Molecular Diagnosis of Infectious Diseases

Thilagavathy

Consultant-Microbiologist, Kauvery Hospital, Cantonment, Trichy

Background

Infectious molecular testing has moved from single plex to multiplex testing.

Syndromic testing is the process of using one test to simultaneously target multiple pathogens with overlapping signs and symptoms.

Major disadvantage of conventional multiplex assays is cross contamination, reagent contamination, repeated freeze thaw leading to deterioration of reagents, and increased false positivity rates.

Disease-Specific Panels provide comprehensive coverage for Syndromic Testing

Biofire® Respiratory 2.1 Plus Panel*

22 Targets

  • 4 bacteria
  • 19 viruses

Biofire® Blood Culture Identification 2 Panel†*

43 Targets

  • 26 bacteria
  • 7 yeast
  • 10 antimicrobial resistance genes

Biofire® Filmarray® Gastrointestinal Panel†‡

22 Targets

  • 13 bacteria
  • 5 viruses
  • 4 parasites

Biofire® Filmarray® Meningitis/Encephalitis Panel†*

14 Targets

  • 6 bacteria
  • 7 viruses
  • 1 yeast

Biofire® Oint Infection Panel†*

39 Targets

  • 29 bacteria
  • 2 yeast
  • 8 antimicrobial resistance genes

Setting Up the Filmarray is easy

The FilmArray pouch

Step 5: Amplification 2nd PCR Singleplex

  • 102 individual (1 mL) 2nd stage PCR wells pre-spotted with primer pairs
  • Each well contains one reaction
  • Multiple PCR cycles performed
  • Nested singleplex reaction setup
    • One primer pair in each array well

Detection

Targets are tested in triplicate

  • 1 & RP2.1plus Panels
  • GI Panel
  • ME Panel

Targets tested in duplicate

  • Pneumonia Panel
  • BCID2 Panel
  • Joint Infection Panel

Respiratory 2.1(RP 2.1) Panel

Sample Type: Nasopharyngeal swab in viral transport media

Sample Volume: 300 µL

VirusesBacteria
AdenovirusBordetella parapertussis
Coronavirus 229EBordetella pertussis
Coronavirus HKU1Chlamydia pneumoniae
Coronavirus NL63Mycoplasma pneumoniae
Coronavirus OC43
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
Human Metapneumovirus
Human Rhinovirus/Enterovirus
Influenza A
Influenza A/H1
Influenza A/H1-2009
Influenza A/H3
Influenza B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4
Respiratory Syncytial Virus

*Overall 97.4% Sensitivity and 99.4% Specificity

Biofire® RP 2.1 Panel Specifications

Nasopharyngeal swab placed in transport media or saline (up to 3mL) à 300 µL

Methods

At room temperature for up to 4 hours (15-25°C)

Refrigerated for up to 3 days (2-8°C)

Frozen (≤ – 70°C for up to 30 days)

Ideal patients for the Biofire RP Panel

At a minimum, many utilize the BioFire RP Panel for these patients:

  • Intensive care (ICU/NICU/PICU)
  • Adults 65+
  • Children under 5
  • Immunocompromised patients (chemo, HIV, bone marrow/organ transplant, hematologic disorders)

Our Experience

  • Respiratory Panels
  • Influenza B -2
  • Influenza A – 4
  • Adenovirus -1
  • Parainfluenza virus – 1
  • Human rhinovirus/enterovirus -4
  • Mycoplasma pneumoniae -1

Blood Culture Identification 2 Panel

Sample Type: Positive blood culture

Sample Volume: 200 µL

Gram-negative BacteriaGram-Positive BacteriaYeastAntimicrobial Resistance Genes
Acinetobacter calcoaceticus-baumannii complexEnterococcus faecalisCandida albicansCarbapenemases
Bacteriodes fragilisEnterococcus faeciumCandida aurisIMP
EnterobacteralesListeria monocytogenesCandida glabrataKPC
Enterobacter cloacae complexStaphylococcusCandida kruseiOxa-48-like
Escherichia coliStaphylococcus aureusCandida parapsilosisNDM
Klebsiella aerogenesStaphylococcus epidermidisCandida tropicalisVIM
Klebsiella oxytocaStaphylococcus lugdunensisCryptococcus neoformans/gattiiColistin Resistance
Klebsiella pneumoniae groupStreptococcusmcr-1
ProteusStreptococcus agalactiaeESBL
SalmonellaStreptococcus pneumoniaeCTX-M
Serratia marcescensStreptococcus pyogenesMethicillin Resistance
Haemophilus influenzaemecA/C
Neisseria meningitidismecA/C and MREJ
Pseudomonas aeruginosaVancomycin Resistance
Stenotrophomonas maltophiliavanA/B

*Overall 99% Sensitivity and 99.8% Specificity

Streamline Workflow and provides fast, actionable results

Advantages

  • Reduced turn around time
  • Biggest advantage is for detection of MDR GNB and VRE –empirical tiherapy is Ineffective
  • Resistant gene detection
  • Can identify polymicrobial infection.

