Herpes Simplex Virus-1 qPCR in the Diagnosis of Lower Respiratory Tract Infections in Organ Transplant Recipients and Critically Ill Patients

Michelle N. Stram, MD, ScM; Christopher N. Suciu, MD, MS; Jansen N. Seheult, MB BCh BAO, MSc, MD(Res); Melissa A. McCullough, MLS(ASCP); Muhamuda Kader, MSc, PhD; Alan Wells, MD, DMSc; A. William Pasculle, ScD; Charles R. Rinaldo, PhD; Nahed Ismail, MD, PhD

Disclosures

Am J Clin Pathol. 2018;150(6):522-532.

Abstract and Introduction

Abstract

Objectives: To determine a quantitative herpes simplex virus (HSV) DNA threshold in lower respiratory tract specimens that correlates with positive viral culture and clinical outcomes.

Methods: Bronchoalveolar lavage and bronchial wash samples from 53 HSV culture-positive and 61 culture-negative matched controls were tested using HSV-1 and HSV-2 quantitative polymerase chain reaction (qPCR).

Results: Median viral culture turnaround time was 21.8 days and 9.9 days for culture-negative and culture-positive specimens, respectively. Using an HSV-1 viral load threshold of 1.62 × 10³ copies/mL, there was 93% agreement with viral culture. An HSV-1 viral load ≥1.3 × 10⁴ copies/mL was associated with worse clinical outcome compared to a viral load <1.3 × 10⁴ copies/mL (hazard ratio [HR] = 4.27, P = .017), and there was a trend of worse outcome compared to patients with undetectable HSV-1 DNA (HR = 1.60, P = .056).

Conclusions: qPCR has clinical utility for rapid accurate identification of HSV-1 in lower respiratory tract specimens.

Introduction

Debate continues as to whether the detection of herpes simplex virus (HSV) in lower respiratory tract specimens, especially from immunosuppressed, critically ill patients, and organ transplant recipients, represents a true clinical infection requiring therapy or innocuous viral shedding secondary to severe underlying illness.^[1–4] Moreover, rendering a confident clinical diagnosis of HSV pneumonia is challenging, particularly because many patients lack easily detectable herpetic lesions, radiographic and bronchoscopic findings are nonspecific, and clinical interpretations are frequently complicated by superimposed coinfections.^[5–8] The morphologic and immunohistochemical evaluation of cellular material for the cytopathic effect of HSV, using bronchoalveolar lavage (BAL) cytology or lung biopsy specimens performed for another reason, constitutes the gold standard for HSV diagnosis due to the high specificity, but suffers from poor sensitivity.^[9] In many medical centers, the preferred method for identifying HSV in BAL specimens has become viral culture. Conventional viral culture, however, has a lengthy turnaround time (TAT) and is thus not the ideal diagnostic modality for guiding therapeutic decisions in critically ill patients.

Detection of HSV in the lower respiratory tract is further complicated by the nonuniformity of lavage specimens. BAL cellularity is affected by environmental exposures and underlying disease, as well as operator experience and technique.^[10–12] Unlike HSV viremia, where the diagnosis is dependent on the detection of cell-free viral DNA in plasma, the diagnosis of HSV-associated lower respiratory tract infections requires the detection of cell-associated viruses. Indeed, HSV has been detected by immunohistochemistry in alveolar macrophages and pulmonary epithelial lining cells in patients who died from acute respiratory distress syndrome or pneumonia; no extracellular virus was observed.^[9]

In this study, we determined a quantitative threshold of HSV DNA in lower respiratory tract specimens that correlated with positive viral culture and clinical outcome. We also investigated the utility of quantitative polymerase chain reaction (qPCR) for a housekeeping gene to account for differences in the cellularity of the lower respiratory tract specimens. Finally, we evaluated the relationship between the HSV viral load and clinically relevant outcomes.

