Evaluation of use of a single intravaginal swab to detect multiple sexually transmitted infections in active-duty military women.

The accuracy and suitability of use of a single intravaginal swab (SIS) for polymerase chain reaction detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and human papillomavirus infection was assessed in a cross-sectional study of 841 active-duty military women. The SIS, compared with standard diagnostic tests, allowed detection of more gonorrhea, more chlamydial infection, and more trichomoniasis. Sensitivity and specificity of SIS detection compared with adjudicated true-positive diagnoses were 95.8% and 97.8%, respectively, for gonorrhea, 94.6% and 99.3% for chlamydial infection, and 92.2% and 98.2% for trichomonal infection. Results with SISs were comparable to those with cervical swabs tested for human papillomavirus. Assay of clinician-collected and self-collected SISs yielded prevalences similar to those of standard diagnostic tests for all sexually transmitted infections. Therefore, the use of SISs is acceptable for the simultaneous diagnosis of multiple sexually transmitted infections and has potential for use as a self-administered diagnostic tool with widespread applicability among women.

Atherosclerosis in apoE knockout mice infected with multiple pathogens.

Cytomegalovirus (CMV) and Chlamydia pneumoniae (CP) possibly contribute to atherosclerosis. Murine CMV (MCMV) and CP increase lesion size in apoE knockout mice. In this study, apoE knockout mice were infected with MCMV and CP to determine whether infection with multiple pathogens increases lesion size to a greater extent than either pathogen alone and whether infection with MCMV changes serum cytokine levels in a manner that could increase lesion development. One group of mice received MCMV at 2 weeks of age, followed by 2 doses of CP at 6 and 8 weeks of age. Additional groups received only MCMV or CP. Animals were killed at 16 weeks of age to determine lesion area. Infection with MCMV alone, CP alone, and both MCMV and CP increased lesion size 84% (P<.001), 70% (P<.0001), and 45% (P<.01), respectively. The MCMV-induced increase in circulating levels of interferon-gamma may have contributed to this increase.

Effect of azithromycin on murine arteriosclerosis exacerbated by Chlamydia pneumoniae.

Chlamydia pneumoniae infection can exacerbate atherosclerosis in animals. To test the hypothesis that antibiotic therapy inhibits the atherogenic effects of C. pneumoniae infection, 10-week-old apolipoprotein E (ApoE) null mice were infected with C. pneumoniae or placebo, were treated for 2 weeks after infection with azithromycin or placebo, and were killed at 20 weeks of age. Infection did not affect the size of the aortic lesion, and antibiotic treatment had no effect. Another group of mice, 12-week-old ApoE mice, were infected with C. pneumoniae or placebo, were treated for 2 weeks after infection with azithromycin or placebo, and were killed at 26 weeks of age. C. pneumoniae infection increased the size of the lesion in infected mice, but azithromycin did not reduce the size of the aortic lesion in infected mice. Therefore, immediate therapy of acute infection may be necessary to prevent the proatherogenic effects of C. pneumoniae infection.

Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes.

Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (kappa, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples.

A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children.

OBJECTIVE: Lactobacillus GG (L-GG), an acid- and bile-resistant strain that colonizes the intestinal mucosa, has been used to manage diarrhea in children. Our objective was to evaluate the prophylactic use of L-GG to prevent diarrhea in children at high risk from a developing country in a randomized, placebo-controlled trial. STUDY DESIGN: Two hundred four undernourished children 6 to 24 months old from an indigent peri-urban Peruvian town received either L-GG or placebo in flavored gelatin once daily, 6 days a week, for 15 months. Episodes of diarrhea were documented by daily home visits, and diagnostic studies were done in a subset of cases. Recovery of L-GG in stool from subjects and from family contacts was examined. RESULTS: Subjects in the L-GG group had significantly fewer episodes of diarrhea (5.21 episodes diarrhea/child/year ['ecy'] L-GG group, 6. 02 ecy placebo group; P =.028). The decreased incidence of diarrhea in the L-GG group was greatest in the 18- to 29-month age group (P =. 004) and was largely limited to nonbreastfed children (Breastfed: 6. 59 ecy L-GG, 6.32 ecy placebo, P =.7; Nonbreastfed: 4.69 ecy L-GG, 5. 86 ecy placebo, P =.005). The duration of diarrhea episodes and the causes of diarrhea were similar in both groups, except adenovirus was more common in the placebo group. CONCLUSION: L-GG supplementation may be useful as a prophylactic measure to control diarrhea in undernourished children at increased risk, especially nonbreastfed children in the toddler age group.

