|Year : 2021 | Volume
| Issue : 4 | Page : 90-94
Impact of reverse transcriptase real-time polymerase chain reaction-cycle threshold values on SARS-CoV-2–positive cancer patients undergoing treatment
Rashmisnata Barman1, Manash Jyoti Taw2, Avdhesh Kumar Rai3, Manigreeva Krishnatreya4, Anupam Sarma5, Sawmik Das5, Mouchumee Bhattacharyya6, Amal Chandra Kataki7, Manoj Kalita4
1 Department of Microbiology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
2 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India
3 Department of Molecular Biology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
4 Department of Cancer Registry and Epidemiology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
5 Department of Pathology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
6 Department of Radiation Oncology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
7 Department of Gyaencological Oncology, Dr. B. Borooah Cancer Institute, Guwahati, Assam, India
|Date of Submission||30-Apr-2021|
|Date of Decision||14-Jun-2021|
|Date of Acceptance||18-Jun-2021|
|Date of Web Publication||16-Oct-2021|
Room No. 116, OPD Building, Department of Microbiology, Dr. B. Borooah Cancer Institute, Guwahati, Assam
Source of Support: None, Conflict of Interest: None
Introduction: Coronavirus disease 2019 (COVID-19) pandemic, being a novel viral infection, has resulted in disruption of health services, including cancer patient's care and treatment. Hence, there was a need for testing and lateral integration of services for cancer patients with COVID-19. Materials and Methods: A total of 1178 samples were collected from cancer patients for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing before undergoing treatment in a tertiary care cancer center. The realtime reverse transcriptase polymerase chain reaction (RTPCR) was done using the E gene for screening, and for the confirmation, any of the three reactions using RdRp, ORF1bnsp14 and RNasePas were run as internal control. Results: Out of the 1178 samples tested, 211 (17.91%) were positive, and of these patients, 863 (73.25%) were men and 342 (29%) were women. Among the 863 men with cancer, 133 (15.41%) were real-time reverse transcriptase PCR (RT-PCR) positive. Of the 342 women with cancer, 78 (22.80%) were positive. Of the 144 SARS-CoV-2–positive cancer patients with cycle threshold (Ct) <25, 112 (84.2%) were asymptomatic and 32 (41.0%) were symptomatic. Sixty-seven patients showed positive results with a Ct >25. Out of them, 21 (31.3%) were asymptomatic and 46 (68.65%) were symptomatic cancer patients (P < 0.001). Of 144 patients with Ct <25, only 4 (2.8%) patients tested negative within 7–9 days, whereas the rest of the 140 (97.22%) became negative in >9 and up to 28 days. In the 67 cancer patients with Ct >25, within 7–9 days, 50 (74.6%) became RT-PCR negative and the remaining 17 patients mostly >60 years age group became RT-PCR negative in >9–28 days. Conclusions: Ct value of qualitative SARS-CoV-2 reverse transcriptase RT-PCR should be an important tool for an oncologist in designing and implementing patient management guidelines for SARS-CoV-2–positive cancer patients without or with symptoms for COVID-19.
Keywords: Cancer, COVID-19, cycle threshold value, RT-PCR
|How to cite this article:|
Barman R, Taw MJ, Rai AK, Krishnatreya M, Sarma A, Das S, Bhattacharyya M, Kataki AC, Kalita M. Impact of reverse transcriptase real-time polymerase chain reaction-cycle threshold values on SARS-CoV-2–positive cancer patients undergoing treatment. Adv Hum Biol 2021;11:90-4
|How to cite this URL:|
Barman R, Taw MJ, Rai AK, Krishnatreya M, Sarma A, Das S, Bhattacharyya M, Kataki AC, Kalita M. Impact of reverse transcriptase real-time polymerase chain reaction-cycle threshold values on SARS-CoV-2–positive cancer patients undergoing treatment. Adv Hum Biol [serial online] 2021 [cited 2021 Dec 4];11:90-4. Available from: https://www.aihbonline.com/text.asp?2021/11/4/90/328403
| Introduction|| |
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as novel coronavirus, emerged in the city of Wuhan in Hubei province in Central China.,, The WHO declared the outbreak as a public health emergency of international concern on 30 January 2020. In India, the first laboratory-confirmed case of COVID-19 was reported from Kerala on 30 January 2020. In India, a total of 10.1 million cases and 146,000 deaths were reported by 22 December 2020. The first case of the COVID-19 pandemic in the Indian state of Assam was reported on 31 March 2020. The outbreak of this COVID-19 viral pandemic is having a serious impact on the health-care system of the entire country and has created an unprecedented challenge for the treatment and management of potentially vulnerable cancer patients., Severe outcomes of the disease were seen in patients older age group and the presence of medical comorbidities, and these risk factors were commonly seen in cancer patients. Implementation of government guidelines and restrictions such as lockdowns and shortages of essentials has affected the patient's management protocols.
