False negative result of polymerase chain reaction in very early stages of acute retinal necrosis.

BACKGROUND: Viral nucleic acid testing of intraocular fluid using polymerase chain reaction (PCR) is a major laboratory examination in the diagnosis of acute retinal necrosis (ARN). Importantly, false negative PCR results may occur in several special situations. We reported a case of ARN with a negative PCR result in the aqueous humour in the very early stages of disease. CASE PRESENTATION: A female patient presented to the ophthalmologist with complaints of blurred vision and redness in her left eye. Her medical history included ARN in her right eye 10 years prior. Although the result of the aqueous viral analysis by PCR in her left eye was negative the first time (one day after the appearance of ocular symptoms), ARN in her left eye was presumed based on the clinical signs. With timely antiviral and anti-inflammatory treatments, the retinal lesions diminished. The viral load of herpes simplex virus (HSV) turned positive (7.25 × 103 copies/mL) one week later, increased to 2.49 × 105 copies/mL after three weeks, and finally turned negative about five weeks after the onset of disease. The initial HSV-IgG level in the aqueous humour was 0.01 U/mL and increased to 222.64 U/mL in the final sampling. CONCLUSIONS: The results of PCR analysis can be negative in the very early stages of ARN. Diagnosis of ARN should be made based on the clinical features, and antiviral treatments should not be delayed. Repeated PCR analysis of the aqueous humour is necessary to confirm the diagnosis and monitor the disease process.

Reasons for truly negative cytology reports preceding the diagnoses of invasive cervical cancer-Results of a false-negative cytology audit in Polish Cervical Cancer Screening Programme.

BACKGROUND: False-negative (FN) results in cervical cancer (CC) screening pose significant risk for participants and should be audited. The aim of the study was to analyse the results of audit of FN slides collected in 2010-2013 in Polish Cervical Cancer Screening Program (CCSP) and to seek for risk factors of obtaining true-negative result (TN; not containing abnormal cells as confirmed in audit) before CC diagnosis. METHODS: Screening database was merged with National Cancer Registry to identify negative slides preceding histologically confirmed CC diagnosis up to 42 months. Two blinding slides were randomly assigned per each FN. The whole set was reassessed independently by three pathologists with 30 years of experience in cytology evaluation. Final audit result was established in the case of ≥2 coherent reports. Agreement rates and kappa (κ) coefficients were calculated. Logistic analysis of risk factors for obtaining TN result was performed. RESULTS: Of 374 included FNs, 204 were considered abnormal (54.6%) and 91 were confirmed negative for intraepithelial neoplasia (24.3%). Agreement between experts was moderate for FNs (κ = 0.266) and fair for blinding slides (κ = 0.142) when grouping abnormal slides. Adenocarcinoma diagnosis elevated the risk of TN result (OR = 3.83); detection of macroscopic changes on the cervix and smoking lowered the risk (OR = 0.39, OR = 0.40 respectively). CONCLUSIONS: Misinterpretation was the main reason for FN cytology in the CCSP which indicated the need of further personnel training to increase screening quality. Rather low agreement between auditors requires further insight. A standardised process of auditors' selection should be planned to increase audit quality.

False-negative rate of SARS-CoV-2 RT-PCR tests and its relationship to test timing and illness severity: A case series.

Objective: To determine the false-negative rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) test results, and to describe the characteristics of coronavirus disease 2019 (COVID-19) patients with false-negative results. Methods: We validated SARS-CoV-2-positive RT-PCR test results performed in hospitalized patients between February 1 and August 31, 2020 and classified the patients according to disease severity. Results: In total, 2038 SARS-CoV-2 RT-PCR tests were performed on 1890 patients. Of these, 145 patients had positive results and were diagnosed with COVID-19. Among the 145 patients with COVID-19, the initial RT-PCR tests were negative in five patients. Of these, three had moderate illness and were initially tested in the early stage of disease, and two had severe illness and were initially tested in the late stage of disease, when RT-PCR testing has lower sensitivity due to viral clearance. Conclusions: These findings suggest that false-negative results can be caused by both observer errors and by low viral RNA levels in the later stages of disease, after the infection has cleared. Clinicians should be aware that patients with COVID-19 can have negative RT-PCR test results in the later stages of infection.

