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OBJECTIVE: To compare resource use by diagnostic outcome among hospital admissions during which tuberculosis (TB) was suspected. DESIGN: Retrospective study based on chart review and microbiology laboratory data. SETTING: The department of medicine in a municipal hospital serving central Brooklyn, New York. PARTICIPANTS: We identified all adult admissions in 1993 during which TB was suspected. We assigned each admission to one of four mutually exclusive groups defined by the results of microbiological tests (acid-fast bacilli [AFB] smear and culture): culture-positive and smear-positive (C+S+); culture-positive and smear-negative (C+S-); culture-negative and smear-positive (C-S+); or culture-negative and smear-negative (C-S-). Each admission was divided into two separate periods to which the utilization of medical resources was assigned: the diagnostic and the postdiagnostic periods, which were separated by the date of receipt of the first definitive culture report. RESULTS: Data on 519 admissions (93 C+S+; 57 C+S-; 30 C-S+; and 339 C-S-) were analyzed. Although C+S+ were more likely than other groups to have an admitting diagnosis of TB, approximately one quarter of the admissions without TB (C-S+, C-S-) were admitted with the principal diagnosis of TB. For the four groups, C+S+, C+S-, C-S+, and C-S-, the respective rates of TB isolation and anti-TB treatment, and median lengths of isolation were 98%, 87%, and 34 days; 74%, 74%, and 7 days; 83%, 83%, and 15 days; and 44%, 29%, and 0 days. During the diagnostic period, the rate and length of isolation were similar in the AFB-smear-positive groups (C+S+ and C-S+). We estimated that admissions without culture-proven TB (C-S+ and C-S-) accounted for 3,174 (36%) of the 8,712 days of TB isolation expended and for 65% of the 16,671 days of anti-TB treatment. The vast majority of this resource consumption (2,737 [86%] of 3,174 days of isolation) occurred during the diagnostic period before a definitive culture result was known. CONCLUSIONS: Our results suggest that prolonged diagnostic uncertainty and misclassification of cases due to false-positive and false-negative smears are associated with substantial medical-resource consumption. New diagnostic modalities that reduce the period of diagnostic uncertainty could reduce the utilization of resources later found to be unnecessary.

Objective: To investigate the effect of human immunodeficiency virus (HIV) infection on vaginal yeast colonization and symptomatic vulvovaginitis and to explore the effects of immune compromise on these conditions in HIV-positive women. Methods: Between September 1991 and May 1993, 223 HIV-positive women without AIDS-defining conditions were enrolled for prospective follow-up and compared with 289 HIV-negative women enrolled in a concurrent study. Standardized gynecologic assessment was carried out. Results: Cultures from 81 of 223 (36%) HIV-positive women and 72 of 289 (25%) HIV-negative women were positive for any yeast. The most commonly isolated yeasts were Candida albicans and Torulopsis glabrata ; the proportion of non- C albicans isolates (26%) did not differ by serostatus. The rates of C albicans colonization and vulvovaginitis among immunocompetent (CD4 count at least 500 cells/mm 3 ) HIV-positive women did not differ from those among HIV-negative women. Among HIV-positive women, risks for colonization and for symptomatic vulvovaginitis were increased approximately threefold and fourfold, respectively, in women with CD4 counts below 200 cells/mm 3 compared with either immunocompetent HIVpositive women or HIV-negative women. Conclusion: The yeast species isolated from HIV-positive and HIV-negative women were similar. Rates of vaginal colonization and vaginitis were similar among nonimmunocompromised HIV-positive women and HIV-negative women. Elevated rates of yeast colonization and vaginitis were not seen among this population of HIV-infected women before immune compromise. Both vaginal colonization and symptomatic vaginitis increased with immune compromise among HIV-positive women, especially at CD4 counts below 200 cells/mm 3 .

