Poor-fit indexes of Kupperman index: a psychometric analysis based on item response theory.

OBJECTIVE: This study aims to test the construct validity of the 11 items of the Kupperman index (KI), which has been pioneering in its attempt to quantify climacteric symptoms. METHODS: Unidimensional confirmatory factor analysis of the 11 graded items (hot flashes, paresthesia, insomnia, nervousness, melancholia, vertigo, weakness, arthralgia or myalgia, headache, palpitations, and formication), using a four-point scale (0, none; 4, severe), was used to evaluate the KI in a sample consisting of 84 women with a mean (SD) age of 54.34 (4.00) years who have been in menopause for a mean (SD) of 4.36 (2.53) years. RESULTS: The KI returned poor results on unidimensional model testing (root-mean-square error adjustment, 0.109; 90% CI, 0.075-0.142; comparative fit index, 0.871; Tucker Lewis index, 0.838; weighted root-mean-square residual, 0.971; χ(2)(44) = 87.599; P < 0.001), indicating that the set of items does not properly evaluate the underlying phenomena (climacteric symptoms). CONCLUSIONS: Our study verifies the poor fit of the KI and provides psychometric evidence that KI items warrant revision and/or that the concept underlying climacteric symptoms should be revisited.

Role of IFN-gamma +874 T/A single nucleotide polymorphism in the tuberculosis outcome among Brazilians subjects.

Several genetic cytokine gene variants have been associated with host susceptibility to infectious diseases, including tuberculosis. Based upon the importance of IFN-gamma in protective immunity against Mycobacterium tuberculosis, and the functional role of the IFN-gamma + 874T/A single nucleotide polymorphism in IFN-gamma production, we genotyped 93 Brazilian tuberculosis patients and 266 asymptomatic health care workers, including 150 individuals with a positive tuberculin skin test, and analyzed the possible association of the +874A low IFN-gamma producer allele with tuberculosis occurrence. Using multivariable logistic regression models, genotype and allele frequencies of the mutant + 874A (low IFN-gamma producer) allele were significantly associated with tuberculosis disease. Heterozygous carriers had a 25% increased chance, while individuals presenting the A/A homozygous genotype had an over two-fold risk of having active tuberculosis (95% CI, 1.16-5.91, P = 0.03). Despite the mixed ethnicity observed in Brazilian populations, the present data agree with observations reported in other populations and thus demonstrate that the functional +874T/A IFN-gamma gene polymorphism is associated with tuberculosis in different populations.

Bronchoalveolar lavage for diagnosis of fungal disease. Five years' experience in a southern United States rural area with many blastomycosis cases.

OBJECTIVE: To determine the relative frequency of Blastomyces dermatitidis among other microorganisms in bronchoalveolar lavage (BAL) specimens. STUDY DESIGN: The study group consisted of 208 BAL specimens received from 192 patients from March 1988 to August 1993. RESULTS: Forty-seven specimens from 42 patients were positive for pathogenic microorganisms, and 2 other specimens were diagnostic of malignancy. Pneumocystis carinii (23 specimens) was the most common microorganism found in the specimens. Candida spp (10 specimens) was the second most common microorganism, and B dermatitidis (5 specimens) was the third. Cryptococcus neoformans (3 patients), Histoplasma capsulatum (2 patients) and Conidiobolus coronatus (1 patient) were the other fungi detected in BAL. Acid-fast bacilli, cytomegalovirus and herpes simplex virus were also found (1 patient each). Several patients had more than one organism. CONCLUSION: B dermatitidis was the third most common microorganism found in BAL specimens at our hospital.

Diagnosing dengue virus infection in archived autopsy tissues by means of the in situ PCR method: a case report.