Limitations

  • 26 bacteria and 7 yeast identified
  • Burkholderia, Ralstonia species and certain anaerobes not identified.
  • Helped in rapid antibiotic escalation but de-escalation was much slower in a study.
  • Reflex testing

Gastrointestinal Panel

Sample Type: Stool in Cary Blair

Sample Volume: 200 µL

BacteriaVirusesParasites
Campylobacter (jejuni, coli, and upsaliensis)Adenovirus F 40/41Cryptosporidium
Clostridium difficile (Toxin A/B)AstrovirusCyclospora cayetanensis
Plesiomonas shigelloidesNorovirus GI/GIIEntamoeba histolytica
SalmonellaRotavirus AGiardia lamblia
Vibrio (parahaemolyticus, vulnificus, and cholerae)Sapovirus (I, II, IV, and V)
Vibrio cholerae
Yersinia enterocolitica
Diarrheagenic E. coli/Shigella
Enteroaggregative E. coli (EAEC)
Enteropathogenic E. coli (EPEC)
Enterotoxigenic E. coli (ETEC)
Shiga-like toxin-producing E. coli (STEC)
E. coli O157
Shigella/Enteroinvasive E. coli (EIEC)

*Overall 98.5% Sensitivity and 99.2% Specificity1

Indications

  • Individuals at high risk of spreading disease to others and during known or suspected outbreaks
  • Cases presenting with:
    • Dysentery
    • Moderate to severe disease
    • Symptoms lasting more than seven days
    • Signs of sepsis
  • Immunocompromised patients, especially those with moderate or severe primary or secondary immune deficiencies
  • Returning travelers, untreated or following treatment failure

Limitations

  • Does not detect all parasitic infection (Cystoisospora ,Strongyloides)
  • The biofire GI panel primarily provides qualitative results (positive or negative for specific pathogens), lacking the ability to quantify the amount of each pathogen present.
  • A positive result does not rule out the possibility of co-infection with other pathogens not included in the panel.
  • False positive norovirus noted.

Clinical Benefits

  • Reduce antibiotic use1
  • Reduce time to antimicrobial therapy2
  • Lead to more targeted therapy2
  • Reduce downstream procedures such as endoscopies and abdominal imaging1

17% more targeted therapy2

Antibiotic prescriptions were 11% less likely1

Challenges in Diagnosing Meningitis and Encephalitis Infections

  • Meningitis and encephalitis often present with similar symptoms, sometimes as a flu-like illness1
  • Since the causative agents are often not distinguishable based on clinical symptoms alone, a specific diagnosis often needs accurate and comprehensive laboratory testing2

Meningitis/Encephalitis Panel

Sample Type: cerebrospinal fluid

Sample Volume: 200 µL

Bacteria (6)Viruses (7)Yeast (1)
Escherichia coli K1Cytomegalovirus (CMV)Cryptococcus neoformans/gattii
Haemophilus influenzaeEnterovirus (EV)
Listeria monocytogenesHerpes Simplex Virus 1 (HSV-1)
Neisseria meningitidisHerpes Simplex Virus 2 (HSV-2)
Streptococcus agalactiaeHuman Herpesvirus 6 (HHV-6)
Streptococcus pneumoniaeHuman Parechovirus (HPeV)
Varicella Zoster Virus (VZV)

*Overall 94.2% Sensitivity and 99.8% Specificity1

Sepsis Panel

Transplant Panel

Pitfalls

  • Discriminate between active infection and asymptomatic colonization
  • Detection of non-viable nucleic acids

Traditional Diagnostic Testing Makes the Clinician Choose Among Speed, Accuracy, and Comprehensiveness

References

  1. Axelrad JE, Freedberg DE, Whittier S, Greendyke W, Lebwohl B, Green DA. Impact of Gastrointestinal Panel Implementation on Healthcare Utilization and Outcomes. J of Clin. Microbiology. 2019; 27;57(3). e01775-18.
  2. Cybulski R, Bateman A, Bourassa L, Bryan A, Beail B, Matsumoto J, Cookson B, Fang FC; Clinical impact of a Multiplex Gastrointestinal PCR Panel in Patients with Acute Gastroenteritis. 2018. Clinical Infectious Diseases, ciy357, https://doi.org/10.1093/cid/ciy357.

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