. Therefore, it is important to recognize early disease in elderly patients with COVID-19 since they are more likely to progress to severe disease with grave prognosis.1 Liu K et al.
reported that advanced age is a crucial risk factor for mortality in COVID.3 In this case report, the patient was diagnosed as COVID-19 moderate illness with uncontrolled type 2 diabetes mellitus and diabetic gangrene, but at the end of the follow-up, she was suspected to have a sudden cardiac death. Her unusual clinical features, acute confusional state, paleness, and weakness were consistent with a geriatric who has severe infection. It is interesting to note that both SARS-CoV-2 IgG and IgM were positive in this case. SARS-CoV-2 IgM develops early in the first week after infection and decays more rapidly than IgG. IgG antibodies develop later and could persist for months.6 IgM and IgG could be detected in serum 1-3 weeks after infection.6 It is possible that the onset of COVID-19 in this case was at least 2 weeks, consistent with the symptoms of 2 weeks low-grade fever before being admitted to the first hospital. Uncontrolled type 2 diabetes mellitus with its complications is a critical comorbidity in COVID-19 and could further worsen the prognosis. During disease progression, the patient complained of gastrointestinal symptoms and dyspepsia.
Gastrointestinal symptoms such as nausea, vomiting, diarrhea, abdominal pain, or GI discomfort are common in COVID-19 since the virus also has a tropism in GI tract.5-8
Specimens from various organs have been evaluated as a valuable source in COVID-19 diagnostic. An anal swab is one of the options as a source of diagnostic specimen in COVID-19
Table 1. Amino acid substitutions in virus genome in matched samples Nasopharyngeal swab Anal swab
RT-PCR Gene
RdRp Ct value: 28.26 Ct value: 27.06 E Ct value: 27.62 Ct value: 26.37
WGS Mutation
Spike D614G D614G, S12F
N T205I T205I
NS3 D155H, Q57H, S220I D155H, Q57H, S220I
NSP2 L217V L217V
NSP6 - D159G
NSP12 P323L P323L
Clade GH GH
Pango Lineage B.1.470 B.1.465
RT-PCR – real time polymerase chain reaction; WGS – whole genome sequencing.
cases.4,5 COVID-19 genetic material in anal swab was detected in patients who were asymptomatic despite negative nasopharyngeal swabs.5 Based on a recent systematic review, its positivity rate is 32.8% (107/300; 95% CI: 15.8‐49.8%).
Moreover, this test still also detected SARS-CoV- 2 for an extended period, as long as 43 days, with a higher positive rate and higher viral load than paired respiratory samples.9 A retrospective study in China found that anal swab positivity and viral load are higher in non-survivors, and a high initial viral load in anal swab was correlated with death and the need for ICU and invasive mechanical ventilation.8 Despite its benefits in diagnostics, fecal viral shedding in anal swab raises important issues. An anal swab positivity and high viral load in asymptomatic cases is evidence that SARS-CoV-2 may use the intestine as a reservoir without interrupting its function.
Higher viral load was detected in rectal swab compared to respiratory samples.8 the gastrointestinal tract may potentially be a long- lasting fomite for SARS-CoV-2 transmission, even for asymptomatic patients that could be a source of infection in the environment.9
A sample from a nasopharyngeal swab yielded a B.1470 variant and classified as clade GH. This variant showed a mutation in spike gene D614G, N gene (T2051); NS3 gene (D155H, Q57H, S220I); NSP2 gene (L217V) and NSP12 gene (P323L). Interestingly, the matched
sample from the anal swab taken on the same day also yielded a positive result. The WGS from the anal swab yielded a different variant, i.e., B.1.465, despite being the same GH clade. It has the same mutation in spike gene D614G (Table 1). The first case was reported in Finland in March 2020 and several countries reported the same variant since then.10 Our study was the first report of B.1.465 variant in Indonesia. The specimen was collected in November 2020 and no additional data was reported for B.1.465 until December 2020. Moreover, this study is the only report of B.1.465 variant obtained from an anal swab in Indonesia.
The difference between the samples from the nasopharyngeal and the anal swab was the additional mutation in spike gene S12F that was not found in the nasopharyngeal sample. Two mutations were detected in the spike gene in the anal swab sample, i.e., D614G and S12F. Spike alteration is the most significant mutation to be concerned with. As shown in Figure 1 the mutation position is not in the RBD region. The D614G spike mutation was reported to increase infectivity of SARS-CoV-2 by assembling a more functional spike in the virion.11 This mutation correlates with stability of the virion and enhances viral load in the upper respiratory tract that may increase virus transmission.10,11 The S12F mutation could impact cell interaction and
is associated with the entry of spike glycoprotein into endoplasmic reticulum in virion assembly.