BACKGROUND: The pathogenesis of the severe form of dengue virus infection, dengue hemorrhagic fever, is still obscure. A major research objective has been to determine which body organs are being damaged by dengue virus in this form of dengue. Research has been difficult because dengue hemorrhagic fever is sporadic and tends to occur in parts of the world where modern facilities are scarce and fresh or frozen patient materials are not available. However, major hospitals in these areas have accumulated libraries of paraffin-embedded surgical and autopsy tissues over the years. These tissues may have been subjected to less than optimal fixation and storage. Attempts to localize dengue virus using antigen detection in the stored tissue have encountered many difficulties. OBJECTIVE: Since viral nucleic acid may be preserved under circumstances which destroy protein antigens, our objective was to detect dengue viral RNA in situ in histologic sections of tissues from patients dying of dengue hemorrhagic fever in Thailand. STUDY DESIGN: Tissues from an 11-year-old boy who died at Ramathibodi Hospital, Bangkok, Thailand in November, 1987 with the clinical diagnosis of dengue hemorrhagic fever were treated by transcribing the dengue viral RNA to DNA followed by amplification using the polymerase chain reaction with subsequent in situ hybridization in order to visualize the cells infected with dengue virus. RESULTS: Viral RNA was detected in hepatocytes in the mid-zonal region of the liver, as well as scattered macrophages in skin and lymph nodes. CONCLUSION: Dengue virus infection can be detected in paraffin-embedded autopsy tissues which have been stored for five years. The same procedure can be used for diagnosing dengue viral infection and for studying the pathogenesis of dengue hemorrhagic fever.

Identification of dengue virus-infected cells in paraffin-embedded tissue using in situ polymerase chain reaction and DNA hybridization.

Using tissue from suckling mice infected with Dengue virus, the following improved method for detecting Dengue viral RNA in tissue sections was devised. Reverse transcription of the viral RNA to DNA was followed by the in situ polymerase chain reaction to amplify the viral nucleic acid. This was followed by DNA hybridization with an alkaline phosphatase-labelled probe. The enzyme was then reacted with Fast red and counterstained with hematoxylin. Viral nucleic acid was readily demonstrated within glial cells and macrophages in brains of animals infected with two different serotypes of Dengue.

Combined treatment with 2'-nor-cGMP and ganciclovir against cytomegalovirus infection in a guinea pig model.

The combination 2'-nor-cGMP/DHPG at fixed ratios 1:5, 1:10 and 1:20 showed synergistic antiviral effects against GPCMV replication in vitro with CI value < 1. In vivo, a fixed ratio of 1:10 at three different dosage levels of 1.25/12.5 mg, 2.5/25 mg and 5/50 mg/kg/day 2'-nor-cGMP/DHPG combination showed only additive results when compared with each drug alone. However, synergistic antiviral effects were obtained when infected guinea pigs were treated with 2'-nor-cGMP/DHPG combination 2.5/10 mg/kg/day (1:4). A significantly lower GPCMV infectivity titer was noted in the salivary gland, lung and spleen of infected guinea pigs treated with the combination of 2'-nor-cGMP/DHPG 2.5/10 mg/kg/day, as compared to animals treated with a corresponding dose of each drug alone. In addition, GPCMV-infected animals treated with the latter combination showed increased body weight than when either drug was used alone. Histopathologically, each drug alone reduced the viral induced changes in the lung and spleen, but the combination therapy reduced these changes still further. Toxic changes seen in the kidney and bone marrow of infected animals treated with 2'-nor-cGMP, 2.5 mg/kg/day were not significantly increased when DHPG 10 mg/kg/day was added to the regimen. Therefore, combined treatment with 2'-nor-cGMP/DHPG in appropriate concentration is more helpful for acute cytomegalovirus infection in guinea pigs than when either drug was used alone.

Phaeohyphomycosis caused by Bipolaris spicifera: an informative case.

A case of phaeohyphomycosis caused by Bipolaris spicifera involving the brain and sinuses is presented. The patient survived following surgery and ketoconazole therapy, which successfully treated both the sinus and the brain infections.

Human immunodeficiency virus infection elicits early antibody not detected by standard tests: implications for diagnostics and viral immunology.

The FDA-approved tests for diagnosis of HIV exposure depend on detection of specific antibody in serum. HIV infection is missed in some individuals because they score seronegative by the standard clinical EIA and Western blot assays. This apparent immunological "silent" period following infection may last for months and has been reported to be as long as 3 years in rare cases. Is there truly a lack of an immune response or is there a more subtle, narrowly focused antibody response in these HIV-infected individuals which is not detected by the current tests? Using a nondenaturing serological assay (immunofluorescence of live infected T-cells), we found that each of four infected individuals "seronegative" by the standard tests did possess antibody against native HIV proteins expressed on infected cells. These antibodies reacting with native HIV antigenic epitopes were of the IgG isotype, they cross-reacted with many, but not all, of seven random HIV-1 isolates, and one of the sera immunoprecipitated HIV gp160 from NP-40-solubilized infected cells. These results show that seronegative, high-risk, infected individuals can actually be seropositive and that different types of assays using native antigenic epitopes may be required for screening. Implementation of these findings thus may decrease HIV transmission. These results also highlight the importance of protein conformation for many natural viral antigenic epitopes.