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Next Section

Abstract and Introduction
Materials and Methods
Results
Discussion
References

Table 1. Demographics, Underlying Disease Parameters, Specimen Collection, and Admission Data of Patient Cohorts With and Without HSV Positive Viral Culture
Characteristic	Cases (n = 53)	Controls (n = 61)	P Value
Age, median y (IQR, range)	62 (53-65, 22–87)	62 (53-69, 23–90)	.695^a
Male:female ratio, No. (%)	30:23 (56.6:43.4)	34:27 (55.7:44.3)	.926^b
White:black:other, No. (%)	48:4:1 (90.6:7.6:1.9)	55:5:1 (90.2:8.2:1.6)	.987^b
Source of admission, No. (%)			.213^b
Emergency department	9 (17.0)	12 (19.7)
Outside hospital	28 (52.8)	29 (47.5)
Surgery	5 (9.4)	13 (21.3)
Internal medicine clinic	8 (15.1)	3 (4.9)
Outpatient bronchoscopy	3 (5.7)	4 (6.6)
Length of hospital stay prior to collection, median d (IQR, range)	7 (1-15, 0–249)	5 (1-12, 0–117)	.295^a
Admission to ICU within the week preceding specimen collection, No. (%)	40 (75.5)	47 (77.1)	.843^b
Sample collected in ICU, No. (%)	38 (71.7)	42 (68.9)	.740^b
Receiving mechanical ventilation at time of specimen collection, No. (%)	35 (66.0)	41 (67.2)	.894^b
Specimen collection after mechanical ventilation for at least 2 days, No. (%)	23 (43.4)	12 (19.7)	.006^b
Transplant recipient, No. (%)	22 (41.5)	24 (39.3)	.814^b
Lung	19 (35.9)	19 (31.2)	.622^b
Heart	1 (1.9)	3 (4.9)	.213^c
Kidney	1 (1.9)	5 (8.2)	.132^c
Liver	2 (3.8)	1 (1.6)	.597^c
Bone marrow	1 (1.9)	0 (0)	.465^c
Multiorgan	2 (3.8)	3 (4.9)	.000^c
Immunosuppression, No. (%)	34 (64.2)	42 (68.9)	.595^b
Nontransplant-related immunosuppression	12 (22.6)	18 (29.5)	.406^b
Transplant-related immunosuppression	22 (41.5)	24 (39.3)	.814^b
Underlying disease, No. (%)
Respiratory	47 (88.7)	51 (83.6)	.437^b
Cardiovascular	44 (83.0)	47 (77.1)	.428^b
Renal	23 (43.4)	26 (42.6)	.934^b
Shock ^d	6 (11.3)	10 (16.4)	.437^b
Neurologic ^e	5 (9.4)	7 (11.5)	.723^b
Autoimmune disease ^f	10 (18.9)	7 (11.5)	.269^b
AIDS	2 (3.8)	1 (1.6)	.597^c
Diabetes	19 (35.9)	22 (36.1)	.981^b
Hypothyroidism	5 (9.4)	11 (18.0)	.187^b
Malignancy	1 (1.9)	4 (6.6)	.370^c

AIDS, acquired autoimmune deficiency virus; HSV, herpes simplex virus; ICU, intensive care unit; IQR, interquartile range.
^aMann-Whitney U test.
^b χ ² test.
^cFisher exact test.
^dIncludes hemorrhagic and septic shock.
^eIncludes stroke, encephalopathy, anoxic brain injury, coma, and toxoplasmosis.
^fIncludes connective tissue disease, inflammatory bowel disease, polymyositis, and rheumatoid arthritis.