Culture is currently considered the "gold standard" for detecting Chlamydia trachomatis infections. We evaluated the Syva MicroTrak enzyme immunoassay (EIA) and Gen-Probe PACE 2 tests, which detect chlamydial antigens and rRNA, respectively. These assays were compared with each other and with culture for the detection of C. trachomatis in cervical specimens obtained from 217 women attending a clinic for sexually transmitted diseases. The prevalence of infection was 22.1% by culture. The sensitivity, specificity, and positive and negative predictive values were 79.2, 98.2, 92.6, and 94.3%, respectively, for EIA. For PACE 2, the respective values were 77.1, 97.6, 90.1, and 93.7%. After corrections for two false-negative cultures, the sensitivities and specificities were 80 and 99.4%, respectively, for the EIA and 78 and 98.8%, respectively, for the probe assay. Quantitative evaluation of the results showed that false-negative results with either assay were associated with cultures that had low inclusion counts or were negative without subpassage. Analysis of nonculture results revealed that 2.3% of the EIA results and 4.6% of the probe assay results were within +/- 30% of the respective assay cutoff values. These included four false-negative (one EIA and three probe) and two false-positive (one EIA and one probe) results. The Syva MicroTrak EIA and the Gen-Probe PACE 2 assay are comparable to but significantly less sensitive than culture. Use of a grey zone may help identify the need for repeat or confirmatory testing.

The recovery of cytomegalovirus from bronchoalveolar lavage (BAL) specimens was compared after inoculation of MRC-5 tube and shell vial cell cultures with four different BAL preparations. Analysis of culture results obtained with 55 cytomegalovirus culture-positive samples showed significant differences in the ability to isolate virus from the supernatant and cellular components of these specimens. There was a 52% reduction in cytomegalovirus recovery and a significant delay in the development of cytopathic effect in cultures inoculated with the cellular component of BAL specimens when compared to cultures inoculated with crude BAL cells and fluid. The mean time for detection of cytopathic effect was 11.8 days in tubes inoculated with crude BAL and 18.2 days for tubes inoculated with BAL cells. A similar effect was observed using a rapid shell vial culture technique. A 39% reduction in the number of isolates and a 57% reduction in the number of positive cells were observed in vials inoculated with cells when compared to cultures inoculated with crude BAL. By contrast, using cell-free BAL supernatant as inoculum did not reduce the number of positive cultures or delay development of cytopathic effect. The results suggest that in most BAL specimens, cytomegalovirus is associated with the cell-free, rather than the cellular, component. Although BAL cell concentrates frequently are used for cultivation of viruses from BAL, our results showed that the use of these preparations results in a significant number of false-negative cytomegalovirus cultures.

To identify the effect of human immunodeficiency virus (HIV) infection on the clinical course of pelvic inflammatory disease (PID).Women hospitalized with PID at an urban hospital serving a population at high risk for HIV were studied cross-sectionally. Data abstracted from medical records of 349 women, admitted between July 1992 and April 1994 were linked anonymously to HIV serology. Main outcome measures were length of hospital stay, prolonged fever, tubo-ovarian abscess, surgery, and change in antibiotics.Among the 349 women with PID, 27 were HIV-positive. These HIV-positive women had lower mean white blood cell counts at admission (7411 versus 11,266, P.01), lower mean lymphocyte counts (1411 versus 1928, P.01), greater febrile morbidity (54 versus 28.3%, P.01), and longer hospital stays (10.5 versus 6.4 days, P.01) than HIV-negative women. Women who were HIV-positive required more time for defervescence and needed to change their antibiotic regimen more frequently (41 versus 12.7%, P.01); differences in tubo-ovarian abscesses (19 versus 14%, P = .52) or surgery (15 versus 6.2%, P = .10) were not significant. The differences in hospital course between HIV-positive and HIV-negative women were modest, and they were resolved largely by the fourth or fifth hospital day. All HIV-positive women were treated successfully with first- or second-line antibiotic regimens.Despite more severe initial presentation and a prolonged hospital course, HIV-positive women with PID, but without other acute illnesses, were treated successfully with standard therapeutic regimens. These observations support current recommendations for hospitalization of HIV-positive women with PID and treatment according to current standards.