Performance characteristics of a rapid western blot assay system for antibody to human immunodeficiency virus type 1.

One hundred and twenty reactive sera were selected from specimens studied by enzyme immunoassay (EIA, Abbott Laboratories, Abbott Park, North Chicago, IL) for antibodies against human immunodeficiency virus (HIV-1). Using these sera, the 'WesPage' system (American Bionetics, Inc., Haywood, CA), was compared to the Western blot evaluation performed by a commercial reference laboratory (Abbott Laboratories, Abbott Park, North Chicago, IL). Using criteria established by the Food and Drug Administration, all major bands representing specific antigens of HIV-1 and their corresponding antibodies were identified on the immunoblot membrane when the strongly reactive control serum was used in the assay. The weakly reactive control serum demonstrated antibodies to the p24 core antigen and the gp120/160 envelope antigen of the virus in addition to others. The non-reactive control serum did not react with any antigen on the immunoblot sheet. All results obtained by our evaluation agreed with the reference laboratory results. The WesPage assay offers a combination of advantages which include rapid turn around time, less direct contact with potentially infectious materials, good resolution of bands and high reproducibility of results.

The effect of an arenavirus infection on liver morphology and function.

Patients with severe Lassa fever have high serum levels of liver enzymes. Studies of the histology of the liver have shown only minor alterations, seemingly insufficient to account for death. Pichinde virus is an arenavirus which causes severe illness similar to Lassa fever in strain 13 guinea pigs, but does not cause severe illness in man. This can serve as a relatively safe model for studying the pathology and pathophysiology of fatal arenaviral infection. We used this infection to evaluate the effect of arenavirus on liver morphology and function. When guinea pigs were infected with Pichinde virus, all developed severe disease and died within 14 days of infection. The animals lost large amounts of weight. Higher levels of virus were detected in the liver than in serum. Aspartate aminotransferase and alanine aminotransferase were elevated late in the course of the disease; no elevations were seen in gamma glutamyl transpeptidase or bilirubin. Alkaline phosphatase, initially high in these growing animals, was markedly decreased early in infection. Prothrombin time and activated partial thromboplastin time were increased late in the disease, and decreased levels of Factors VIII and IX were seen relatively early. Fatty metamorphosis, indicating problems in lipid processing, occurred by day 11, but necrosis was minor and occurred late. Pichinde virus infection results in significant alterations in the metabolic and synthetic capacities of the hepatocytes early in infection in the absence of significant necrosis.

Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) against guinea pig cytomegalovirus infection in cultured cells and in guinea pigs.

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC, and two HPMPC-related nucleoside analogs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, HPMPA, and (2-phosphonylmethoxyethyl)guanine, PMEG, were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in guinea pig embryo (GPE) cells and human cytomegalovirus (HCMV) infection in human diploid fibroblast (MRC-5) cells. DHPG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, was used for comparison. The antiviral activity of HPMPC against GPCMV infection in vivo and its toxicity to Hartley guinea pigs were also evaluated. The 50% antiviral effective doses (ED50) of HPMPC, HPMPA, PMEG and DHPG against GPCMV infection in GPE cells were 0.22, 1.4, 0.07 and 62 microM, respectively; and against HCMV infection in MRC-5 cells, the ED50s were 0.51, 0.72, 0.01 and 17.5 microM, respectively. Their cytotoxic doses (CyD50) in GPE replicating cells were 84, 35, 1.4 and 700 microM, respectively and in MRC-5 cells were approximately 114, 31, 0.86 and 750 microM, respectively. Based on their calculated therapeutic indexes, HPMPC was the most potent and selective of the four compounds tested. In vivo, during acute infection, the spleen indexes of all infected animals that were treated with 1.25 to 5.0 mg/kg/day of HPMPC for 5 days were significantly reduced as compared with sham-treated animals. Virus infectivity titers in blood and various tissues of infected animals treated with HPMPC, 2.5 or 1.25 mg/kg/day were not significantly lower than those of the infected, sham-treated animals; with 5 mg/kg/day, infectivity titers in the blood, spleen, and salivary gland were significantly lower in HPMPC-treated than in sham-treated animals. However, HPMPC was toxic to guinea pigs especially at doses of 5 to 10 mg/kg/day. These data showed that HPMPC was highly active and selective in cultured guinea pig cells and human fibroblast cells against CMV infection but did not effectively inhibit GPCMV infection in guinea pigs at minimum toxic concentrations.