Table 2. HSV-1 Quantification by qPCR, Radiologic and Bronchoscopic Findings, and Antiviral Treatment in Culture-Positive and -Negative Patients
	Cases (n = 53)	Controls (n = 61)	P Value
qPCR results
HSV copy number/mL
Median	3.36 × 10⁵	0	<.001^a
IQR	1.44 × 10⁴-2.54 × 10⁶	0–0
Range	0–6.43 × 10⁷	0–5 × 10⁶
GAPDH copy number/mL
Median	2.69 × 10⁴	4.14 × 10⁴	.090^a
IQR	1.32 × 10⁴-6.56 × 10⁴	2.02 × 10⁴-9.09 × 10⁴
Range	5.59 × 10⁰-2.61 × 10⁵	5.61 × 10⁰-2.74 × 10⁵
HSV copy number/10 ⁴ GAPDH
Median	4.01 × 10⁵	0	<.001^a
IQR	1.25 × 10⁴-1.70 × 10⁶	0–0
Range	0–3.72 × 10⁸	0–1.77 × 10⁶
Bronchoscopy, No. (%)
Secretions	29 (54.7)	27 (44.3)	.265^b
Abnormal mucosa and secretions	16 (30.2)	16 (26.2)	.639^b
Negative	8 (15.1)	18 (29.5)	.067^b
Bronchial fluid cytology or lung biopsy performed	37 (69.8)	37 (60.7)	.307^c
Cytologic diagnosis of HSV	3 (5.7)	0 (0)	.097^c
Chest imaging, No. (%)	50 (94.3)	59 (96.7)	.662^c
Opacities or consolidations	36 (72.0)	36 (61.0)	.230^b
Only effusions or edema	7 (14.0)	14 (23.7)	.201^b
Negative	7 (14.0)	9 (15.3)	.854^b
Interpretation of pneumonia	21 (42.0)	25 (42.3)	.973^b
Antiviral treatment, No. (%)
Prophylaxis prior to BAL collection	5 (9.4)	17 (27.9)	.013^b
Due to symptoms after BAL collection	4 (7.5)	5 (8.2)	.898^c
Died prior to culture result	8 (15.1)	14 (22.9)	.291^b
Treatment due to culture result	21 (39.6)	0 (0)	<.001^c
No treatment after culture result	15 (28.3)	25 (39.7)	.204^b

BAL, bronchoalveolar lavage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSV, herpes simplex virus; IQR, interquartile range; qPCR, quantitative polymerase chain reaction.
^aMann-Whitney U test.
^b χ ² test.
^cFisher exact test.