India's response to COVID-19 has been 'pre-emptive, pro-active and graded'; the testing strategy included people who had international travel history with symptoms, symptomatic contacts of laboratory-confirmed COVID-19 patients and symptomatic health-care workers managing respiratory distress/severe acute respiratory illness. During this period of a viral pandemic, patients with a history of or active malignancy due to their immunocompromised state might be at increased risk of contracting the virus and developing COVID-19-related complications.,, Cancer patients with haematopoietic stem cell malignancies are usually immunocompromised due to antineoplastic therapy, supportive medications such as steroids and the immunosuppressive properties of cancer itself. Furthermore, these patients with haematopoietic malignancies might also have an augmented immune response to infection secondary to immunomodulatory drugs, etc. Moreover, age was an important criterion in both general patient and cancer patient, and it was observed that patients >60 years of age with one or more major comorbidities were at increased risk for COVID-19–related morbidity and mortality. Cancer care and management require a frequent and regular visit to the hospital as uninterrupted treatment plays a crucial role in cancer patient's recovery and final prognosis. Thus, considering the high transmissibility of SARS-CoV-2 among the cancer patients, proactive screening, detection and management of these patients with SARS-CoV-2 infection were the urgent need for any cancer care centre. A viral diagnostics laboratory was set up in our hospital to test for SARS-CoV-2 for all cancer patients requiring admission for cancer treatment and related management. A separate COVID ward was created for such admission of symptomatic and asymptomatic SARS-CoV-2–positive individuals. The objective of the present study was to determine the profile of cancer patients undergoing testing for SARS-CoV-2 and to examine the impact of SARS-CoV-2 reverse transcriptase real-time polymerase chain reaction (PCR)-cycle threshold (Ct) values on SARS-CoV-2–positive cancer patients.
| Materials and Methods|| |
This observational retrospective study was approved by the scientific committee and ethics committees of the institute vide letter no. Ref No. BBCI-TMC/Misc-01/MEC/201/2020, dated 25 September 2020.
The viral diagnostic laboratory where this study was undertaken is functioning under the broad umbrella of the Indian Council of Medical Research (ICMR). The RNA extraction and transcriptase real-time PCR (RT-PCR) kits are supplied by the ICMR. Following the ICMR guidelines, a combination of one throat swab and one nasal swab was collected from each of the patients and transported in viral transport media to the laboratory. The BioRad CF ×96 Touch Real-Time PCR machine was used, and the PCR assay was done for the E gene, RDRP gene, N gene and ORF gene. Since our PCR reagents and consumables were supplied by the ICMR, so our target assays were done from a mixed basket of the ICMR approved test kits of 16 domestic and overseas companies for sale in India. The real-time reverse transcriptase PCR was done using E gene for screening and confirmation of either of the three reactions. This strategy ensures the quality of the clinical sample as well as the testing process and identification of true positives. This two-step diagnostic protocol for SARS-CoV-2 detection is followed by the Virus Research and Diagnostic Laboratory Network.,, A total of 1178 samples were collected from cancer patients undergoing treatment in a tertiary care cancer centre and processed using Biosafety Cabinet Class II A2 in a Biosafety Level 2 level laboratory.
