Value of polymerase chain reaction (PCR) for diagnosis of tuberculoid meningitis

The prognosis of tuberculous meningitis (TBM) depends on early therapy based on rapid diagnosis. To study the clinical value of PCR in diagnosis of TBM, we investigated CSF specimens from 49 patients. After cell lysis and DNA preparation following a standard protocol, we performed a half-nested PCR with primers able to detect mycobacterial DNA. PCR results were evaluated according to clinical features, histopathological data, and bacteriological results. PCR detected four of five cases of confirmed TBM, corresponding to a sensitivity of 80%. Positive PCRs were also obtained in 25% CSF samples of non-TBM patients. Most of these false positive results were due to amplification of Mycobacteria fortuitum (M. fortuitum) as determined by direct sequencing analysis. To enhance specificity of our half nested protocol, the oligonucleotide primers that were specific for several mycobacterial subspecies were substituted by a primerpair, which allows selective amplification of DNA from Mycobacteria tuberculosis (M. tuberculosis). By using the altered PCR protocol, the screening of CSF samples revealed a much higher specificity (97%) and constant sensitivity (80%) in diagnosis of TBM. These findings indicate, that M. fortuitum, as an ubiquitous mycobacterial subtype of low pathogenicity, can potentially contaminate clinical specimens and account for false positive PCR results. Therefore, the clinical value of PCR in diagnosis of TBM strongly depends on appropriate oligonucleotide primers, that allow to differentiate between mycobacterial subtypes.     

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis. The European Union Concerted Action on Viral Meningitis and Encephalitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the "gold standard" were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Tuberculosis meningitis in Hong Kong: experience in a regional hospital

Tuberculous meningitis (TBM) remains common in Hong Kong. From January 1996 to June 1997, 11 adult patients with TBM presented to Queen Mary Hospital, a regional hospital in Hong Kong. The annual incidence of TBM was estimated at 1.8 per 100,000 population. Nine patients were local Chinese, and only one patient had the acquired immune-deficiency syndrome (AIDS). In contrast to the classical presentation as a chronic indolent disease, our patients presented acutely: the mean duration from onset of symptoms to presentation was 4.8 days (range 0-10). The most common presenting symptoms were headache (64%), fever (46%), or both (36%), with focal deficits occurring in 64% of patients. Cerebrospinal fluid (CSF) culture and polymerase chain reaction (PCR) were positive in 30% and 29% of cases. Mean CSF cell count, protein and glucose levels were 340 x 10(6)/L, 267 mg/dL, and 2.3 mmol/L, respectively. Extra-neural tuberculosis occurred in 46% of cases. All patients survived and responded to treatment. Drug-induced hepatotoxicity was common; 64% of patients developed biochemical hepatitis.     

Laryngotracheal stenosis in children after intubation. Report of five cases

In this study we present a modified diagnostic routine PCR (polymerase chain reaction) technique for the detection of specific enteroviral nucleic acid sequences in human body fluids, the use of which would reduce the number of false-positive results, the costs and the concentration of reagents required in PCR. Cerebrospinal samples, pharyngeal swabs and faeces were tested. To this end, general primers, selected in the highly conserved 5' non-coding region were used, with a closed-tube RT semi-nested PCR protocol, and the PCR product was analysed using a hybridization in solid phase. A total of 32 patients with suspected enteroviral infection, 17 with suspected viral meningitis and 15 with a possible acute enteroviral infection different from meningitis were analysed, and 80% of the patients with possible acute enteroviral infection were PCR-positive. A broad range of enteroviruses can be detected and false-positive results can be reduced. The availability of results in less than 12 h and the lack of need for radiolabelled probes also increase the convenience of this protocol.     

Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.     

