cobas® HIV-1/HIV-2 Qualitative nucleic acid test for use on the cobas® 5800/6800/8800 systems

BK261349 · Roche Molecular Systems, Inc. Pleasanton, CA 94588 · QST · Jun 17, 2026 · Microbiology

Device Facts

• Record ID: BK261349
• Device Name: cobas® HIV-1/HIV-2 Qualitative nucleic acid test for use on the cobas® 5800/6800/8800 systems
• Applicant: Roche Molecular Systems, Inc. Pleasanton, CA 94588
• Product Code: QST · Microbiology
• Decision Date: Jun 17, 2026
• Decision: SESE
• Regulation: 21 CFR 866.3957
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

cobas® HIV-1/HIV-2 Qualitative for use on the cobas® 5800/6800/8800 systems is an in vitro nucleic acid amplification test for the qualitative detection and differentiation of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) nucleic acid in human serum and plasma. The test is intended to be used as an aid in diagnosis of HIV-1/HIV-2 infection. Detection of HIV-1 or HIV-2 nucleic acid is indicative of HIV-1 or HIV-2 infection, respectively. The presence of HIV-1 or HIV-2 nucleic acid in the plasma or serum of individuals without antibodies to HIV-1 or HIV-2 is indicative of acute or primary infection. The cobas® HIV-1/HIV-2 Qualitative may also be used as an additional test to confirm the presence of HIV-1 or HIV-2 infection in an individual with specimens reactive for HIV-1 or HIV-2 antibodies or antigens. The assay may also be used as an aid in the diagnosis of infection with HIV-1 and/or HIV-2 in pediatric subjects and pregnant women. The test is also intended for early infant diagnosis (EID) of HIV-1 or HIV-2 infection in infants ≤18 months using dried blood spot (DBS) specimens. This assay is not intended to be used for monitoring patient status, or for screening donors of blood, plasma, or human cells, tissues, and cellular and tissue-based products (HCT/Ps) for HIV.

Device Story

Automated in vitro nucleic acid amplification test (NAAT) for qualitative detection/differentiation of HIV-1 and HIV-2; utilizes cobas® 5800/6800/8800 systems. Input: human serum, plasma, or dried blood spot (DBS) specimens. Process: automated nucleic acid extraction/purification using magnetic glass particles; real-time PCR amplification of HIV-1 gag/LTR and HIV-2 LTR regions; thermostable DNA polymerase; AmpErase enzyme for carryover contamination control. Output: non-reactive, reactive, or invalid results displayed on system screen or PDF report. Used in clinical laboratories; operated by trained personnel. Benefits: aids in diagnosis of HIV-1/HIV-2 infection, including early infant diagnosis, facilitating timely clinical decision-making.

Clinical Evidence

Clinical study (2016, South Africa) compared DBS performance of subject device against a comparator assay with an existing EID claim. Sample size: 279 valid paired results. Results: 127/127 positive samples correctly identified (100% sensitivity, 95% CI: 97.13-100%); 151/152 negative samples correctly identified (99.34% specificity, 95% CI: 97.5-100%). McNemar p-value = 1. Bench testing established LoD, precision, specificity (100%), cross-contamination (0%), and stability for DBS samples.

Technological Characteristics

Automated NAAT using real-time PCR and TaqMan detection chemistry. Targets: HIV-1 (gag, LTR) and HIV-2 (LTR). Platform: cobas® 5800/6800/8800 systems. Sample prep: automated magnetic glass particle extraction. Reagents: armored RNA internal control, proteinase, lysis buffer, master mix with dUTP and AmpErase. Connectivity: system-integrated data management, PDF export. Sterilization: N/A (reagent kits).

Indications for Use

Indicated for individuals suspected of active HIV-1/HIV-2 infection and for early infant diagnosis (EID) in infants ≤18 months. Used as an aid in diagnosis, confirmation of reactive antibody/antigen tests, and diagnosis in pediatric subjects and pregnant women. Not for monitoring patient status or screening blood/tissue donors.

Regulatory Classification

Identification

Human immunodeficiency virus (HIV) nucleic acid (NAT) diagnostic and supplemental tests are prescription devices for the qualitative detection of HIV nucleic acid in human body fluids or tissues. The tests are intended for use as an aid in the diagnosis of infection with HIV and are for professional use only. The test results are intended to be interpreted in conjunction with other relevant clinical and laboratory findings. These tests are not intended to be used for monitoring patient status, or for screening donors of blood or blood products, or human cells, tissues, or cellular or tissue-based products (HCT/Ps).

