Compliance

PMDA Compliance

PMDA guidelines for xenotransplantation pathogen screening and the 91 designated pathogens.

91 Pathogen Overview

PMDA Designated Pathogens

Complete coverage across 5 categories

41
Viruses
27
Bacteria
19
Parasites
2
Fungi
5
Special Mgmt
91
Total Designated Pathogens

Detection Methods

  • • Kraken2 classification
  • • BLAST sequence alignment
  • • Diamond protein search
  • • PERV-specific PCR primers

Reporting Requirements

  • • All 91 pathogens tested
  • • PERV-specific analysis
  • • Quantitative results
  • • Quality control metrics

Validation Standards

  • • Sensitivity: >95%
  • • Specificity: >98%
  • • LOD documented
  • • Reproducibility >90%

Sample Preparation Protocol

Protocol 12 v2.1: Unified Workflow (Recommended)

TRUE 100% coverage - includes circular and ssDNA viruses

Workflow Specifications

Workflows:2 (DNA + RNA)
Hands-on time:15.5 hours
Cost/sample:¥162,000
Coverage:91/91 (TRUE 100%)

Key Features

  • Universal poly(A) selection for all RNA viruses
  • CpG methylation-based host depletion
  • Step 2.5: Circular DNA linearization + ssDNA conversion
  • LOD: 100-500 copies/mL (screening-sufficient)

Step 2.5: Circular & ssDNA Virus Support

Added to enable detection of 4 critical PMDA pathogens that were incompatible with standard ligation-based library preparation:

Affected Pathogens:
  • • PCV2 - Porcine Circovirus 2 (circular ssDNA, 1.7kb) - Special Mgmt
  • • PCV3 - Porcine Circovirus 3 (circular ssDNA, 2.0kb) - Special Mgmt
  • • TTV - Torque teno virus (circular ssDNA, 3.8kb)
  • • PPV - Porcine parvovirus (linear ssDNA, 5.0kb)
Process Steps:
  • • Sub-step 2.5.1: DNase I linearization (0.005 U, 30 min)
  • • Sub-step 2.5.2: Klenow Fragment second-strand synthesis (2 hours)
  • • Time added: +2.5 hours (+19%)
  • • Cost added: +¥5,000 (+3.2%)

Conditional Screening Strategy

Two-Tier Detection Approach

Universal screening + conditional follow-up for low-probability pathogens

While most PMDA pathogens are screened universally (100% of samples), certain low-probability pathogens trigger conditional follow-up testing only when initial metagenomic screening detects relevant signatures.

Universal Screening (90 pathogens)

All samples undergo metagenomic analysis via Protocol 12

  • • PERV (A/B/C subtypes)
  • • All 41 DNA/RNA viruses
  • • All 27 bacterial pathogens
  • • All 19 parasites
  • • All 2 fungi

Conditional Testing (1 pathogen)

Triggered only when metagenomic data shows relevant signatures

Porcine Spumavirus
Protocol 13: Nested PCR + Phylogenetic analysis
Trigger rate: 5-10% of samples
Detection probability: <0.1%
Reason: 0 detections in 70 years, tested precautionarily

Critical Pathogens

High-Priority Screening

7 pathogens requiring immediate action

PERV-A
Porcine Endogenous Retrovirus A
CRITICAL
PERV-B
Porcine Endogenous Retrovirus B
CRITICAL
PERV-C
Porcine Endogenous Retrovirus C
CRITICAL
ASFV
African Swine Fever Virus
CRITICAL
CSFV
Classical Swine Fever Virus
CRITICAL
FMDV
Foot-and-Mouth Disease Virus
CRITICAL
PRION
Transmissible Spongiform Encephalopathy
CRITICAL

PERV Detection

Porcine Endogenous Retrovirus (PERV)

Most critical pathogen for xenotransplantation

Detection Strategy

  • Targeted primers: PERV-specific PCR primers for env gene
  • NGS screening: Metagenomic reads aligned to PERV reference sequences
  • Typing: Differentiation between PERV-A, PERV-B, and PERV-C subtypes
  • Quantification: Copy number per mL plasma with 95% confidence intervals

Action Thresholds

Detection Threshold
1 read
Action
Immediate Quarantine
Notification
Immediate SNS Alert
Follow-up
PCR Confirmation

Example Detection Report

Sample PMDA Report

Run ID: RUN-2024-001 | Date: 2024-01-15

Detection Summary

Total PMDA Pathogens
91
Detected
5
Not Detected
86
Critical Detected
0

PERV Analysis

Detected Pathogens (Non-Critical)

HEV - Hepatitis E Virus
450 reads | Confidence: 96.0% | 8.5e+4 copies/mL
MEDIUM
PCV2 - Porcine Circovirus 2
1250 reads | Confidence: 98.0% | 5.2e+4 copies/mL
MEDIUM
SS - Streptococcus suis
320 reads | Confidence: 92.0% | 2.1e+3 copies/mL
MEDIUM
EC - Escherichia coli
180 reads | Confidence: 88.0% | 1.5e+3 copies/mL
MEDIUM
CA - Candida albicans
95 reads | Confidence: 85.0% | 8.2e+2 copies/mL
MEDIUM

JSON Report Format

pmda_report.jsonjson
{
  "run_id": "RUN-2024-001",
  "analysis_date": "2024-01-15T18:00:00Z",
  "pmda_compliance": {
    "total_pathogens": 91,
    "detected": 5,
    "not_detected": 86,
    "critical_detected": 0,
    "detection_rate": 0.055
  },
  "perv_analysis": {
    "detected": false,
    "perv_a": false,
    "perv_b": false,
    "perv_c": false,
    "pass": true
  },
  "detected_pathogens": [
    {
      "code": "PCV2",
      "name": "Porcine circovirus 2",
      "category": "virus",
      "reads": 1250,
      "confidence": 0.98,
      "risk_level": "MEDIUM",
      "is_pmda": true,
      "quantification": {
        "copies_per_ml": 52000,
        "log10_copies": 4.72
      }
    }
  ],
  "quality_metrics": {
    "total_reads": 50000,
    "mean_quality": 10.5,
    "depletion_efficiency": 0.95
  },
  "compliance_status": "PASS"
}

Validation Requirements

Analytical Validation

  • Limit of Detection (LOD): Documented for each pathogen
  • Reproducibility: CV <20% across replicates
  • Specificity: No cross-reactivity with host genome
  • Linearity: R² > 0.90 for quantification

Quality Control

  • Positive Control: Spiked pathogen standards
  • Negative Control: Pathogen-free plasma
  • Spike-in Control: PhiX174 for normalization
  • Data Integrity: ALCOA+ principles

Regulatory Resources

PMDA Guidelines

  • Xenotransplantation Guidelines (2024):
    Comprehensive guidelines for donor animal screening and pathogen testing
  • 91 Designated Pathogens List:
    Complete list of pathogens requiring screening for xenotransplantation
  • NGS Validation Guidance:
    Technical requirements for NGS-based pathogen detection methods