Cytomegalovirus (CMV) seroprevalence and genital tract shedding in human immunodeficiency virus (HIV)-seronegative and HIV-seropositive women from an urban minority community were investigated. CMV seropositivity was high in both groups : 181(95.2%) of 190 HIV-negative and 158 (90.3%) of 175 HIV-positive subjects. Cervicovaginal shedding was detected in 8 (4.4%) CMV-positive HIV-negative subjects and 31 (19.6%) HIV-positive subjects (odds ratio [OR], 5.28 ; P < .001). Multiple logistic regression analysis revealed that CMV shedding was independently associated with younger age (OR = 0.90 ; P

The prevalence of untreated sexually transmitted diseases (STDs) was assessed in a cohort of 372 sexually active inner-city women (92% black, 49% US-born) with no history of injection drug use who were recruited in Brooklyn, New York, in 1990 and 1991. The presence of STDs was assessed via culture, serologic analyses, and medical history. Sexual and drug-use histories were obtained, as was a urine sample for toxicologic analysis. Thirty-five percent of the women had at least one STD (27% Trichomonas vaginalis, 6.8% Chlamydia trachomatis, 5.2% syphilis, 2.4% human immunodeficiency virus (HIV), and 1.4% Neisseria gonorrhoeae). US-born women were more likely than foreign-born (96% Caribbean) women to have an STD (50% vs. 22%; p < 0.001). Among US-born women, 61% of crack and/or cocaine users had an STD as opposed to 34% of non-users (OR = 2.9, 95% CI 1.6-5.5). Recent crack cocaine use was the strongest predictor of syphilis infection (OR = 12.8, p = 0.019), and was reported by each of the seven HIV-positive women. This study, based on a large sample with laboratory confirmation of both STDs and drug use, documents that women who use crack cocaine are at substantially higher risk of contracting an STD than other women. STD/HIV prevention programs in inner cities should target US-born women, particularly crack cocaine users.

In the United States there have been recent outbreaks of multidrug-resistant tuberculosis. These outbreaks have primarily involved persons infected with the human immunodeficiency virus (HIV).We collected clinical information on 17 patients seen at a New York City hospital who had repeatedly positive cultures for Mycobacterium tuberculosis. Analysis of restriction-fragment--length polymorphisms (RFLPs) was performed on serial isolates of M. tuberculosis obtained from these patients.Six patients had isolates that remained drug-susceptible, and the RFLP patterns of these isolates did not change over time. Eleven patients had isolates that became resistant to antimicrobial agents. The RFLP patterns of the isolates from six of these patients remained essentially unchanged (two strains showed one additional band) despite the development of drug resistance. In five other patients, however, the RFLP patterns of the isolates changed dramatically at the time that drug resistance was detected. The change in the RFLP pattern of the isolate from one patient appeared to be the result of contamination during processing in the laboratory. In the remaining four patients, all of whom had advanced HIV disease, the clinical and microbiologic evidence was consistent with the presence of active tuberculosis caused by a new strain of M. tuberculosis.Resistance to antituberculous drugs can develop not only in the strain that caused the initial disease, but also as a result of reinfection with a new strain of M. tuberculosis that is drug-resistant. Exogenous reinfection with multidrug-resistant M. tuberculosis can occur either during therapy for the original infection or after therapy has been completed.

Context In laboratory trials, nucleic acid amplification tests for the diagnosis of tuberculosis (TB) are more accurate than acid-fast bacilli (AFB) smear microscopy and are faster than culture. The impact of these tests on clinical diagnosis is not known. Objective To assess the performance of a nucleic acid amplification test, the enhanced Mycobacterium tuberculosis Direct (E-MTD) test, against a uniform clinical standard stratified by level of clinical suspicion. Design Prospective multicenter trial conducted between February and December 1996, documenting the clinical suspicion of TB at enrollment and using final comprehensive diagnosis as the criterion standard. Setting Six urban medical centers and 1 public health TB clinic. Patients A total of 338 patients with symptoms and signs consistent with active pulmonary TB and complete clinical diagnosis were stratified by the clinical investigators to be at low ( 75%) relative risk of having TB. Main outcome measures Sensitivity, specificity, and positive and negative predictive values of the E-MTD test in clinical suspicion of groups with low (n = 224); intermediate (n = 68); and high (n = 46) clinical suspicion of TB. Results Based on comprehensive clinical diagnosis, sensitivity of the E-MTD test was 83%, 75%, and 87% for low, intermediate, and high clinical suspicion of TB, respectively, and corresponding specificity was 97%, 100%, and 100% (P = .25). Positive predictive value of the E-MTD test was 59% (low), 100% (intermediate), and 100% (high) compared with 36% (low), 30% (intermediate), and 94% (high) for AFB smear. Corresponding negative predictive values were 99%, 91%, and 55% [corrected] (E-MTD test) vs 96%, 71%, and 37% (AFB smear). Conclusions For complex diagnostic problems like TB, clinical risk assessments can provide important information regarding predictive values more likely to be experienced in clinical practice. For this series, a clinical suspicion of TB was helpful in targeting areas of the clinical spectrum in which nucleic acid amplification tests can make an important contribution.