Immunomodulatory activity of thunberginol A and related compounds isolated from Hydrangeae Dulcis Folium on splenocyte proliferation activated by mitogens

We have prospectively studied 27 adult patients attending the Department of Infectious Diseases, G?teborg, Sweden, between October 1992 and October 1996 with a diagnosis of acute viral encephalitis. In addition to cerebrospinal fluid (CSF) virus isolations and antibody analyses against herpes simplex virus, cytomegalovirus, varicella zoster virus, Epstein-Barr virus (EBV), enterovirus, adenovirus, tick-borne encephalitis virus, and mycoplasma, polymerase chain reaction test (PCR) to 5 viruses from the family of human herpes viridae, and to adenovirus as well as to enterovirus were analysed in CSF. 10 patients had herpes simplex virus type-1 (HSV-1), 1 had varicella zoster virus, 1 had tick-borne encephalitis, and 2 had Influenza A infections. In 13 patients the aetiology remained unclear. Eight patients with HSV-1 encephalitis and clinical symptoms for 2-11 d before admission were PCR-positive, while 2 patients with a < or = 2 d history of disease were negative for HSV-1 DNA on admission. These 2 patients became positive for HSV-1 DNA in CSF samples taken 4 d later in 1 case and 7 d later in the other. In 4 patients with HSV-1 encephalitis, in 1 patient with Influenza A complicated by encephalitis, and in 1 patient with encephalitis of unknown origin EBV DNA was found in CSF samples during the study. The clinical significance of these findings is unclear. The study shows that HSV-1 was the most common etiological agent in patients with viral encephalitis in the G?teborg area. In spite of improved diagnostic procedures, a large proportion of patients with symptoms and laboratory findings compatible with viral encephalitis still have an unclear aetiology.     

EMLA cream provides rapid pain relief for the curettage of molluscum contagiosum in children with atopic dermatitis without causing serious application-site reactions

BACKGROUND: Determination of the etiology of pneumonia in young children is difficult because blood culture, the usual method of diagnosis, is positive in only a small proportion of cases. For this reason vaccine trials that include bacterial pneumonia as an endpoint must be large. OBJECTIVES: To determine whether a diagnostic test based on a polymerase chain reaction could be used as an alternative to conventional blood culture for diagnosis of invasive Haemophilus influenzae type b (Hib) infections in young children investigated during the course of a large vaccine trial. METHODS: DNA was extracted from blood culture supernatants and probed for the presence of Hib DNA with a PCR assay with primers derived from the cap gene locus of Hib. Results of the PCR assay were compared with those obtained by conventional culture techniques. RESULTS: Blood cultures were obtained from 1544 children with suspected pneumonia, meningitis or septicemia and from 31 healthy control children who were contacts of cases. Blood culture supernatants were tested for Hib DNA in the PCR test. The sensitivity and specificity of a positive PCR test in blood culture supernatant as against culture of Hib from any normally sterile site were 100 and 99%, respectively. Eleven children had positive Hib PCR tests on blood culture supernatants but were negative by culture. In one of these cases Hib was isolated from a lung aspirate and in two other patients H. influenzae strains other than Hib were obtained from the cerebrospinal fluid. Eight of these 11 children were in the control group. When the results of the PCR assay were used to determine vaccine efficacy, a value of 86% was obtained compared with a figure of 95% obtained when conventional culture techniques were used. CONCLUSIONS: An Hib PCR assay on blood culture supernatants proved to be sensitive and specific for the diagnosis of Hib disease in children. The distribution of PCR-positive, culture-negative cases between Hib-vaccinated and control groups paralleled that of culture-positive cases, suggesting that most of these children had been infected with Hib. A trial of a highly efficacious vaccine provides a novel way for evaluating new diagnostic tests for which there is no standard diagnostic test of 100% reliability.     