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) For all HIV NAT diagnostic and/or supplemental tests (i) The labeling must include: (A) An intended use that states that the device is not intended for use for screening donors of blood or blood products, or HCT/Ps. (B) A detailed explanation of the principles of operation and procedures used for performing the assay. (C) A detailed explanation of the interpretation of results and recommended actions to take based on results. (D) Limitations, which must be updated to reflect current clinical practice and disease presentation and management. The limitations must include, but are not limited to, statements that indicate: ( *1* ) The matrices with which the device has been cleared, and that use of this test kit with specimen types other than those specifically cleared for this device may result in inaccurate test results.( *2* ) The test is not intended to be used to monitor individuals who are undergoing treatment for HIV infection.( *3* ) A specimen with a reactive result should be investigated further following current guidelines.( *4* ) All test results should be interpreted in conjunction with the individual's clinical presentation, history, and other laboratory results.( *5* ) A test result that is nonreactive does not exclude the possibility of exposure to or infection with HIV. Nonreactive results in this assay may be due to analyte levels that are below the limit of detection of this assay.(ii) Device verification and validation must include: (A) Detailed device description, including the device components, ancillary reagents required but not provided, and an explanation of the methodology. Additional information appropriate to the technology must be included, such as design of primers and probes. (B) For devices with assay calibrators, the design and nature of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a reference material. In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance, or when there is a transition to a new calibration standard. (C) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including, but not limited to, limit of blank, limit of detection, cutoff determination, precision, endogenous and exogenous interferences, cross reactivity, carryover, quality control, matrix equivalency, and sample and reagent stability. Samples selected for use in analytical studies or used to prepare samples for use in analytical studies must be from subjects with clinically relevant circulating genotypes in the United States. The effect of each claimed nucleic-acid isolation and purification procedure on detection must be evaluated. (D) Multisite reproducibility study that includes the testing of three independent production lots. (E) Analytical sensitivity of the test must be the same as or better than that of other cleared or approved tests. Samples tested must include appropriate numbers and types of samples, including real clinical samples near the lower limit of detection. Analytical specificity of the test must be as the same as or better than that of other cleared or approved tests. Samples must include appropriate numbers and types of samples from patients with different underlying illnesses or infections and from patients with potential endogenous interfering substances. (F) Detailed documentation of performance from a multisite clinical study. Performance must be analyzed relative to an FDA cleared or approved comparator. This study must be conducted using appropriate patient samples, with appropriate numbers of HIV positive and negative samples in applicable risk categories. Additional subtype, strain, or types must be validated using appropriate numbers and types of samples. The samples may be a combination of fresh and repository samples, sourced from within and outside the United States, as appropriate. The study designs, including number of samples tested, must be sufficient to meet the following criteria: ( *1* ) Clinical sensitivity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 99 percent.( *2* ) Clinical specificity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 99 percent.(G) Strategies for detection of new strains, types, subtypes, genotypes, and genetic mutations as they emerge. (H) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on test performance. (I) Final release criteria to be used for manufactured test lots with appropriate evidence that lots released at the extremes of the specifications will meet the claimed analytical and clinical performance characteristics as well as the stability claims. (J) All stability protocols, including acceptance criteria. (K) Appropriate and acceptable procedure(s) for evaluating customer complaints and other device information that determine when to submit a medical device report. (L) Premarket notification submissions must include the information contained in paragraph (b)(1)(ii)(A) through (K) of this section. (iii) Manufacturers must submit a log of all complaints. The log must include the following information regarding each complaint, if available: The type of event ( *e.g.,* false negative/false nonreactive or false positive/false reactive), lot, date, population, and whether or not the complaint was reported under part 803 of this chapter (Medical Device Reporting). The log must be submitted annually on the anniversary of clearance for 5 years following clearance of a traditional premarket notification.(2) If the test is intended for Point of Care (PoC) use, the following special controls, in addition to those listed in paragraph (b)(1) of this section, apply: (i) The PoC labeling must include a statement that the test is intended for PoC use. (ii) The PoC labeling must include the following information near the statement of the intended use: (A) That the test is for distribution to clinical laboratories that have an adequate quality assurance program, including planned systematic activities that provide adequate confidence that requirements for quality will be met and where there is assurance that operators will receive and use the instructional materials. (B) That the test is for use only by an agent of a clinical laboratory. (C) Instructions for individuals to receive the “Subject Information Notice” prior to specimen collection and appropriate information when test results are provided. (iii) PoC labeling must include instructions to follow current guidelines for informing the individual of the test result and its interpretation. (iv) The instructions in the labeling must state that reactive results are considered preliminary and should be confirmed following current guidelines. (v) Device verification and validation for PoC use must include: (A) Detailed documentation from a well-conducted multisite clinical study conducted at appropriate PoC sites. Performance must be analyzed relative to an FDA cleared or approved comparator. This study must be conducted using patient samples, with appropriate numbers of HIV positive and HIV negative samples in applicable risk categories. Additional subgroup or type claims must be validated using appropriate numbers and types of samples. The samples may be a combination of fresh and repository samples, sourced from within and outside the United States, as appropriate. If the test is intended solely for PoC use, the test must meet only the performance criteria in paragraphs (b)(2)(v)(A)( *1* ) and (*2* ) of this section and not the criteria in paragraph (b)(1)(ii)(F) of this section:( *1* ) Clinical sensitivity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 98 percent.( *2* ) Clinical specificity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 98 percent.(B) Premarket notification submissions must include the information contained in paragraph (b)(2)(v)(A) of this section. (3) If the test is intended for supplemental use in addition to use as an aid in initial diagnosis, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, as appropriate, apply: (i) The labeling must include a statement that the test is intended for use as an additional test to confirm the presence of HIV viral nucleic acid in specimens found to be repeatedly reactive by a diagnostic screening test. (ii) Device validation and verification for supplemental use must include a clinical study, including samples that were initially reactive and repeatedly reactive on a diagnostic test but were negative or indeterminate on a confirmatory test. Premarket notification submissions must include this information. (4) If the test is intended solely as a supplemental test, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, except those in paragraphs (b)(1)(ii)(F) and (b)(2)(v)(A) of this section, as appropriate, apply: (i) The labeling must include a statement that the test is intended for use as an additional test to confirm the presence of HIV viral nucleic acid in specimens found to be repeatedly reactive by a diagnostic screening test. (ii) The labeling must clearly state that the test is not for use for initial diagnosis or is not intended as a first-line test. (iii) Device validation and verification must include a clinical study including samples that were initially reactive and repeatedly reactive on a diagnostic test but were negative or indeterminate on a confirmatory test. Premarket notification submissions must include this information. (5) If the test is intended to differentiate different HIV types, the following special controls, in addition to those listed in paragraphs (b)(1) through (4) of this section, as appropriate, apply: (i) The labeling must include the statement that the test is intended for the confirmation of initial results and differentiation of different HIV types. (ii) The results interpretation in the labeling must include instructions for the user on how to interpret the results, including un-typeable and co-infection results. (iii) Device validation and verification must include evaluation of analytical and clinical sensitivity and specificity for each of the types, strains, and subtypes of HIV intended to be differentiated. Premarket notification submissions must include this information.