In two previously well nonsmokers fatal pneumonia developed with extensive abscess formation. Legionnaires' bacillus was the only pathogen isolated. These cases indicate that Legionnaires' bacillus is capable of causing extensive necrosis of the lung.

Medical patients receiving IV therapy were randomly assigned to one of two IV tubing change groups. One group had a 48-hour tubing change and the other had no tubing change for the remainder of the cannula placement. A daily IV fluid specimen was processed microbiologically. To complete the study, a minimum of 3 continuous days of therapy and three fluid specimens was required. There were two contaminated specimens, one in each tubing change group. The contamination rate in the 48-hour change group was 0.87% and 0.96% in the no change group. The rate difference of 0.09% has a 95% confidence interval (−0.035 to +0.036) which includes zero. Survival analysis also revealed no significant difference in the cumulative probability of survival, however the mean duration of continuous tubing use of 4.3 days in the no change group and 1.8 days in the 48 hour change group were significantly different (p

A low-molecular-weight protein synthesized in yeast mitochondria was purified. The protein was identified as a component of the rutamycin-sensitive ATPase. The amino-acid composition of the purified protein shows an extremely large preponderance of nonpolar residues, which may account for its solubility in chloroform-methanol. Indirect evidence suggests that this component is involved in the conferral of rutamycin sensitivity and may also have a function in the assembly of the ATPase complex.

In an eight year period (1972–1979), 158 cases of enteric gram-negative bacillary meningitis and 53 cases of Listeria meningitis were reported to the New York City Health Department. These two forms of bacterial meningitis were the fourth and fifth most common ones reported to the Department, respectively. The cumulative total of reported cases of gram-negative rod meningitis was only slightly less than the reported cases due to Neisseria meningitis over this period. In contrast to most previous studies of Listeria and enteric gram-negative meningitides, the majority of cases occurred in adults. Listeria meningitis occurred more often in immunosuppressed hosts and the elderly than in the newborn; gram-negative bacillary meningitides were seen more often in elderly septic patients and in patients with traumatic skull fracture than in infants during the first months of life. The over-all mortality of Listeria meningitis was 62 percent; that of the combined gram-negative meningitides 71.3 percent. The mortality in adults with Escherichia coli and Klebsiella meningitis who were treated with chloramphenicol as the major antibiotic was 83 percent. In comparison, the only appreciable survival rates noted were in those with Listeria, Escherichia coli, Proteus mirabilis and Salmonella meningitis who were treated with ampicillin alone in whom the over-all mortality was 51 percent.

The rutamycin-insensitive ATPase (F1) of yeast mitochondria was found in previous studies to be synthesized on the cytoplasmic ribosomes and to accumulate in the postribosomal supernatant (Tzagoloff, A. (1969) J. Biol. Chem. 244, 5027). The enzyme has been purified from the postribosomal fraction of yeast grown in the presence of chloramphenicol and compared with F1 isolated from normal yeast mitochondria. The subunit protein compositions of the two preparations are identical, indicating that all of the component proteins of F1 are products of the cytoribosomal system of protein synthesis. Following removal of chloramphenicol and further incubation of the cells in growth medium (derepression), an increase of the ATPase activity of mitochondria and a loss of F1 activity in the postribosomal supernatant are observed. Evidence is presented indicating that during derepression the postribosomal F1 is incorporated into the larger rutamycin-sensitive ATPase complex of mitochondria.

The activities of cefotaxime, moxalactam, MK 0787 ( N -formimidoyl thienamycin), ampicillin, oxacillin, vancomycin, and clindamycin were compared against gram-positive cocci. MK 0787 was the most active and moxalactam was the least active of these drugs, except against methicillin-resistant Staphylococcus aureus , where vancomycin was most active, and penicillin-resistant pneumococci, where cefotaxime was more active.