Value of the polymerase chain reaction for the detection of Toxoplasma gondii in cerebrospinal fluid from patients with AIDS

To investigate whether the polymerase chain reaction (PCR) on the BI gene of Toxoplasma gondii could contribute to the diagnosis of cerebral toxoplasmosis in patients with AIDS, we retrospectively tested CSF samples from 20 patients with AIDS suspected of having cerebral toxoplasmosis for the presence of T. gondii. Suspicion of cerebral toxoplasmosis was based on accepted criteria. Nine patients with AIDS with IgG antibodies to T. gondii but who were not suspected of having cerebral toxoplasmosis and four patients with AIDS seronegative for T. gondii served as negative control patients. T. gondii was demonstrated by PCR in the CSF from 13 of the 20 patients with AIDS suspected of having cerebral toxoplasmosis but was not demonstrated in the CSF samples from the nine control patients seropositive for T. gondii and the four control patients seronegative for T. gondii. The data were statistically evaluated. This study shows the value of PCR for the detection of T. gondii in CSF for the diagnosis of cerebral toxoplasmosis in patients with AIDS.     

Rapid and sensitive detection of enteroviruses in specimens from patients with aseptic meningitis

A 5-h PCR assay (Amplicor enterovirus test) was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid. Of the cerebrospinal fluid specimens collected during a summer outbreak of aseptic meningitis, 34% were positive by viral culture whereas 66% were positive by the Amplicor PCR, suggesting that this technique improves the diagnosis of enteroviral meningitis.     

Hong Kong reverts to Chinese rule but won''t introduce China''s strict birth-control policies

A previously healthy, 37-year-old immunocompetent man presented with disseminated cutaneous zoster and aseptic meningitis. Varicella zoster virus DNA was recovered from the cerebrospinal fluid (CSF) by the polymerase chain reaction. Cytological evaluation of the CSF revealed 'reactive, highly atypical lymphocytosis'. The patient fully recovered after treatment with aciclovir.     

Distribution of desmin and fibronectin in chick embryo gonad during testicular cord formation

In order to compare PCR with rapid virus culture for the early detection of CMV in bronchoalveolar lavage (BAL) after bone marrow transplantation, 26 asymptomatic patients were routinely evaluated for the presence of CMV on day 35 using these two techniques. Concurrent blood samples were also analyzed in all cases. CMV was detected synchronously by both culture and PCR in six of 26 (23%) BAL and in five of 26 (19%) blood specimens. Among these positive specimens, three BAL and blood samples were positive in the same patients. Five (19%) BAL and five (19%) blood samples were culture-negative but PCR-positive. No BAL or blood specimens were positive by culture alone. When considering matched BAL-blood samples, five were positive in only one fluid, BAL (n = 3) or blood (n = 2) using culture, while seven were positive in only one fluid, BAL (n = 4) or blood (in = 3) using PCR. Overall, six of 26 (23%) patients had culture-negative but PCR-positive results. Three of these six patients were positive only in BAL and two of them subsequently received antiviral therapy for development of symptoms suggestive of CMV infection. We suggest that asymptomatic patients with negative-culture but PCR-positive results on day 35 in BAL should be subsequently closely monitored for the presence of CMV.     

Effects of dopaminergic drugs on food- and cocaine-maintained responding. IV: Continuous cocaine infusions

We modified and optimized a new microplate hybridization assay to detect the varciella-zoster virus (VZV) PCR product, and studied cerebrospinal fluid (CSF) samples of 287 patients with meningitis, encephalitis or other neurological diseases or symptoms. Specific antibodies to VZV and reference antigens were determined by enzyme immunoassay from serum and CSF, they were then compared with clinical findings and with the results obtained by VZV-PCR using different detection methods for VZV-specific amplified DNA. VZV DNA was found in the CSF of 25 patients using the microplate hybridization assay and chemiluminescence detection for amplified DNA. All 25 CSF samples were also positive in Southern blotting. Among the patients, 10 had chickenpox, 4 had shingles, and 11 had no rash at all. The detection rate of VZV-specific DNA by microplate hybridization was 30% higher than that obtained by conventional agarose gel electrophoresis. In most patients the diagnosis was confirmed by demonstrating specific intrathecal antibody production to VZV but not to other viruses. These results indicate the presence of VZV in the central nervous system (CNS) in many patients with chickenpox or shingles, and even in patients without a rash. The microplate hybridization assay based on chemiluminescence detection improves considerably the detection rate of the VZV-PCR product compared to agarose gel electrophoresis and will add to the list of recognized VZV infections in the CNS. It is especially useful in cases where there is no cutaneous manifestation.     

