Performances of four Nucleic Acid Amplification Tests for the identification of SARS-CoV-2 in Ethiopia.

Since Coronavirus Disease-2019 (COVID-19) outbreak was reported, many commercial Nucleic Acid Amplification Tests (NAAT) have been developed all over the world, and it has been the standard method. Even though several assays were rapidly developed and applied to laboratory diagnostic testing, the performance of these assays was not evaluated in different contexts. Thus, this study aimed to assess the performance of Abbott SARS-CoV-2, Daan Gene, BGI and Sansure Biotech assays by using Composite Reference Standard (CRS). The study was conducted at the Ethiopian Public Health Institute (EPHI) from December 1 to 30/2020. Of the 164 nasopharyngeal samples were extracted by using a QIAamp RNA mini kit and Abbott DNA sample preparation system. Out of 164 samples, 59.1% were positive and 40.9% were negative by CRS. Sansure Biotech positivity was significantly low compared to CRS (p < 0.05). The overall agreement of the four assays compared to CRS was 96.3-100%. The performance of the four assays had almost comparable diagnostic performance, except for a low positive rate of Sansure Biotech assay. Hence, Sansure Biotech assay [Research Use Only (RUO)] needs further verification on its use in Ethiopia. Finally an additional study should be considered for evaluating assays with respective manufacturer claims.

Effect of sample management on quantitative HIV-1 viral load measurement at Saint Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia.

PURPOSE: This study was meant to determine the effect of time to plasma separation, storage duration, freeze-thawing cycle and dilution proportion on the HIV-1 viral load level. METHODS: Experimental study design was employed by collecting 10mL whole blood samples into two EDTA tubes from 88 eligible HIV infected patients at St Paul's Hospital Millennium Medical College. The viral load test was done using Abbott m2000sp/rt analyzer. Data was entered into Microsoft excel and analyzed by SPSS version 20. Repeated measure analysis of variance was used to compare HIV RNA viral load mean difference between different time to plasma separation, storage, freeze-thawing cycles and dilution levels. Post-hoc analysis was employed to locate the place of significant differences. P value less than 0.05 was used to declare statistical significance while viral RNA level of 0.5 log copies/ml was used to determine clinical significance. RESULTS: There was significant HIV-1 RNA viral load log mean difference between plasma separation time at 6 hours (hrs) and 24hrs (p<0.001). There was also significant HIV-1 RNA viral load log mean difference between plasma tested within 6hrs and those stored at 2-8°C for 15 days (p = 0.006), and between plasma stored at 2-8°C for 6 days versus 15 days (p<0.001). There was significant log mean difference between plasma that was exposed to fourth cycle of freeze-thawing after storage at -20°C when compared with plasma tested within 6hrs (p = 0.013). CONCLUSION: Plasma separated at 24hrs, stored at 2-8°C for 15 days or freeze-thawed for four cycles had significant effect on HIV viral load level. However, the differences were not clinically significant at a cut-off viral load level of 0.5 log copies/ml. Avoiding delays to plasma separation beyond 24 hrs, storing at 2-8°C for 15 days and freeze-thawing for no more than 4 cycles is recommended to improve the result quality.

Effect of heat inactivation for the detection of severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) with reverse transcription real time polymerase chain reaction (rRT-PCR): evidence from Ethiopian study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been a major public health importance and its specimen needs to be handled safely due to concerns of potential transmissibility to health care workers. Heat inactivation of the sample before nucleic acid isolation might permit safe testing processes. Hence, it is important to assess the effect of heat inactivation on SARS-CoV-2 RT-PCR detection in resource limited settings. METHODS: An experimental study was conducted at Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing. One batch of the sample was inactivated at 56 °C heat for 30 min, and the other batch was stored at 4 °C for a similar period of time. RNA extraction and detection were done by DAAN Gene kit protocols. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. p-value < 0.05 was considered as statistically significant. RESULTS: Out of 188 total samples, 119 (63.3%) were positive and 69 (36.7%) were negative in the non-inactivated group. While, 115 (61.2%) of samples were positive and 73 (38.8) were negative in heat inactivated sample batch. Rate of positivity between groups did not have statistically significant difference (p > 0.05). The mean Cycle threshold (Ct) value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95% CI - 0.247-0.331; t = 0.28; p = 0.774) and 0.38 (95% CI 0.097-0.682; t = 2.638; p = 0.010) respectively. CONCLUSION: Heat inactivation at 56 °C for 30 min did not affect the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding showed that there was statistically significant Ct value increment after heat inactivation compared to untreated samples. Therefore, false-negative results for high Ct value (Ct > 35) samples were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.