The laboratory performs the tests using ICMR-supplied RNA extraction kits.
Nucleic acids were recovered from clinical specimens by conventional manual RNA extraction method using [email protected] RNA Mini kits.
Carrier RNA 5.6 μl were mixed with 560 μl buffer a viral lysis (AVL). Then, 140 μl clinical sample from viral transport medium (VTM) was added. Vortexing and intermittent centrifugations were done using spin columns and adding 500 μl AW1 and 500 μl AW2 buffers stepwise, and finally, 60 μl buffer a viral elution (AVE) was added before a final centrifugation step. Then, the delusion is kept in 1.5 ml microcentrifuge tubes and stored in − 800°C.
Reverse transcriptase real-time polymerase chain reaction
Two key steps determine the accurate detection of SARS-CoV-2 in patient samples. The first is RNA extraction from patient samples, and the second is RT-PCR. Proper execution of both is key for accurate testing and reporting.
RT-PCR detects levels of target RNA against which primers have been designed. RNA, once extracted from patient samples, is reverse transcribed into complementary DNA (cDNA). The number of starting cDNA for the target transcript corresponds to the number of starting RNA in the sample. During the PCR, the number of copies of cDNA doubles with each thermocycling. All new copies are tagged with fluorescent nucleotides. The RT-PCR machine detects the number of new copies per cycle through this fluorescence. The number of cycles at which this fluorescence signal exceeds background levels is called the threshold cycle (Ct). Lower Ct values imply high levels of target RNA in the patient sample. Conversely, high Ct values imply low levels of target RNA in the patient sample.
SARS-CoV-2 transcripts E, N and RdRP genes are being probed for inpatient samples. In the first step, RT-PCR is performed for the transcript for E, and if present, a second confirmatory RT PCR is done for N or RdRP transcript. Controls to ensure proper RNA extraction, primer sensitivity and accuracy are necessary. Recording the results of control samples is as important as recording the results of test samples to definitively decide whether the patient sample had SARS CoV2 or not.
RT-PCR system used
- Bio-Rad CFX96 Touch™ Real-Time PCR Detection System.
RT-PCR kits used
- 3B Blackbio TRUPCR SARS-CoV-2 RT Qpcr kit
- Thermo Fisher TaqPath SARS-CoV-2 RTPCR
- Thermo Fisher Scientific AgPath-ID™ One-Step RT-PCR Reagents
- GCC Biotech DiAgSure nCoV-19 detection assay
- Meril COVID-19 One-step RT-PCR Kit.
(All are multiplex RTPCR kits. S and E genes were tested for screening, and N/ORF/RDRP genes were screened for confirmation of SARS CoV 2 as per each kit's specifications.).
The laboratory is functioning under the broad umbrella of the ICMR. The RNA extraction and RT-PCR kits are supplied by the ICMR. The interpretation of the laboratory test results was based on the state of Assam suggestions which were made on the basis of the manufacture's guideline for considering cut-off Ct value [Table 1].
| Results|| |
Over the 2 months' period, from July 2020 to August 2020, 1178 nasopharyngeal and oropharyngeal swabs were collected from cancer patients undergoing treatment at our institute. Reverse transcriptase real-time PCR was done in the Molecular Virology Laboratory of the Institute. Of 1178 samples tested, 211 (17.91%) were positive. Out of the 1178 cancer patients tested for SARS CoV-2 RT-PCR, 863 (73.25%) were male, and 342 (29.03%) were female. Among the 863 men cancer patients, 133 (15.41%) were RT-PCR positive, and out of the 342 women with cancer, 78 (22.80%) were positive. The difference in SARS-CoV-2 positivity between men and women was significant (P = 0.0118).