A gradual change of chromosome mosaicism from placenta to fetus leading to T18 false negative result by NIPS.

BACKGROUND: Noninvasive prenatal screening (NIPS) has higher sensitivity and specificity compared to traditional prenatal screening. Nevertheless, the discordant results between the NIPS and prenatal diagnosis were occasionally reported. In current study, we investigated the genetic basis of a T18 fetus with a discordant trisomy 5 (T5) positive and trisomy 18 (T18) negative NIPS result. METHODS: NIPS was used to detect fetal DNA in maternal circulating plasma based on semiconductor sequencing platform. The aneuploidies of the fetus and different part of placental tissues were investigated by copy number variation sequencing (CNV-seq) and chromosome microarray analysis (CMA). RESULTS: The positive result of T5 was detected for the pregnant woman in NIPS, while T18 was found in the fetal karyotyping analysis after amniocentesis. Furthermore, placental mosaicism of T5 and T18 was found by CNV-seq and CMA, which revealed the mosaic ratio of T5 was gradually increased from umbilical cord to the placenta center, while that of T18 was gradually decreased. CONCLUSION: For the reason of cell-free fetal DNA (cff DNA) in the maternal circulation originates from trophoblast cells of placenta, the level of placental mosaicism could cause false negative NIPS result in multiple aneuploidies. The present study proved that a discordant T5 positive and T18 negative NIPS result was caused by placental mosaicism. This study highlights placental mosaicism as a significant risk factor for discordant NIPS results. The result will be helpful for genetic counseling and clinical management of such pregnant woman.

Negative Biopsy of Focal Hepatic Lesions: Decision Tree Model for Patient Management.

OBJECTIVE: The purpose of this study was to investigate patient- and procedure-related variables affecting the false-negative rate of ultrasound (US)-guided liver biopsy and to develop a standardized patient-tailored predictive model for the management of negative biopsy results. MATERIALS AND METHODS: We retrospectively included 389 patients (mean age ± SD, 62 ± 12 years old) who had undergone US-guided liver biopsy of 405 liver lesions between January 1, 2013, and June 30, 2015. We collected multiple patient- and procedure-related variables. By comparing pathology reports of biopsy and the reference standard (further histology or imaging follow-up), we were able to categorize the biopsy results as true-positive, true-negative, and false-negative. Diagnostic accuracy and diagnostic yield were measured. Univariate and multivariate analyses were performed to identify variables predicting false-negative results. A standardized patient-tailored predictive model of false-negative results based on a decision tree was fitted. RESULTS: Diagnostic accuracy and diagnostic yield were 93.8% (380/405) and 89.4% (362/405), respectively. The false-negative rate was 6.5% (25/387). Predictive variables of false-negative results at univariate analysis included body mass index, lesion size, sample acquisition techniques, and immediate specimen adequacy. The only independent predictors at multivariate analysis were patient age and Charlson comorbidity index. By combining lesion size and location with patient age and history of malignancy, we developed a decision tree model that predicts false-negative results with high confidence (up to 100%). CONCLUSION: False-negative results are not negligible at US-guided liver biopsy. The combination of selected lesion- and patient-specific variables may help predict when aggressive management is warranted in patients with likely false-negative results.

18F-fluorodeoxyglucose positron emission tomographic scan in solid-type p-stage-I pulmonary adenocarcinomas: what can produce false-negative results?