Thirty-four semen samples from fertile men were subjected to bacteriologic evaluation before and after freezing to -196 degrees C using 10% glycerol as the protective medium. Cultures of the fresh portions from all ejaculates examined showed the presence of aerobic bacteria. Staphylococcus epidermidis, Corynebacterium sp., alpha-streptococci, and nonhemolytic streptococci were found so commonly that they were considered commensals. Enterococci, Escherichia coli, S. aureus, and beta-hemolytic streptococci group B, as well as Pseudomonas aeruginosa, P. maltophilia, and P. fluorescens, were also recovered from a few semen samples of these fertile men. Practically all bacteria isolated from the fresh ejaculate were also recovered from the portions frozen in liquid nitrogen.

Pseudomonas aeruginosa is the most common cause of osteomyelitis following puncture wounds of the feet of children. The source of the initial inoculum is unknown. Only one strain of P. aeruginosa was cultured from paired samples of the heel or corresponding shoe's surface or both obtained from 100 children. Neither the skin of the heel nor the shoe appears to be the source of the initial inoculum.

The antimicrobial susceptibilities were determined for 14 strains of Yersinia enterocolitica, 28 strains of Eikenella corrodens, 10 strains of penicillin-resistant Streptococcus pneumoniae, and 10 strains of penicillin-susceptible S. pneumoniae. Y. enterocolitica was found to be susceptible to the aminoglycosides tested and to chloramphenicol, moxalactam, cefoperazone, and cefotaxime but resistant to ampicillin and variably susceptible to carbenicillin, cefoxitin, and cefazolin. On a weight basis, cefotaxime and moxalactam were the most active agents against E. corrodens. Most strains of E. corrodens were resistant to the aminoglycosides tested: gentamicin, tobramycin, kanamycin, and amikacin. Penicillin-resistant S. pneumoniae was most susceptible to cefotaxime and moxalactam. Cefotaxime was consistently active against all tested strains of Y. enterocolitica, E. corrodens, and penicillin-resistant S. pneumoniae, three unusual but clinically significant pathogens.

Two hundred seventy nasopharyngeal aspirates were tested in duplicate with the Ortho Diagnostics, Inc. (Raritan, N.J.), respiratory syncytial virus antigen enzyme-linked immunosorbent assay. The test was sensitive (80 to 82%) and specific (96%) when compared with cell culture. The enzyme-linked immunosorbent assay detected seven antigen-positive specimens not among the 71 specimens that were positive for respiratory syncytial virus in cell culture. A blocking test confirmed those specimens as true positives (specificity, 100%).

A brain abscess caused by Listeria monocytogenes developed in an immunosuppressed renal transplant patient. Meningitis and meningoencephalitis from this organism were encountered in three other renal transplant recipients at this medical center during the past 4 years. Focal neurologic deficits occurred in patients with either Listeria abscess or meningoencephalitis. Computerized tomography was a rapid aid to the diagnosis of abscess. Immunosuppression has increased the incidence of central nervous system Listeria infections, but ampicillin still provides effective treatment, even when immunosuppressive therapy is continued. Limited experience with Listeria brain abscess suggests that surgical intervention improves the prognosis.

Cefoperazone was used to treat patients with complicated urinary tract infections due to multiple antibiotic-resistant gram-negative rods who had failed prior courses of intravenous antibiotic therapy. Cure was achieved in 44% (4/9) of cases; 44% of patients improved but relapsed and 11% (1/9) of patients were reinfected. Relapse and reinfection were associated with Pseudomonas aeruginosa and/or with conditions not normally responsive to medical therapy alone including prostatitis, reflux and chronic indwelling Foley catheters. The pharmacokinetics of cefoperazone were studied in renal transplant recipients. Peak serum concentrations (range 146-241 micrograms/ml) and 2-hour noncumulative urine concentrations (range 161-291 micrograms/ml) exceeded the minimal inhibitory concentrations of the bacteria in all cases. There was no accumulation of cefoperazone despite the presence of impaired renal function.