Predicate Devices

• cobas® HIV-1/HIV-2 Qualitative nucleic acid test for use on the cobas® 5800/6800/8800 systems (BK251233)

Submission Summary (Full Text)

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# **cobas® HIV-1/HIV-2 Qualitative
510(k) Summary**

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

| **510(k) Owner's Name** | Roche Molecular Systems, Inc. |
| --- | --- |
| **Company Address** | 4300 Hacienda Drive Pleasanton, CA 94588-2722 |
| **Phone Number** | 925-366-0647 |

# **Device**

| Proprietary Name | **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test for use on the **cobas®** 5800/6800/8800 systems |
| --- | --- |
| Common Name | **cobas®** HIV-1/HIV-2 Qualitative |
| Classification Name | Human immunodeficiency virus (HIV) nucleic acid (NAT) diagnostic and/or supplemental test |
| Regulation Number | 21CFR 866.3957 |
| Product Code | QST |
| Establishment Registration | 2243471 |

# **Predicate Device**

| Predicate Trade Name | **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test for use on the **cobas®** 5800/6800/8800 systems |
| --- | --- |
| Predicate Common Name | **cobas®** HIV-1/HIV-2 Qualitative |
| Predicate Classification Name | Human immunodeficiency virus (HIV) nucleic acid (NAT) diagnostic and/or supplemental test |
| Regulation Number | 21CFR 866.3957 |
| Product Code | QST |
| Predicate 510(k) Number | BK251233 |

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# 1. DEVICE DESCRIPTION

cobas® HIV-1/HIV-2 Qualitative is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 system is designed as one integrated instrument. The cobas® 6800/8800 systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 5800 or cobas® 6800/8800 systems software which assigns test results for all tests as non-reactive, reactive, or invalid. Results can be reviewed directly on the system screen, exported, or printed as a PDF report.

Nucleic acid from patient samples and added armored RNA internal control (IC) molecules (which serve as the sample preparation and amplification/detection process control) is simultaneously extracted. In addition, the test utilizes three external controls: two positive and one negative control. In summary, viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature.

Selective amplification of target nucleic acid from the sample is achieved by the use of target virus-specific forward and reverse primers which are selected from highly conserved regions of the HIV-1 and HIV-2 genomes. The HIV-1 gag gene, the HIV-1 LTR region (dual target for HIV-1) and HIV-2 LTR region are amplified by cobas® HIV-1/HIV-2 Qualitative.

Selective amplification of IC is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the HIV-1 or HIV-2 genomes. A thermostable deoxyribonucleic acid (DNA) polymerase enzyme is used for both reverse-transcription and PCR amplification. The target and IC sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicons from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

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**cobas®** HIV-1/HIV-2 Qualitative master mix contains two detection probes specific for the HIV-1 target sequences, one for HIV-2 target sequences and one for the IC. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of HIV-1 target, HIV-2 target and IC in three different target channels. When not bound to the target sequence, the fluorescent signal of the intact probes is suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and IC, respectively.