A correlation of SARSCoV2 RTPCR positive cases with the age group of the patient was done. In the age group of 0–18 years, a total of 55 patients were tested for SARS-CoV-2. Of them, 16 (29.0%) were SARSCoV2 positive. In the age group of 19–40 years, 391 patients were tested, and of them, 55 (14.0%). Similarly, in the 41–60 years age group, 573 were tested, and of them 65 (11.34%) were tested RTPCR positive [Table 2]. In 170 cancer patients of >60 years, 75 (44.11%) of them tested positive. There was a statistically significant (P = 0.0001) difference in the number of SARS-CoV-2–positive cases in 0–18 years and more than 60 years when compared with other age groups. Out of the 1178 cancer patients undergoing SARS CoV-2 RT-PCR testing, 917 were patients presented with solid tumours, and 261 were patients with haematological malignancies (including pediatric oncology patients). Test positivity in patients with solid tumour was 178 (19. 41%). Out of the 261 haematological malignancies, 33 (12.64%) patients were tested RT-PCR positive. Sixteen patients with haematological malignancies were in the 0–18 years of age group. There was a statistically significant difference in the number of RT PCR–positive cancer patients with solids tumour and haematopoietic stem cell malignancies, as shown in [Table 2].
|Table 2: Patient profile with reference to cancer type and age of the patients|
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We correlated Ct values with asymptomatic SARS-CoV-2–positive cancer patients and with typical symptoms for COVID-19 such as fever, shortness of breath, cough, body ache/myalgia, loss of smell and weakness. There were 144 SARS-CoV-2 cancer patients with positive results of Ct <25. Of 144 patients, 112 (77.7%) were asymptomatic for COVID-19, and 32 (22.2%) were COVID-19, symptomatic cancer patients. Sixty-seven patients showed positive results with a Ct >25. Of them, 21 (31.3%) were asymptomatic for COVID-19, and 46 (68.6%) were COVID-19, symptomatic cancer patients. This co-relation was significant (P = 0.0001) as more patients with <25 Ct value were asymptomatic for COVID-19 when compared with patients with Ct >25 [Table 3].
|Table 3: Correlation of cycle threshold value with the presence or absence of symptoms and duration for reverse transcriptase-polymerase chain reaction negative results on repeat testing|
Click here to view
The second test for RT-PCR was done on day 9–day 12 from the first positive report and on a rare instance on the 28th day. The correlation of Ct values with the duration for the second test of RT-PCR to turn negative showed that, out of 144 patients with Ct <25, only 4 (2.8%) patients tested negative on the 9th day. When the first test Ct <25, 140 (97.22%) patients tested negative for SARSCoV2 after 9 days and one patient tested negative on the 28th day. In the 67 cancer patients with Ct >25, within 7–9 days, 50 (74.6%) patients showed RT-PCR–negative results, and the remaining 17 patients, mostly above 60 years age group, showed RT-PCR–negative results on the 9th day. The Ct values have a significant impact on the duration of the second RT-PCR–negative test report (P = 0.0001).
During the early period of testing in mid-July, on average, 20–25 beds per day were occupied in the COVID-19 Ward. During this study period, there were six SARS-CoV-2–positive cancer patients admitted to the COVID-19 Ward. Of the 211 patients who tested positive for SARS-CoV-2, nine (4.2%) patients succumbed till the drafting of this paper. It was seen that, all nine deaths were due to underlying cancer and not due to COVID19 (acute respiratory distress syndrome) due to pneumonia as the cause of death defined by the World Health Organization.
| Discussion|| |
Out of the 1178 samples tested, the test positivity report was around 18%. Similar observations were made by Yu et al., where they found that SARS-CoV-2 infection rate was much higher in cancer patients compared with the general population, which is around 5%–10% in the general population of India., Our institutional policy of screening all the patients undergoing treatment in the institute has also played an important role in the early detection of most of the cases. In our study, there was a significant difference in SARS-CoV-2 positivity rate between men and women, which can be accounted to limited outdoor activities and maintenance of social distancing among the female cancer patients. Most cancer patients were adults in the age group of 41–60 years (n = 573), and the number of COVID-positive cases among them was 65 (11.34%). However, among the cancer patients above 60 years of age group, the proportion of positive cases was higher 44.1%. In cancer patients in the age group of 0–18 years, the positivity rate was around 18%, which was comparable to the study by Robilotti et al.