Arenavirus infection in the guinea pig model: antiviral therapy with recombinant interferon-alpha, the immunomodulator CL246,738 and ribavirin.

Human arenaviral infections have a high mortality, and are dangerous to work with in the laboratory. There is a need for good antiviral agents to treat these infections. Pichinde virus infection of the inbred strain 13 guinea pig is a relatively safe, good animal model for human arenavirus infections. Mortality is consistently 100% between days 12 and 25 (mean 14.8) days after infection. When infected animals were treated with recombinant human interferon alpha A, or with CL246,783, an immunomodulator known to induce interferon, no beneficial effect was noted. When animals received ribavirin, 25 mg/kg/day for the first 14 days of infection, the course of infection was prolonged, with death occurring a mean of 22.5 days after infection. If ribavirin was administered for 28 days, mortality was reduced to 25%, with those animals dying a mean of 21.0 days after infection. These results confirm the studies that indicate that ribavirin is a useful agent for treating arenaviral infections. However, treatment with this agent must be prolonged. They also demonstrate the potential usefulness of Pichinde virus infection in strain 13 guinea pigs as an animal model of human disease.

Effect of 2'-nor-cyclic GMP against guinea pig cytomegalovirus infection.

Cyclic phosphate derivative of DHPG, 2'-nor-cGMP [9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]-guani ne phosphate-oxide] was evaluated for activity against guinea pig cytomegalovirus (GPCMV) infection in cultured guinea pig embryo cells and in guinea pigs. By virus yield reduction and plaque reduction assays, 2'-nor-cGMP was demonstrated to be 15- to 20-fold more potent against GPCMV infection than its parental drug DHPG. The selectivity index of 2-nor-cGMP was 110, which was 10-fold higher than that of DHPG. In cultured cells, 2'-nor-cGMP attained maximal antiviral activity when added to the cells within 12 h postinfection. In the studies on GPCMV infection in guinea pigs, 2'-nor-cGMP administered subcutaneously once daily (5 mg/kg per day) for 8 days, starting 24 after virus inoculation, significantly suppressed GPCMV infectivity titers in the blood, spleen, lung, and salivary gland during acute infection (10 days postinfection) as compared with sham-treated infected animals. A greater reduction of GPCMV infectivity titers in the salivary gland was noted during chronic infection (i.e., 24 days postinfection). Clinically, splenomegaly and peripheral lymphocytosis were significantly modified as compared with the sham-treated animals (P less than 0.05). The drug, administered at this dosage, was reasonably tolerated by the guinea pigs and showed clinical benefit.

Role of the virology laboratory in diagnosis and management of patients with central nervous system disease.

A number of viruses cause acute central nervous system disease. The two major clinical presentations are aseptic meningitis and the less common meningoencephalitis. Clinical virology laboratories are now more widely available than a decade ago; they can be operated on a modest scale and can be tailored to the needs of the patients they serve. Most laboratories can provide diagnostic information on diseases caused by enteroviruses, herpesviruses, and human immunodeficiency virus. Antiviral therapy for herpes simplex virus is now available. By providing a rapid diagnostic test or isolation of the virus or both, the virology laboratory plays a direct role in guiding antiviral therapy for patients with herpes simplex encephalitis. Although there is no specific drug available for enteroviruses, attention needs to be paid to these viruses since they are the most common cause of nonbacterial meningitis and the most common pathogens causing hospitalization for suspected sepsis in young infants in the United States during the warm months of the year. When the virology laboratory maximizes the speed of viral detection or isolation, it can make a significant impact on management of these patients. Early viral diagnosis benefits patients with enteroviral meningitis, most of whom are hospitalized and treated for bacterial sepsis or meningitis or both; these patients have the advantage of early withdrawal of antibiotics and intravenous therapy, early hospital discharge, and avoidance of the risks and costs of unnecessary tests and treatment. Enteroviral infection in young infants also is a risk factor for possible long-term sequelae. For compromised patients, the diagnostic information helps in selecting specific immunoglobulin therapy. Good communication between the physician and the laboratory will result in the most benefit to patients with central nervous system viral infection.

Group A streptococcal rapid test. Antigen detection after 18-24 hours of penicillin therapy.