PIP: This article features a placebo-controlled trial of Lactobacillus GG (L-GG) for diarrhea prevention in undernourished children in Peru. The purpose of the study was to evaluate the use of L-GG as prophylactic treatment for diarrhea. The study population included 204 undernourished children aged 6-24 months, 99 of which were on L-GG and 105 on placebo. Subjects were followed by daily home visits to document diarrhea episodes and diagnostic studies were conducted. Results revealed that children receiving L-GG experienced fewer episodes of diarrhea, which were more pronounced among 18-29 month old children and largely limited to non-breast-fed children. Moreover, the duration of diarrhea episodes and its causes were similar in both groups, except that adenovirus was detected more frequently in the placebo group. In conclusion, L-GG supplementation would decrease diarrhea incidence in high-risk children.

Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples.

Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.

Use of lambda phage DNA as a hybrid internal control in a PCR-enzyme immunoassay to detect Chlamydia pneumoniae.

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification.

Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay.

A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian Mycobacterium tuberculosis isolates (including both pansensitive and multidrug-resistant strains) and the H37Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.

Pooling urine samples for ligase chain reaction screening for genital Chlamydia trachomatis infection in asymptomatic women.

The accuracy of pooling urine samples for the detection of genital Chlamydia trachomatis infection by ligase chain reaction (LCR) was examined. A model was also developed to determine the number of samples to be pooled for optimal cost savings at various population prevalences. Estimated costs included technician time, laboratory consumables, and assay costs of testing pooled samples and retesting individual specimens from presumptive positive pools. Estimation of population prevalence based on the pooled LCR results was also applied. After individual urine specimens were processed, 568 specimens were pooled by 4 into 142 pools and another 520 specimens were pooled by 10 into 52 pools. For comparison, all 1,088 urine specimens were tested individually. The sample-to-cut-off ratio was lowered from 1.0 to 0.2 for pooled samples, after a pilot study which tested 148 samples pooled by 4 was conducted. The pooling algorithm was 100% (48 of 48) sensitive when samples were pooled by 4 and 98.4% (61 of 62) sensitive when samples were pooled by 10. Although 2.0% (2 of 99) of the negative pools of 4 and 7.1% (1 of 14) of the negative pools of 10 tested presumptive positive, all samples in these presumptive-positive pools were negative when retested individually, making the pooling algorithm 100% specific. In a population with 8% genital C. trachomatis prevalence, pooling by four would reduce costs by 39%. The model demonstrated that with a lower prevalence of 2%, pooling eight samples would reduce costs by 59%. Pooling urine samples for detection of C. trachomatis by LCR is sensitive, specific, and cost saving compared to testing individual samples.

Epidemiology and treatment of Cyclospora cayetanensis infection in Peruvian children.