## 2. INDICATIONS FOR USE

**cobas®** HIV-1/HIV-2 Qualitative for use on the **cobas®** 5800/6800/8800 systems is an in vitro nucleic acid amplification test for the qualitative detection and differentiation of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) nucleic acid in human serum and plasma.

The test is intended to be used as an aid in diagnosis of HIV-1/HIV-2 infection. Detection of HIV-1 or HIV-2 nucleic acid is indicative of HIV-1 or HIV-2 infection, respectively. The presence of HIV-1 or HIV-2 nucleic acid in the plasma or serum of individuals without antibodies to HIV-1 or HIV-2 is indicative of acute or primary infection. The **cobas®** HIV-1/HIV-2 Qualitative may also be used as an additional test to confirm the presence of HIV-1 or HIV-2 infection in an individual with specimens reactive for HIV-1 or HIV-2 antibodies or antigens. The assay may also be used as an aid in the diagnosis of infection with HIV-1 and/or HIV-2 in pediatric subjects and pregnant women.

The test is also intended for early infant diagnosis (EID) of HIV-1 or HIV-2 infection in infants ≤18 months using dried blood spot (DBS) specimens.

This assay is not intended to be used for monitoring patient status, or for screening donors of blood, plasma, or human cells, tissues, and cellular and tissue-based products (HCT/Ps) for HIV.

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### 3. TECHNOLOGICAL CHARACTERISTICS

The primary technological characteristics and intended use of the **cobas®** HIV-1/HIV-2 Qualitative are substantially equivalent to other legally marketed nucleic acid amplification tests intended for the qualitative detection and differentiation of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) nucleic acid in human serum, plasma, and dried blood spots (DBS).

As indicated in Table 1, **cobas®** HIV-1/HIV-2 Qualitative is substantially equivalent to significant characteristics of the identified predicate device, **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test for use on the **cobas®** 5800/6800/8800 systems (**cobas®** HIV-1/HIV-2 Qualitative) (BK251233).

**Table 1: Similarities Between the Subject and the Predicate Device.**

| Comparator | Subject Device: cobas® HIV-1/HIV-2 Qualitative | Predicate Device: BK251233 cobas HIV-1/HIV-2 Qualitative |
| --- | --- | --- |
| Proprietary Name | **cobas®** HIV-1/HIV-2 Qualitative Nucleic acid test for use on the **cobas®** 5800/6800/8800 systems | Same |
| Regulation Number | 21 CFR 866.3957 | Same |
| Regulation Name | Human immunodeficiency virus (HIV) nucleic acid (NAT) diagnostic and/or supplemental test | Same |
| Regulatory Class | Class II | Same |
| Product Code | QST | Same |

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| Comparator | Subject Device: cobas® HIV-1/HIV-2 Qualitative | Predicate Device: BK251233 cobas HIV-1/HIV-2 Qualitative |
| --- | --- | --- |
| Intended Use | **cobas®** HIV-1/HIV-2 Qualitative for use on the **cobas®** 5800/6800/8800 Systems is an in vitro nucleic acid amplification test for the qualitative detection and differentiation of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) nucleic acid in human serum and plasma. The test is intended to be used as an aid in diagnosis of HIV-1/HIV-2 infection. Detection of HIV-1 or HIV-2 nucleic acid is indicative of HIV-1 or HIV-2 infection, respectively. The presence of HIV-1 or HIV-2 nucleic acid in the plasma or serum of individuals without antibodies to HIV-1 or HIV-2 is indicative of acute or primary infection. The **cobas®** HIV-1/HIV-2 Qualitative may also be used as an additional test to confirm the presence of HIV-1 or HIV-2 infection in an individual with specimens reactive for HIV-1 or HIV-2 antibodies or antigens. The assay may also be used as an aid in the diagnosis of infection with HIV-1 and/or HIV-2 in pediatric subjects and pregnant women. The test is also intended for early infant diagnosis (EID) of HIV-1 or HIV-2 infection in infants ≤18 months using dried blood spot (DBS) specimens. This assay is not intended to be used for monitoring patient status, or for screening donors of blood, plasma, or human cells, tissues, and cellular and tissue-based products (HCT/Ps) for HIV. | **cobas®** HIV-1/HIV-2 Qualitative for use on the **cobas®** 5800/6800/8800 Systems is an in vitro nucleic acid amplification test for the qualitative detection and differentiation of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) RNA in human serum and plasma. The test is intended to be used as an aid in diagnosis of HIV-1/HIV-2 infection. Detection of HIV-1 or HIV-2 nucleic acid is indicative of HIV-1 or HIV-2 infection, respectively. The presence of HIV-1 or HIV-2 nucleic acid in the plasma or serum of individuals without antibodies to HIV-1 or HIV-2 is indicative of acute or primary infection. The **cobas®** HIV-1/HIV-2 Qualitative may also be used as an additional test to confirm the presence of HIV-1 or HIV-2 infection in an individual with specimens reactive for HIV-1 or HIV-2 antibodies or antigens. The assay may also be used as an aid in the diagnosis of infection with HIV-1 and/or HIV-2 in pediatric subjects and pregnant women. This assay is not intended to be used for monitoring patient status, or for screening donors of blood, plasma, or human cells, tissues, and cellular and tissue-based products (HCT/Ps) for HIV. |
| Conditions for use | For prescription use | Same |
| Analyte Targets | HIV-1 HIV-2 | Same |
| Sample Preparation Procedure | Automated by cobas 5800/6800/8800 systems | Same |
| Amplification Technology | Real-time PCR | Same |
| Detection Chemistry | Paired reporter and quencher fluorescence labeled probes (TaqMan Technology) | Same |
| Controls used | RNA Internal Control (internal control) **cobas®** HIV-1/HIV-2 Qualitative Control Kit (external positive control) **cobas®** NHP Negative Control Kit (external negative control) | Same |