Table 3. Clinical Information and Normalized qPCR Results for Cases With Discrepant Culture-qPCR Results and Cases With Dual Positivity for HSV-1 and HSV-2 by qPCR
Patient	HSV qPCR Result		Clinical Description
Patient	In-house Laboratory	Reference Laboratory	Clinical Description
Culture-negative/qPCR positive
Patient 1	8.08 × 10² HSV copies/mL	6.20 × 10³ HSV copies/mL	76-year-old man with acute respiratory distress and Staphylococcus aureus pneumonia following cardiac arrest in the ICU on mechanical ventilation
Patient 2	5.73 × 10³ HSV copies/mL	5.44 × 10⁵ HSV copies/mL	79-year-old man in the ICU on mechanical ventilation with clinical and radiographic findings consistent with pneumonia and no bacterial, fungal, or other viral pathogens identified in the diagnostic BAL specimen
Patient 3	1.28 × 10³ HSV copies/mL	3.85 × 10⁴ HSV copies/mL	61-year-old woman in the ICU on mechanical ventilation with clinical and radiographic findings consistent with pneumonia and no bacterial, fungal, or other viral pathogens identified in the diagnostic BAL specimen, who was receiving acyclovir for possible HSV meningitis
Patient 4	5.00 × 10⁶ HSV copies/mL	3.80 × 10⁷ HSV copies/mL	72-year-old woman in the ICU on mechanical ventilation receiving acyclovir treatment for systemic HSV-1 infection, HSV-1 1.91 × 10³ copies/mL blood, and possible HSV-1 encephalitis 5 days before BAL collection
Culture-positive/qPCR negative
Patient 5	Not detected	Not detected	57-year-old male lung transplant recipient with positive enterovirus identified in the BAL sample by RVP
Patient 6	Not detected	Not detected	58-year-old man in the ICU on mechanical ventilation with positive Proteus mirabilis and respiratory syncytial virus identified in the BAL sample by RVP
Patient 7	Not detected	Not detected	64-year-old female lung transplant recipient with a history of recurrent episodes of acute cell mediated rejection, most recently treated with IV methylprednisolone
Patient 8	Not detected	Not detected	62-year-old male lung and liver transplant recipient with a history of cystic fibrosis who was revived after cardiac arrest secondary to hyperkalemia
Patient 9	Not detected	7.00 × 10² HSV copies/mL	79-year-old man in the ICU on mechanical ventilation positive for adenovirus identified in the BAL sample by RVP
Culture-positive/HSV-1 + HSV-2 qPCR positive
Patient 10	2.77 × 10³ HSV-1 copies/mL	9.79 × 10² HSV-2 copies/mL	52-year-old man with HIV-1, viral load 1.31 × 10⁵ copies/mL plasma, a CD4 count of 8 cells/mm³ receiving ganciclovir treatment for systemic CMV infection, CMV 1.17 × 10⁶ copies/mL blood, and a BAL also positive for CMV by early antigen detection and Aspergillus fumigatus
Patient 11	1.98 × 10⁵ HSV-1 copies/mL	3.97 × 10³ HSV-2 copies/mL	52-year-old male lung transplant recipient with sepsis in the ICU on mechanical ventilation receiving valganciclovir with urine and sputum specimens positive for Legionella pneumophila
Patient 12	2.65 × 10⁶ HSV-1 copies/mL	1.90 × 10⁵ HSV-2 copies/mL	62-year-old woman with sepsis and ground-glass opacities in the ICU on mechanical ventilation who had a negative CSF qPCR for HSV-1 collected 2 days prior to BAL

BAL, bronchoalveolar lavage; CMV, cytomegalovirus; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; HSV, herpes simplex virus; ICU, intensive care unit; IV, intravenous; qPCR, quantitative polymerase chain reaction; RVP, respiratory viral panel (Genmark).

Table 4. Clinical Outcome Data of Patients With and Without HSV-Positive Viral Culture
	HSV ≥1.3 × 10⁴ Copies/mL (n = 41)	HSV <1.3 × 10⁴ Copies/mL (n = 73)^a	P Value^b
Death within 21 days after sample collection, No. (%)	11 (26.8)	15 (20.6)	.443
Days between specimen collection and death, median (IQR, range)	11 (5-25, 2–127)	7 (4-14, 1–85)	.374
Length of hospital stay following specimen collection, median d (IQR, range)	21 (7-30, 2–127)	9 (6-22, 1–85)	.027
Length of stay more than 21 days after sample collection, No. (%)	21 (51.2)	20 (27.4)	.011
Length of stay in ICU following specimen collection if specimen collection occurred within 48 hours of ICU admission, median d (IQR, range)	11 (7-21, 2–127)	10 (5-16, 1–85)	.298
ICU stay for more than 14 days after sample collection , No. (%)	16 (39.0)	18 (24.%)	.108
Subsequent continuous days of mechanical ventilation following specimen collection, median (IQR, range)	8.5 (5-17, 2–127)	8.5 (4-16, 1–85)	.612
Mechanical ventilation for more than 14 days after sample collection, No. (%)	11 (26.8)	11 (15.1)	.127
Worse combined clinical outcome,^c No. (%)	33 (80.5)	38 (52.1)	.003

^aIncludes 11 patients with detectable but low HSV1 viral loads (<1.3 × 10⁴ copies/mL) and 62 patients with undetectable HSV-1 DNA.
HSV, herpes simplex virus; ICU, intensive care unit; IQR, interquartile range.
^b χ ² test or Mann Whitney U test, where appropriate.
^cCombined clinical outcome includes death within 21 days after specimen collection, length of stay more than 21 days after sample collection, and ICU stay for more than 14 days after sample collection.

