False-Positive PCR Detection of Tropheryma whipplei in Cerebrospinal Fluid and Biopsy Samples from a Child with Chronic Lymphocytic Meningitis

Abstract

We report the case of a teenager with chronic lymphocytic meningitis for whom Tropheryma whipplei 16S rRNA PCR results were positive in two cerebrospinal fluid samples and one duodenal biopsy specimen. PCR targeting another specific sequence of Tropheryma whipplei and sequencing of the initially amplified 16S rRNA fragment did not confirm the results. CASE REPORTA 13-year-old boy went to the emergency room with a persistent headache which lasted for 2 weeks and nausea, vomiting, and dizziness. His past medical history included Helicobacter pylori infection (which was treated successfully) and unspecific abdominal upper pain lasting for 2 years. Upon examination, the patient was afebrile, and the neurological exam disclosed only bilateral papilledema and meningeal syndrome. Routine laboratory analysis and a computed tomography scan of the brain were normal, so a lumbar puncture was performed. The opening pressure was 390 mm H 2 O with a white blood cell count of 195 cells/ml (98% mononuclear cells), protein level of 93 mg/dl, and glucose level of 55 mg/dl. Samples were sent for Gram and Ziehl-Neelsen staining; viral, bacterial, mycobacterial, and fungal cultures; and PCR testing for herpes simplex virus and enterovirus. All results, as well as those of serologies, were negative for human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus, cysticercosis, toxoplasmosis, Borrelia burgdorferi, Brucella abortus, and Mycoplasma pneumoniae. After 24 hours, the patient remained asymptomatic without specific treatment (just analgesics and antiemetics). Cranial MRI was normal, and rheumatologic testing showed no abnormalities. He was diagnosed with subacute lymphocytic meningitis of unknown origin. Two weeks after being discharged, the headaches and vomiting recurred, so another lumbar puncture was performed, revealing 125 white blood cells/ml (98% mononuclear cells), protein level of 62 mg/dl, and glucose level of 59 mg/dl. The new assessment of cerebrospinal fluid (CSF) samples included cytology, adenosine deaminase activity, cryptococcus antigen, and VDRL and serology tests for human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus, cysticercosis, and toxoplasmosis. These tests were negative once again.Even though the suspicion for Whipple's disease was very low, PCR for Tropheryma whipplei was performed with the two CSF samples. The amplification by PCR was performed in a LightCycler thermocycler (Roche), using the primers Whip1 (5Ј-AGAGATACGCCCCCCGCAA-3Ј) and Whip2 (5Ј-ATT CGCTCCACCTTGCGA-3Ј), which targeted a 266-bp sequence of the 16S rRNA gene (11). The PCR was performed using the following protocol: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 62°C for 7 s, extension at 72°C for 10 s, and a melting curve. The PCR of the CSF samples yielded an apparently positive result, based on the amplification and melting curve and the agarose gel electrophoresis of the PCR product, in the two CSF samples. In order to confirm the Whipple's disea...

Clinical and Neuropathological Variability in Clinically Isolated Central Nervous System Whipple's Disease

“…In contrast, Case 3 who had a positive PCR in CSF in the context of highly suggestive clinical symptoms, and without clinically apparent neurological disease at the time of death had two negative PCR and immunohistochemistry on post-mortem brain tissue, suggesting a good response to an adequate treatment. Although we cannot rule out a false positive result in the latter patient, as it has been previously reported in subjects with other CNS diseases or in healthy individuals (1% to −30% depending on the sample and PCR technique used) (1,8,10,18), the clinical improvement associated with long-term appropriate antibiotic therapy makes this possibility unlikely. The most probable explanation for the posttreatment in vivo and the two post-mortem negative PCR results is to consider that the patient was cured in life by antibiotics without disease reactivation prior to death.…”

Progressive dementia associated with ataxia or obesity in patients with Tropheryma whipplei encephalitis

“…PCR testing of cerebrospinal fluid specimens for T. whipplei in neurological patients is clinically reasonable, as the disease is fatal without specific treatment. As is the case for all diagnoses performed using PCR, caution is necessary, and a rigorous strategy should be applied when performing and interpreting the analyses due to the risk of false-positive results [ 58 ]. A carefully checked positive PCR in cerebrospinal fluid specimens is sufficient for diagnosis.…”

Clinical Manifestations, Treatment, and Diagnosis of Tropheryma whipplei Infections

“…They concluded that qPCR on stool and saliva samples is a good initial screening method (40). However, it is critical to validate the specificity of the PCR, as false-positive PCR results on saliva has been reported, especially when using 16S rRNA gene-based primers as the T. whipplei 16S rRNA genes are very similar to those of other bacteria that can be present in the oral cavity (183,187). Wholegenome sequencing of T. whipplei identified more-specific PCR targets like the repetitive sequences, which when used in addition to other targets helped to rule out false-positive results (186,188).…”

“…It is advisable that in atypical cases of T. whipplei, two different specific target genes are tested and that the results for both target genes should be positive in order to rule out potential false-positive results (51,183). The occurrence of false-positive PCR results has been reported, e.g., in cerebrospinal fluid samples and duodenal biopsy specimens (187). In order to minimize the chance of false-positive results caused by contamination or due to a nonspecific PCR, qPCR should preferably be performed on more than one sample and whenever possible also include invasive samples, for instance, blood or biopsy specimen (40).…”