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| Comparator | Subject Device: cobas® HIV-1/HIV-2 Qualitative | Predicate Device: BK251233 cobas HIV-1/HIV-2 Qualitative |
| --- | --- | --- |
| Instrument Platform | **cobas**® 5800/6800/8800 systems | Same |
| Control Scheduling | Positive control and negative control included on every amplification/detection plate | Same |

Table 2: Differences Between the Subject and the Predicate Device

| Parameter | Subject Device: cobas HIV-1/HIV-2 Qualitative | Predicate Device: BK251233 cobas HIV-1/HIV-2 Qualitative |
| --- | --- | --- |
| Intended Population | Individuals suspected of active HIV-1/HIV-2 infection and for infants (≤18 months) for early infant diagnosis (EID). | Individuals suspected of active HIV-1/HIV-2 infection |
| Sample Types | Human serum, plasma, and dried blood spots (DBS) for infants (≤18 months) for early infant diagnosis (EID). | Human serum and plasma |
| Minimum amount of Sample required | Serum and Plasma: 650 μL DBS: 70 μL dried blood/spot in 1,150 μL Specimen Pre-extraction Reagent (SPER) | Serum and Plasma: 650 μL |
| Sample Process Volume | Serum and Plasma: 500 μL DBS: 850 μL | Serum and Plasma: 500 μL |

#### 4. NON-CLINICAL PERFORMANCE EVALUATION

Non-clinical performance for serum and plasma sample types has been established in the initial PMA submission (BP190360). Non-clinical performance for DBS for use in Early Infant Diagnosis is summarized below.

#### 4.1. Limit of Detection (LoD) for DBS

The limit of detection for DBS on Munktell-TFN with the cobas® HIV-1/HIV-2 Qualitative is shown in Table 3, Table 4, and Table 5.

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**Table 3: Results of 95% PROBIT analysis on LoD data collected with viral standards in DBS**

| Matrices | Analyte | Measuring units | LoD | Lower 95% confidence limit | Upper 95% confidence limit |
| --- | --- | --- | --- | --- | --- |
| DBS | HIV-1 group M | copies/mL | 255.1 | 223.7 | 299.1 |
| | HIV-2 | copies/mL | 984.2 | 856.2 | 1169.0 |

**Table 4: Reactivity rates summary for HIV-1 group M in DBS**

| Matrices | HIV-1 group M RNA concentration (cp/mL) | Number of reactive | Number of valid replicates | % Reactive |
| --- | --- | --- | --- | --- |
| DBS | 750 | 252 | 252 | 100% |
| | 600 | 252 | 252 | 100% |
| | 360 | 246 | 250 | 98% |
| | 180 | 220 | 249 | 88% |
| | 90 | 163 | 252 | 65% |
| | 45 | 109 | 250 | 44% |
| | 0 | 0 | 107 | 0% |

**Table 5: Reactivity rates summary for HIV-2 in DBS**

| Matrices | HIV-2 RNA concentration (cp/mL) | Number of reactive | Number of valid replicates | % Reactive |
| --- | --- | --- | --- | --- |
| DBS | 3000 | 252 | 252 | 100% |
| | 1450 | 241 | 247 | 98% |
| | 725 | 226 | 246 | 92% |
| | 362 | 167 | 248 | 67% |
| | 181 | 103 | 250 | 41% |
| | 0 | 0 | 108 | 0% |

#### 4.2. Precision – within laboratory

Precision of **cobas**$^{®}$ HIV-1/HIV-2 Qualitative with DBS was determined for HIV-1 group M and HIV-2 at ~3x LoD, ~1x LoD, and ~0.6x LoD using three reagent lots. The number of replicates and reactive samples tested, along with the precision results of **cobas**$^{®}$ HIV-1/HIV-2 Qualitative with DBS are summarized in Table 6. The results support the conclusion that the **cobas** HIV-1/HIV-2 Qualitative test is reproducible over multiple days, reagent lots, instruments, and replicates per run.