Table S1. Primers and Probes
Primers and Probes	5' → 3'
HSV-1
Forward primer	GGC GAC CTG TAT AAC GTG
Reverse primer	GTC ATA GCG CGG GTT C
Probe	56–FAM/CTT GGC CAA ACG CCT CCG TTC CAT GCA CGT CTT TA/3BHQ_1
HSV-2
Forward primer	ATG GTG AAC ATC GAC ATG TAC GG
Reverse primer	CGC GGT AGC TCA GAT CCT TCT
Probe	56–FAM/TCA AAC TCT CCA GCT ACA AGC TGA ACG CCG TCG CC/3BHQ_1
GAPDH
Forward primer	CAA CTA CAT GGT CTA CAT GTT C
Reverse primer	TCG CTC CTG GAA GAT G
Probe	6–FAM/CGG CAC AGT CAA GGC CGA GAA TGG GAA GC/MGB/BHQ
IPC
Forward primer	GGC ACT GCA TGA CCT GAT GA
Reverse primer	TCC ACC CTT CGT CCG TCT T

Abbreviations: HSV, herpes simplex virus; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; IPC, internal positive control.

Table S2. Infections within 7 days of viral culture specimen collection
	Cases (n=53)	Controls (n=61)	p-value
Pathogens by source detected within 7 days preceding viral culture specimen collection
Respiratory	8 (15.1%)	11 (18.0%)	0.675^a
B:F:V	7:1:2	8:0:3
Disseminated	3 (5.7%)	2 (3.3%)	0.536^b
B:F:V	2:0:1	0:0:2
Urine	3 (5.7%)	1 (1.6%)	0.245^b
B:F:V	2:1:0	0:1:0
Cerebrospinal fluid	0 (0%)	1 (1.6%)	0.349^b
B:F:V	0:0:0	0:0:1
Wound	4 (7.6%)	0 (0%)	0.029^b
B:F:V	4:1:0	0:0:0
Other	1 (1.9%)	0 (0%)	0.281^b
B:F:V	1:1:0	0:0:0
Coinfections in clinical sample submitted for HSV viral culture
Any coinfection	27 (50.9%)	30 (49.2%)	0.851^a
B:F:V	18:4:13	16:10:7
Bacteria	18 (34.0%)	16 (26.2%)	0.368^a
mycobacterium	2 (3.8%)	0 (0%)
gram + bacteria	5 (9.4%)	10 (16.4%)
gram – bacteria	13 (24.5%)	8 (13.1%)
Fungus	4 (7.5%)	10 (16.4%)	0.252^b
Virus	13 (24.5%)	7 (11.5%)	0.068^a
cytomegalovirus	5 (9.4%)	0 (0%)
parainfluenza	3 (5.7%)	0 (0%)
rhinovirus	4 (7.6%)	4 (6.6%)
influenza	2 (3.8%)	3 (4.9%)
respiratory syncytial virus	1 (1.9%)	0 (0%)
adenovirus	1 (1.9%)	0 (0%)
enterovirus	1 (1.9%)	0 (0%)
metapneumovirus	0 (0%)	1 (1.6%)
Pathogens by source detected within 7 days following viral culture specimen collection
Respiratory	24 (45.3%)	23 (37.7%)	0.412^a
B:F:V	17:3:12	17:3:8
Disseminated	4 (7.6%)	2 (3.3%)	0.414^b
B:F:V	2:0:3	1:1:0
Urine	3 (5.7%)	0 (0%)	0.097^b
B:F:V	3:0:0	0:0:0
Wound	1 (1.9%)	0 (0%)	0.465^b
B:F:V	1:0:0	0:0:0