The N gene encoded nucleocapsid serves to package the viral genome RNA into a helical ribonucleocapsid (RNP) and has a critical role during virion assembly. It has an important role in enhancing the efficiency of subgenomic viral RNA transcription as well as viral replication.
The nucleocapsid mutation T205I was identified with high frequency in B.1.351 variants circulating in South Africa and England.
Fortunately, this mutation does not affect the antigenic based virus detection. Mutation in NSP6 (D159G) also arose in the anal sample.
Non-structural protein 6 (NSP6) plays a role in the initial induction of autophagosomes from the host’s endoplasmic reticulum. But mutation in this gene rarely causes viral virulence. Nagy et al.
observed the direct correlation of mutation in viral genes to mild and severe clinical outcomes.12 The mild outcome may be associated with mutations located in ORF8, NSP6, ORF3a, NSP4 and nucleocapsid phosphoprotein N. The severe outcome with low prevalence found alterations in ORF3A and NSP7. Our study had mutation in NSP6 but still has grave prognosis.
The additional mutation found in the anal swab showed that SARS-CoV-2 could mutate within the individual. Based on the pathogenesis, immunosenescence/immunocompromised state and comorbidities in the elderly tend to promote dysregulated immune response, i.e. cytokine storms, resulting in multiorgan failure and life- threatening condition.1,2 Immunocompromised individuals also have a longer viral clearance than normal and sometimes persistent infection.13,14 There are some reports of persistent infection driving accelerated viral evolution in immunocompromised hosts, such as immunocompromised and patients with cancer.13,15 In this case report, we showed that an elderly patient with comorbidities could serve as a suitable host for developing further mutation.
This case report highlights challenges in managing an immunocompromised host such as an elderly patient, who may act as a potential host for viral evolution and sources of community transmission. Genetic sequencing
mitigation is important in predicting new virus variants in the immunocompromised host.
Conclusions
In an immunocompromised host, such as an elderly person with comorbidities, clinical signs and symptoms of COVID-19 varied greatly and could be mistaken as a mild disease in a severe COVID-19 case. Anal excretion of virus should be considered as a source of transmission. An immunocompromised elderly host could act as a potential environment for viral evolution, since SARS-CoV-2 mutates naturally over time in an individual patient.
Authors’ contributions statement: JZ, ACP, RAN, AMR, ARU conceived and designed the article. ACP, JZ, RAN, AMR, ABD, AP, SS, SBI, AW made substantial contributions in reviewing the design of the article and acquiring the data. ACP, RAN, AMR, ZS, BH coordinated sample collection and oversaw data collection. ABD, AP, AA, HH, IN, SBI, SS, AW, conducted and analyzed the laboratory results. JZ, RAN, AMR, ABD, SBI, AW analysed and interpreted the data. JZ, RAN, AMR, ABD, AW, AP, SS conducted the literature review and drafted the manuscript.
JZ, ACP, RAN contributed by revising the manuscript critically for important intellectual content. All authors read and approved the final version of the manuscript.
Conflicts of interest: All authors – none to declare.
Funding: This research was supported by Indonesian Ministry of Finance Funding for SARS CoV-2 Surveillance Genome with contract number 07/FI/P-KCOVID- 19.2B3/X/2020 and B-9734/IPH/KS.02.04/X/2020. The funding source did not have a role in: design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication.
Acknowledgment: The authors would like to thank to COVID-19 Detection Team from BSL-3 Laboratory, Research Organization for Life Sciences, National Research and Innovation Agency, Republic of Indonesia, Cibinong.
Ethics approval: This case study has been reviewed and was approved by Persahabatan Hospital Ethic Committee. Ref no: 81/KEPK-RSUPP/8/2020.
Availability of data: The NGS data of the 2 samples were submitted to GISAID. Sample from nasopharyngeal swab hCoV-19/Indonesia/JK-LIPI219/2020 (EPI_ISL_3186064), sample from anal swab: hCoV-19/Indonesia/JK- LIPI233/2020 (EPI_ISL_3374425).
Consent: Written informed consent was obtained from the next-of-kin for publication of their case report and the accompanying images.
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