In the present study, solid organ tumour was the most frequent type of cancer (917 patients) with SARS-CoV-2–positive report, as solid tumours are more common in our study population. These findings were similar to Robilotti et al.'s study, where most patients presented with solid organ malignancies. Of the 917 solid tumour patients, around 20% were SARS-CoV-2 positive. One hundred and seventy-eight (19.41%) were SARS-CoV-2 RT-PCR positive. In our study, the positivity rate among haematological malignancies was 13%, and the positivity rate for pediatric patients with haematological malignancies was 48%.
The Molecular Virology Laboratory is functioning under the broad umbrella of the ICMR. The RNA extraction and RT-PCR kits are supplied by the ICMR, and we are doing a qualitative assay. The interpretation of the laboratory test results the state of Assam suggestions which were made based on the manufacture's guideline for considering cut-off Ct value. A high Ct >25 generally correlates with lower viral loads, although Ct value may not be directly proportional because of several pre-analytical and analytical factors and the potential presence of inhibitory factors within clinical samples. In our study, out of the 67 patients with Ct value more than 25, 21 (15.8%) were asymptomatic SARS-CoV-2 positive and 46 (59.0%) were symptomatic for COVID-19. The probable reason for this finding is a proactive institutional screening of all cancer patients for admission, and undergoing treatment has led to early detection of SARS-CoV-2 cases during the initial stage of viremia. This may also account for most of the asymptomatic SARS-CoV-2–positive cases with a low Ct <25. Our findings were significant when compared with patients with Ct values of more than 25. There are studies where there was no significant difference in Ct values of symptomatic and asymptomatic COVID-19 patients., A high Ct value of more than 25 generally correlates with lower viral load, although Ct value may not be directly proportional because of several pre-analytical factors and the potential presence of inhibitory factors within clinical samples. In our study, it was seen that out of the 144 patients with Ct <25, 140/144 (97%) took a longer duration to test negative on repeat testing in comparison with 17 (25%) of patients with more than 25 Ct value. In the present study, cancer patients above 60 years, the RT-PCR second test negative results were delayed. Our findings were similar to a study that showed symptomatic COVID-19 patients with low Ct values can have prolonged positive SARS-CoV-2 real-time PCR results and up to several weeks.
The SARS-CoV-2–positive persons with cancer should be taken for treatment and management of cancer and associated problem. Limited findings of low death due to SARS-CoV-2 infection in our cohort of cancer patients suggest that SARS-CoV-2 infection is not the end of the road for cancer patients, including patients with haematological cancer who were under immunosuppressive medications. Cancer treatment or management must not wait or delayed due to SARS-CoV-2 infection.
Limitations of the study
The laboratory is functioning under the broad umbrella of the ICMR. The RNA extraction and RTPCR kits are supplied by the ICMR. There are numerous challenges of keeping a molecular diagnostic laboratory during this viral pandemic. There were drawbacks in our study, but considering the current situation, these were unavoidable, hopefully, overlooked and considered. Since our Ct value was recorded for each of the three genes, so the samples with a detected result for all three genes, or a single target detected just the RdRP, ORF or N gene, were interpreted as SARS-CoV-2 PCR positive in line with the manufacturer's guidance. This study was based on the qualitative RT-PCR, and the test kits were from a mixed basket of ICMR reagents, and the confirmatory genes used for reporting of test results were variable.
| Conclusions|| |
The Ct value of qualitative SARS-CoV-2 reverse transcriptase real-time PCR should be an important tool for an oncologist in designing implementable patient management guidelines for SARS-CoV-2–positive cancer patients without or with symptoms of COVID-19. The goal should be uninterrupted treatment during the viral pandemic, as cancer treatment delayed for weeks or months will have a serious impact on these patients. This present viral pandemic is neither the last one nor the worst one. In future, for new viral pandemics, the Ct value of qualitative reverse transcriptase real-time PCR test can be considered as a guide for lateral integration of healthcare, including cancer care. There should be no interruption of cancer treatment due to a high expectant mortality due to COVID-19.
Authors would like to thank National Health Mission, Assam for setting up the Molecular Virology Laboratory at our institute.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]