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**Table 6: cobas® HIV-1/HIV-2 Qualitative reagent lot-to-lot precision summary (DBS)**

| Analyte | Concentration | Reagent lot | % Reactive (reactive/valid replicates) | Lower limit of 95% confidence interval | Upper limit of 95% confidence interval |
| --- | --- | --- | --- | --- | --- |
| HIV-1 group M | ~0.6 × LoD | 1 | 88.0% (73/83) | 79.0% | 94.1% |
| HIV-1 group M | ~0.6 × LoD | 2 | 83.3% (70/84) | 73.6% | 90.6% |
| HIV-1 group M | ~0.6 × LoD | 3 | 88.1% (74/84) | 79.2% | 94.1% |
| HIV-1 group M | ~1 × LoD | 1 | 96.4% (81/84) | 89.9% | 99.3% |
| HIV-1 group M | ~1 × LoD | 2 | 96.4% (81/84) | 89.9% | 99.3% |
| HIV-1 group M | ~1 × LoD | 3 | 95.2% (80/84) | 88.3% | 98.7% |
| HIV-1 group M | ~3 × LoD | 1 | 100% (84/84) | 95.7% | 100% |
| HIV-1 group M | ~3 × LoD | 2 | 100% (83/83) | 95.7% | 100% |
| HIV-1 group M | ~3 × LoD | 3 | 97.6% (82/84) | 91.7% | 99.7% |
| HIV-2 | ~0.6 × LoD | 1 | 88.1% (74/84) | 79.2% | 94.1% |
| HIV-2 | ~0.6 × LoD | 2 | 91.7% (77/84) | 83.6% | 96.6% |
| HIV-2 | ~0.6 × LoD | 3 | 85.7% (72/84) | 76.4% | 92.4% |
| HIV-2 | ~1 × LoD | 1 | 97.6% (82/84) | 91.7% | 99.7% |
| HIV-2 | ~1 × LoD | 2 | 97.6% (82/84) | 91.7% | 99.7% |
| HIV-2 | ~1 × LoD | 3 | 98.8% (83/84) | 93.5% | 100% |
| HIV-2 | ~3 × LoD | 1 | 100% (83/83) | 95.7% | 100% |
| HIV-2 | ~3 × LoD | 2 | 100% (84/84) | 95.7% | 100% |
| HIV-2 | ~3 × LoD | 3 | 100% (83/83) | 95.7% | 100% |

#### 4.2.1. Group/subtype verification

The performance of **cobas®** HIV-1/HIV-2 Qualitative on HIV-1 group M subtypes, group O, group N, and HIV-2 group B was evaluated by verification of the detectability at the limit of detection for HIV-1 group M subtypes, group O (verified by dilution in whole blood for DBS), group N, and HIV-2 group B.

Clinical or cultured HIV samples for HIV-1 group M (A, C, D, F, G, H) and circulating recombinant forms (CRF01_AE, CRF02_AG), HIV-1 group N and HIV-2 group B, and HIV-1 group O were diluted in HIV-negative whole blood, spotted onto Munktell TFN DBS cards to the LoD concentration of the predominant group/subtype (HIV-1 group M subtype B or HIV-2 group A) based on the LoD determined with 95% PROBIT analysis over all lots combined. The reactive rate determination was performed with 42 replicates processed according to the DBS workflow. Testing was conducted with one lot of **cobas®** HIV-1/HIV-2 Qualitative reagents. The

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results from HIV-1 are shown in Table 7 and the results from HIV-2 are shown in Table 8. These results demonstrate that cobas® HIV-1/HIV-2 Qualitative detected HIV for HIV-1 group M (A, C, D, F, G, H, CRF01_AE, CRF02_AG), HIV-1 group O, HIV-1 group N and HIV-2 group B at the claimed concentration for DBS on Munktell-TFN cards with an upper 95% confidence interval being equal to or greater to the expected reactivity rate of 95%.

Table 7: LoD verification of HIV-1 group M subtypes, group O, and group N in whole blood for DBS

| Group | Subtype | DBS: 255 cp/mL Number of valid replicates | DBS: 255 cp/mL Number of Reactive | % Reactive (95% CI*) |
| --- | --- | --- | --- | --- |
| M | A | 41 | 37 | 90% (97%) |
| M | C | 42 | 42 | 100% (100%) |
| M | D | 42 | 39 | 93% (99%) |
| M | F | 42 | 40 | 95% (99%) |
| M | G | 42 | 42 | 100% (100%) |
| M | H | 42 | 41 | 98% (100%) |
| M | CRF01_AE | 42 | 41 | 98% (100%) |
| M | CRF02_AG | 42 | 42 | 100% (100%) |
| O | N/A | 41 | 39 | 95% (99%) |
| N | N/A | 41 | 40 | 98% (100%) |