The activities of moxalactam and cefotaxime, alone and combined with ampicillin or penicillin, against 40 isolates of group B streptococci were assessed by using the microtiter broth dilution, checkerboard, and time-kill techniques. Penicillin and cefotaxime were bactericidal for all isolates at concentrations of 0.06 micrograms/ml or less. Ampicillin was slightly less active. Moxalactam was bactericidal for all strains at concentrations of 4 to 8 micrograms/ml. The ampicillin- moxalactam combination was partially synergistic for 60% of the isolates tested; the ampicillin-cefotaxime combination was partially synergistic for 35% of these isolates. No instances of antagonism were observed. In time-kill evaluations, ampicillin (3.0 micrograms/ml) and penicillin (0.75 micrograms/ml) effected 2.5 to 3.5 log10 reductions in numbers of colony-forming units. The addition of 4 micrograms of cefotaxime per ml or 8 to 16 micrograms of moxalactam per ml to penicillin or ampicillin did not alter killing kinetics. Moxalactam and cefotaxime neither enhanced nor decreased the activity of ampicillin or penicillin against group B streptococci.

We report three cases of serious non-group b Haemophilus influenzae infections. The significance of these isolates with respect to both biotypes and serotypes is discussed.

Vaginal colonization of mothers with Group B beta-hemolytic streptococcus (GBS) has been recognized as a risk factor for neonatal morbidity. The relationship of GBS colonization to risks for the mother who undergoes cesarean section has not been defined. In this study, we found that, among patients who underwent cesarean section, the 19% of them who were colonized with GBS had a higher incidence of standard fever (66.6% vs. 30.5%), clinical diagnosis of endomyometritis (61.1% vs. 12.5%), and use of antibiotics (61.1% vs. 26.3%) in relationship to a significantly increased frequency of premature rupture of the membranes (50.0% vs. 14.8%). Reasons for the association between vaginal colonization and increased morbidity are discussed.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children. Rapid identification of RSV infections would allow for specific chemotherapy. We evaluated a means of rapid diagnosis, the Abbott enzyme immunoassay (EIA), by using 314 stored nasopharyngeal aspirates. RSV antigens were identified in 62 of 66 RSV culture-positive specimens. An additional 37 specimens from which RSV was not isolated were positive in the EIA. Of these, 29 were confirmed as truly positive by a blocking assay, for a total of 95 (66 + 29) positive specimens. The sensitivity of the EIA for total positive samples was 96% (91/95) versus 69% (66/95) for cell culture. The specificity of the EIA was 96% (211/219). In these stored specimens, Abbott EIA was superior to cell culture for the detection of RSV.

The activities of 11 antimicrobial agents against 36 strains of Campylobacter fetus subsp. jejuni were studied by a broth microdilution method. All strains were susceptible to 7 of the 11 antimicrobial agents. Of the newer agents tested N-formimidoyl thienamycin (MK0787) and rosaramicin had very good activity, whereas cefotaxime, moxalactam, and cefoperazone had poorer activity.

Immediate inoculation of HEp-2 cells is generally advocated for the isolation of respiratory syncytial virus. However, delayed laboratory inoculation of properly transported specimens obtained by aspiration of nasopharyngeal mucus provided an isolation rate similar to that obtained with immediate inoculation.

Starting in 1975, an abrupt decline in ampicillin resistance of Salmonella typhimurium (the most common and antibiotic-resistant serotype) occurred in New York City. The present lower rate of 5% to 7.9% at our hospitals represents a return to the 1965 level. At the same time, the ampicillin resistance in S typhimurium from calves and other farm animals from upper New York State, which has been rising since 1972, has reached 75%. Substantial differences between levels of resistance in Escherichia coli from animals and humans were also noted. The divergence of the trends in S typhimurium from these two sources in New York State suggests a substantial degree of separation of the respective reservoirs of Salmonella and of antibiotic resistance. ( JAMA 242:439-442, 1980)

A comparison of the susceptibility patterns of M. tuberculosis isolated from children and adults treated for tuberculosis from 1971 through June, 1984 was performed. There were 126 strains isolated from children and 2735 from adults. The study was divided into 2 periods, 1971-1977 and 1978-June, 1984. During period 1, 13 of 95 (13.7%) children strains and 301 of 1745 (17.22) adult strains were resistant to isoniazid (INH). In period 2, 2 of 31 (6.5%) children strains and 106 of 1008 (10.5%) adult strains were resistant to INH. The decrease in resistance to INH was significant for adults. In period 1, none of 81 children strains and 10 of 1745 (0.6%) adult strains were resistant to rifampin (RF). In period 2, 5 of 31 (16.2%) children strains and 127 of 1008 (12.6%) adult strains were resistant to RF. The increase in resistance to RF in period 2 was significant for both children (P=