Objective: False-negative (FN) uptake of 18F-fluorodeoxyglucose (FDG) can be divided into those cases related to technological limitations of positron emission tomography (PET) and those related to inherent properties of neoplasms. Our goal was to clarify possible factors causing FN PET results in patients with solid-type pulmonary adenocarcinomas (PAs). Methods: From January 2007 to December 2014, of the 255 patients with p-stage-1 non-small-cell lung cancer observed and treated (surgically) in our institution, we retrospectively reviewed the PET/computed tomography (CT) records, the clinical information, the preoperative thin-section CT images, and the pathological features [classified by the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) subtyping criteria] of 94 consecutive solid-type p-stage-1 PAs. Univariable and multivariable logistic analyses were used to identify and weigh the independent predictors of the PET findings using the following variables: body weight, blood glucose level, tumour size, tumour location, and histological classification. Results: There were 58 men and 36 women (mean age = 68.7 ± 8.9 years, range 42-85). Considering the maximum standardized uptake value (SUVmax) ≥ 2.5 as a 'PET-positive' result, 77 lesions (81.9%) proved PET positive and 17 lesions (18.1%), PET negative (with SUVmax < 2.5). Overall, the median SUVmax value was 5.7 [interquartile range (IQR) 2.8-10.3]. Higher SUVmax values ( P < 0.001) were observed in those PAs larger than 2 cm in their major axis (median SUVmax = 9.0; IQR 4.6-14.6); in PAs < 2 cm, the median SUVmax was 4.1; IQR 2.2-5.9. When clustering the cohort in two histological classes (class A, colloid/mucinous/lepidic versus class B, micropapillary/solid/acinar/papillary), the radiometabolic patterns were significantly different (median SUVmax = 2.8; IQR 1.7-4.9 in class A vs median = 7.4 IQR 4.5-13.9 in class B, P < 0.001). Significant PET FN rates were reported in (i) PAs measuring < 2 cm in their major axis (27.9%), (ii) lesions located in the lower zones of the lung (31.0%), and (iii) class A tumours (37.5%). In the multivariable logistic analysis, histological type (IASLC/ATS/ERS aggregated clusters) proved to be the only independent relevant factor for determining whether PET results were negative or positive (OR:7.23, 95% CI: 2.05-25.43, P = 0.002). Conclusions: The IASLC/ATS/ERS pattern significantly influences FDG uptake in solid-type p-stage-1 PAs. The fact that colloid/mucinous/lepidic adenocarcinomas have a notable tendency to produce negative findings on PET scans warrants particular attention.

Dynamics of immune parameters during the treatment of active tuberculosis showing negative interferon gamma response at the time of diagnosis.

OBJECTIVES: In the performance of interferon gamma release assays (IGRA) for the diagnosis of tuberculosis (TB) infection, false-negative results are a major obstacle. In active TB patients, treatment-dependent changes of the negative test results remain unknown. METHODS: The treatment course of 19 smear-positive/culture-confirmed TB patients who had IGRA-negative results by QuantiFERON-TB in-tube (QFT-IT) method at the time of diagnosis (month 0) in a previous study, were monitored in the present study. Blood was further collected at months 2 and 7, and the concentrations of 27 immune molecules were measured in the plasma supernatants remaining after performing the IGRA, using a suspension array system. RESULTS: After initiating treatment, eight of the 19 QFT-IT-negative patients showed positive conversion, whereas the remaining 11 (58%) did not; the interferon gamma (IFN-γ) response was restored to levels higher than 1 IU/ml in only three of the eight patients with positive conversion. Plasma concentrations of interleukin 1 receptor antagonist, interleukin 2, and interferon gamma-induced protein 10 remained low after Mycobacterium tuberculosis-specific antigen stimulation at months 2 and 7 in the continuously QFT-IT-negative group, whereas the parameters were elevated only in the transiently QFT-IT-negative group. CONCLUSIONS: It was demonstrated that a majority of active TB patients showing negative IGRA results did not regain sufficient levels of immune responsiveness despite successful treatment.

Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis.

BACKGROUND: Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite's DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite's DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis. RESULTS: Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD - 567 bp) as well as of small quantities (10 pg) of the target parasite's DNA, detected by amplification of a 138 bp product. CONCLUSIONS: The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.

Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis

Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.

Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis

Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.