Measuring lateral skin temperature profile of premature infants in incubators with thermography

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.     

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.     

Diagnosis of extrapulmonary tuberculosis by a commercial polymerase chain reaction kit

The Roche Amplicor Mycobacterium tuberculosis PCR test was compared with mycobacterial culture for direct detection of M. tuberculosis in extrapulmonary specimens. From January 1995 to October 1996, 124 clinical specimens from 112 patients were assessed, including 47 body fluids, 61 tissue specimens and 16 abscesses. The sensitivity and specificity of Amplicor PCR compared to culture were 63.6% and 93.1% respectively. Analysis of 7 PCR-positive, culture-negative specimens confirmed that all were from patients with recently diagnosed tuberculosis under treatment. Eight specimens were PCR negative-culture positive, including a pleural fluid containing inhibitory substances. On acid-fast bacilli (AFB) smear-negative specimens, sensitivity and specificity were 53 and 100% respectively. The best results for Amplicor PCR were obtained with abscesses and biopsies. It is concluded that this test, highly specific for the diagnosis of tuberculosis, is at least as sensitive on extrapulmonary specimens as on smear-negative respiratory specimens.     

PCR-Enzyme immunoassay for detection of Streptococcus pneumoniae DNA in cerebrospinal fluid samples from patients with culture-negative meningitis

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of Streptococcus pneumoniae and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of S. pneumoniae tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.     

Preoperative diagnosis of Mycobacterium avium lymphadenitis in two immunocompetent children by polymerase chain reaction of gastric aspirates

BACKGROUND: Analysis of gastric aspirates is a routine procedure for detection of Mycobacterium tuberculosis in pediatric pulmonary tuberculosis. However, identification of nontuberculous mycobacteria in gastric aspirates of immunocompetent children is not thought to be clinically significant. METHODS: A PCR method was devised for the detection of M. avium in clinical specimens. The method is based on the amplification of a M. avium-specific DNA fragment present in the 3'-end of the repetitive element IS1245. Surgically removed lymphatic tissue was analyzed prospectively by microscopy, culture and PCR in 13 children admitted to our hospital with suspected mycobacterial lymphadenitis. In 4 of these children 1 to 4 gastric aspirates were obtained before surgical treatment and submitted to the same analysis. RESULTS: We report the detection of M. avium in the gastric aspirates of two children with cervical lymphadenitis before surgical intervention by a novel PCR method. The subsequently surgically removed lymph nodes were also positive by PCR and culture. In one child cultures of both sources grew M. avium. The isolates could be identified as the same strain by DNA fingerprinting. The PCR assay was almost twice as sensitive as culture in detecting M. avium. CONCLUSIONS: Our findings suggest the possibility for noninvasive diagnosis of cervical lymphadenitis caused by nontuberculous mycobacteria before surgery. In addition detection of M. avium in gastric aspirates without evidence of fistula formation provides new insights into the pathogenesis of mycobacterial infection and disease in immunocompetent children.     

Impact of deltamethrin impregnated mosquito nets on the transmission of malaria in the coastal lagoon area, Benin]

Neopterin has been determined in blood as a marker of cellular immune system activation. We studied cerebrospinal fluid (CSF) neopterin levels in children with neurologic diseases, and the following results were obtained: (1) CSF neopterin levels markedly increased at the acute phase of bacterial meningitis, aseptic meningitis, and encephalitis as compared with those in patients without neurologic diseased. (2) In the CSF of patients with bacterial meningitis and aseptic meningitis, neopterin levels decreased more rapidly than the total cell count and 2'5' oligoadenylate synthetase (2-5 AS) did. (3) CSF neopterin in patients with non-infectious neurologic diseases was almost equal to that in patients without neurologic diseases. (4) There was no correlation between CSF neopterin and other CSF values, such as total cell count, mononuclear cell count, protein, and 2-5 AS. These results suggest that CSF neopterin is a useful marker of inflammatory central nervous diseases.