* Upper 95% confidence interval

Table 8: LoD verification of HIV-2 group B in whole blood for DBS

| Group | DBS: 984 cp/mL Number of valid replicates | DBS: 984 cp/mL Number of valid reactive | % Reactive (95% CI*) |
| --- | --- | --- | --- |
| B | 42 | 42 | 100% (100%) |

* Upper 95% confidence interval

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### 4.3. Analytical Specificity

The specificity of cobas® HIV-1/HIV-2 Qualitative was determined by analyzing HIV-negative DBS samples from individual blood donors. A total of 604 individual Munktell-TFN DBS samples were tested with three lots of cobas® HIV-1/HIV-2 Qualitative reagents. The study was performed following the DBS sample preparation workflow. All samples tested were found non-reactive for HIV-1 and HIV-2. The specificity of cobas® HIV-1/HIV-2 Qualitative in DBS samples was 100% (95% confidence limit: 99.5% – 100.0%).

### 4.4. Cross contamination

The cross-contamination rate for cobas® HIV-1/HIV-2 Qualitative was determined by testing 240 replicates of an HIV-negative DBS sample and 225 replicates of a high titer HIV-1 DBS sample at 2.0E+07 cp/mL. The study was performed following the DBS sample preparation workflow. In total, five runs were performed with positive and negative samples in a checkerboard configuration.

All 240 replicates of the negative sample were non-reactive, resulting in a cross-contamination rate of 0%. The 95% confidence interval was 0% for the lower bound and 1.5% for the upper bound.

### 4.5. Endogenous Interference

The effect of potentially interfering endogenous substances in DBS on the performance of cobas® HIV-1/HIV-2 Qualitative test was evaluated by testing 10 individual whole blood donor samples spiked with the potential interferents at the levels recommended by the CLSI guideline Interference Testing in Clinical Chemistry (EP07-A2). The potential interferents tested were albumin (60,000 mg/L), conjugated bilirubin (342 μmol/L), unconjugated bilirubin (342 μmol/L), hemoglobin (2,000 mg/L), triglycerides/intralipid (37 mmol/L), and human DNA (2,000 mg/L). Twelve HIV-negative whole blood specimens spiked with the HIV targets and interferents were spotted on Munktell-TFN DBS cards and used as positive samples for the target analyte. All target negative DBS samples generated valid negative results indicating that the sensitivity and specificity of the cobas® HIV-1/HIV-2 Qualitative was not affected by the potential endogenous interfering substances.

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### 4.6. Onboard Kit Stability

The onboard stability of Roche Manufactured Controls (RMCs) for the **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test using the Dried Blood Spot (DBS) workflow on the **cobas®** 6800/8800 Systems was evaluated. Three RMCs were tested: the HIV-1M/HIV-2 co-formulated positive control, the HIV-1 Group O positive control, and the **cobas®** NHP Negative Control (NC). To simulate stressed onboard conditions, the positive controls were incubated for 9 hours at 37°C and the negative control for 11 hours at 37°C; each was compared against an unstressed counterpart stored at 2–8°C. All stressed and unstressed RMCs demonstrated a 100% reactivity for the positive controls or 100% non-reactivity for the negative control.

### 4.7. Open Kit Stability

The open kit stability of the **cobas®** Specimen Pre-Extraction Reagent (SPER) used with the **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test on the **cobas®** 6800/8800 Systems was evaluated, with a minimum stability target of 30 days. Testing was conducted at four time points — Day 0, Day 10, Day 20, and Day 32 — using one reagent lot and one **cobas®** 8800 System. Between time points, the reagent bottle was subjected to cumulative thermal stress totaling 14 hours of incubation at 30°C (in seven 2-hour intervals) with the bottle open, then re-closed and stored at 2–8°C. At each time point, 11 replicates of both a co-formulated HIV-1/HIV-2 DBS panel at approximately 3× LoD and a HIV-negative DBS panel were tested. Both the HIV-1 and HIV-2 sensitivity panels demonstrated 11/11 reactive results (100%), and the HIV-negative panel demonstrated 11/11 non-reactive results (100%).

### 4.8. DBS Specimen Stability

This study evaluated the clinical specimen stability of DBS samples for the **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test on the **cobas®** 6800/8800 Systems. Whole blood from five HIV-negative donors was spiked with five HIV-1 and three HIV-2 clinical specimens at approximately 3x LoD, then spotted onto two types of specimen collection cards – Munktell-TFN and Whatman 903 - and stored at three temperatures (15°C, 23°C, and 30°C) for up to 12 weeks, with testing occurring at 0, 8, and 12 weeks. Each donor specimen was also used unspiked and tested at time point zero to confirm HIV negativity. Across all storage conditions, time points, and card types, the HIV-1 and HIV-2 spiked samples demonstrated a 100% reactivity rate (20/20 valid replicates per condition). Unspiked donor samples were 100% non-reactive for both HIV-1 and HIV-2 at baseline. These results demonstrate that DBS specimens stored on either card type at

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temperatures ranging from 15°C to 30°C for up to 12 weeks remain stable and detectable by the **cobas®** HIV-1/2 Qualitative Test without any loss of sensitivity.