We studied 29 children, aged 19 months to 16 years, prior to and after 18-24 hours of oral penicillin therapy to confirm the rapid disappearance of detectable pharyngeal antigen and to determine whether the antigen detectable by commercially available kits was excreted into the urine. Patients were recruited based on the presence of pharyngitis, no antibiotic therapy in the preceding 2 weeks, and a positive latex agglutination (LA) for group A beta hemolytic streptococci (GABHS) antigen on pharyngeal swab. Diagnosis was confirmed by positive GABHS culture on blood agar plates. Twenty-five of these children were also tested for GABHS antigen by enzyme-linked immunoassay (EIA). After 18-24 hours of oral antibiotic therapy, only 10 patients had a positive test for GABHS on throat swab. Five of 29 subjects (17%) remained positive by blood agar plate (BAP) culture, eight of 29 (29%) by LA, and four of 23 (17%) by EIA. GABHS antigen was undetectable by LA or EIA in the urines of any of these patients, either prior to or after initiation of treatment, even in specimens concentration as high as 100 fold. Clinicians should routinely seek a history of prior antibiotic therapy in assessing pharyngitis. Neither of the kits tested are reasonably accurate for GABHS disease by detection of antigen in the pharynx after partial treatment or in the urine at any time.

Comparison of cell cultures for rapid isolation of enteroviruses.

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.

Detection of human immunodeficiency virus antigen in vitreous humor.

The vitreous humor from 11 patients with acquired immunodeficiency syndrome was obtained at postmortem examination and tested for human immunodeficiency virus antigen and antibody by using the Abbott enzyme-linked immunosorbent assay procedures. Five patients had detectable antigen, supporting the recent observation that the virus may directly infect the retina.

Efficacy of S26308 against guinea pig cytomegalovirus infection.

Prophylactic use of antiviral agents against cytomegalovirus (CMV) is particularly indicated for the immunocompromised host because morbidity and mortality due to CMV occur most frequently following immunosuppression. We have evaluated the new Riker compound S26308 for its therapeutic and prophylactic antiviral activity against CMV in guinea pigs. The efficacy of the compound was assessed in vitro in guinea pig embryo cells and in vivo in both immunocompetent and immunocompromised guinea pigs. Guinea pig CMV plaque formation was reduced only in cells treated with S26308 prior to virus infection. The antiviral activity remained even when the compound was removed after virus absorption and was due to neither virus destruction nor inhibition of cell growth. The frequency of viremia was reduced in guinea pigs for which S26308 therapy was initiated 24 h prior to virus inoculation compared with sham-treated animals. This reduction in the frequency of viremia did not prevent virus spread to target tissues but did result in a reduction of the severity of CMV-induced disease in immunocompromised guinea pigs. Low levels of interferon were detected in supernatants of S26308-treated cells, and interferon was detected in the serum of guinea pigs given S26308. These results indicate that S26308 can induce interferon and reduce CMV infectivity in vivo and in vitro when used prophylactically. This antiviral activity, although modest, was accompanied by beneficial effects on CMV-induced morbidity and mortality. Prophylactic use of S26308 in combination with other therapeutic agents may be a useful strategy against CMV infections.

Acridine orange as a screen for organisms in clinical specimens and comparison with gram's stain.

We compared the result of acridine orange and Gram's stains with the results of culture for 202 wound swabs and 188 fluid specimens. Cerebrospinal fluid was excluded from the study. Acridine orange was more sensitive and less specific than Gram's stain compared with findings that have been previously reported. A difference in the sensitivity was observed between the two stains and between the types of specimens examined themselves. The sensitivity of acridine orange and Gram's stains was 83% and 49% for swabs and 66% and 45% for fluids, respectively. The negative predictive values for acridine orange and Gram's stains were 60% and 40% for swabs and 84% and 81% for fluids, respectively. Overall, the sensitivity for acridine orange and Gram's stains was 75% and 64% with negative predictive values of 75% and 63%, respectively; specificity was 75% (acridine orange) and 97% (Gram's stain) and did not differ significantly between the two specimen types. Acridine orange was cleaner, faster, easier to perform and read, and less costly than Gram's stain for screening purposes. Slides that were positive by acridine orange staining should be stained with Gram's stain for specificity and for the Gram's-stain reaction report. Acridine orange is recommended for screening smears, with positive results confirmed by Gram's stain.