### 4.9. Specimen Card Collection Equivalency

This study evaluated whether the type of DBS specimen collection card, the Munktell-TFN and Whatman 903 cards, had any effect on the performance of the **cobas®** HIV-1/HIV-2 Qualitative test. DBS specimens were prepared from whole blood collected from 25 individual donors, with each donor's blood spotted onto both card types; samples were either left unspiked (serving as negative controls) or co-spiked with HIV-1 and HIV-2 at approximately 3x LoD. Testing was conducted across three valid runs using a single reagent lot and one **cobas®** 6800 System, with one replicate per donor per card type. The results demonstrated equivalency between the two card types: all spiked samples yielded a 100% reactivity rate for both HIV-1 and HIV-2, and all unspiked samples yielded a 100% negativity rate, with internal controls performing as expected across all conditions.

### 4.10. Lot Interchangeability

Lot interchangeability for the **cobas®** HIV-1/HIV-2 Qualitative nucleic acid test was evaluated using DBS samples on the **cobas®** 6800 and 8800 Systems. Three unique kit lot combinations of test-specific reagent cassettes (96T), **cobas®** omni reagents, **cobas®** Specimen Pre-Extraction reagents, and Roche Manufactured Controls (RMC) were tested using a DBS-specific functional testing procedure and an established RMC release procedure. Test panels included a co-formulated HIV-1/HIV-2 DBS panel at approximately 3x LoD, an HIV-negative DBS panel, and RMC (HIV-1M/HIV-2, HIV-1 Group O, and negative controls), each run with 8 – 11 replicates. All three kit lot combinations demonstrated 100% sensitivity and 100% negativity across all DBS panel test cases, and all RMC test cases demonstrated 100% reactivity.

### 5. CLINICAL PERFORMANCE EVALUATION

Clinical performance for serum and plasma sample types has been established in the initial PMA submission (BP190360). Clinical performance for DBS for EID is summarized below.

A clinical study was conducted in 2016 in Johannesburg, South Africa to evaluate the performance of the **cobas®** HIV1/HIV-2 Qualitative nucleic acid test for use on the **cobas®** 6800/8800 Systems using DBS for EID in an external laboratory and compared with a

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comparator device with a claim for DBS for EID. Two filter papers were used in the study: the Whatman 903 and the Munktell-TFN filter papers.

The study was performed at one external testing site and one sample-providing site from November to December 2016.

Out of 281 valid results with the **cobas**® HIV-1/2 Qual Test and 280 valid results on the comparator assay, 279 samples generated valid results on both systems and are included in the evaluation for the comparison study.

The results of the comparative analysis of the **cobas**® HIV-1/2 Qual Test compared to the comparator assay using DBS are presented in Table 9.

**Table 9: Detailed results of the comparison study**

| Assay | Result | Comparator Assay Positive | Comparator Assay Negative |
| --- | --- | --- | --- |
| **cobas**® HIV-1/2 Qual Test for use on **cobas**® 6800/8800 Systems | Positive | 127 | 1 |
| **cobas**® HIV-1/2 Qual Test for use on **cobas**® 6800/8800 Systems | Negative | 0 | 151 |

Clinical Sensitivity = **100%** (127/127) [95%CI: 97.13 - 100%]

Clinical Specificity = **99.34%** (151/152) [95%CI: 97.5 - 100%]

Overall Percent Agreement = **99.64%** [95%CI 98.02% - 99.99%]

The McNemar exact test showed a p value of 1.

In total, 127 HIV-1 positive DBS samples from infants were analyzed in single determinations with the **cobas**® HIV-1/2 Qual Test and generated a positive result. The clinical sensitivity of the **cobas**® HIV-1/2 Qual Test was determined to be 100% [95% CI: 97.13 - 100%].

## 6. CONCLUSIONS

The **cobas**® HIV-1/HIV-2 Qualitative assay reagents have not changed with the inclusion of DBS as an additional sample type for Early Infant Diagnosis of HIV infection. Non-clinical studies were used to evaluate the performance of DBS for use with **cobas**® HIV-1/HIV-2

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Qualitative Assay on the **cobas**® 5800/6800/8800 systems. Clinical performance for DBS was evaluated by comparing performance with an HIV-1/HIV-2 assay with a claim for DBS for EID. The **cobas**® HIV-1/HIV-2 Qualitative with the added claim for DBS for Early Infant Diagnosis has the same technology, similar intended use, and the data presented demonstrates that the device is as safe, as effective, and performs as well as the predicate.

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