Cyclospora cayetanensis was detected in fecal specimens from 63 (1.1%) of 5,836 Peruvian children studied over 2 years; the protozoan was detected by modified acid-fast staining and autofluorescence under ultraviolet light. The highest prevalence occurred among children between 2 and 4 years of age. Thirty-two percent (20) of the 63 C. cayetanensis-infected children were symptomatic. Nineteen infected children were enrolled in a double-blind, placebo-controlled trial of a 3-day course of trimethoprim-sulfamethoxazole (TMP-SMZ; 5/25 mg/[kg x d]). Children were followed up with daily stool examinations (mean number of samples examined per child +/- SE, 19 +/- 4). The mean duration of oocyst excretion +/- SE was 4.8 +/- 1.2 days for TMP-SMZ recipients compared with 12.1 +/- 6.1 days for placebo recipients (P < .02). The prevalence of C. cayetanensis infection decreases during winter months and as children age; it decreases precipitously by adulthood. In children in areas of endemicity, C. cayetanensis usually causes mild disease that is often asymptomatic. TMP-SMZ therapy significantly decreases the duration of oocyst excretion.

Active surveillance for Vibrio cholerae O1 and vibriophages in sewage water as a potential tool to predict cholera outbreaks.

The 1991 Peruvian cholera epidemic has thus far been responsible for 600,000 cholera cases in Peru. In an attempt to design a cholera surveillance program in the capital city of Lima, weekly sewage samples were collected between August 1993 and May 1996 and examined for the presence of Vibrio cholerae O1 bacteria and V. cholerae O1 bacteriophages (i.e., vibriophages). During the 144 weeks of surveillance, 6,323 cases of clinically defined cholera were recorded in Lima. We arbitrarily defined an outbreak as five or more reported cases of cholera in a week. The odds of having an outbreak were 7.6 times greater when V. cholerae O1 was present in sewage water during the four previous weeks compared with when it was not (P < 0.001). Furthermore, the odds of having an outbreak increased as the number of V. cholerae O1 isolations during the previous 4 weeks increased (P < 0.001). The odds of having an outbreak were 2.4 times greater when vibriophages were present in sewage water during the four previous weeks compared with when they were not, but this increase was not statistically significant (P = 0.15). The odds of having an outbreak increased as the number of vibriophage isolations during the previous 4 weeks increased (P < 0.05). The signaling of a potential cholera outbreak 1 month in advance may be a valuable tool for implementation of preventive measures. In Peru, active surveillance for V. cholerae O1 and possibly vibriophages in sewage water appears to be a feasible and effective means of predicting and outbreak of cholera.

Rates of tetanus protection and transplacental tetanus antibody transfer in pregnant women from different socioeconomic groups in Peru.

In developing countries, neonatal tetanus causes significant mortality. Using a new competitive enzyme-linked immunosorbent assay to measure anti-tetanus toxin antibody levels, we compared rates of protection, total antibody levels, and maternal-to-fetal antibody ratios between different socioeconomic groups in Peru. Upper-middle-class women 25 years and older had significantly lower rates of protection and mean anti-tetanus toxin antibody levels than did lower-class women of the same age. Nevertheless, the former had higher fetal-to-maternal antibody ratios, independent of maternal age, total antibody levels, or parity. We conclude that future vaccination programs in Latin America must target upper-middle-class as well as lower-class women.

The role of pulse oximetry. Its use as an indicator of severe respiratory disease in Peruvian children living at sea level. Respiratory Group in Peru.

OBJECTIVE: To evaluate pulse oximetry as a technique for diagnosing pneumonic and nonpneumonic acute lower respiratory tract infection (ALRI) in Peruvian children. DESIGN: Children with acute respiratory infection were diagnosed with hypoxemia by pulse oximetry, with ALRI by the World Health Organization (WHO) algorithm and clinical examination, and with pneumonia by radiographic examination. Diagnoses were compared using kappa analysis. SETTING: Pediatric emergency department. PATIENTS: Peruvian pediatric patients with acute respiratory infection (n = 269) and well children (n = 162). MAIN OUTCOME MEASURES: Hypoxemia (arterial oxygen saturation < 96.6% of the mean arterial oxygen saturation of well children -2 SD). RESULTS: Children with pneumonic and nonpneumonic ALRI (59%, 160/269) had a mean (+/- SD) arterial oxygen saturation significantly lower than well children (93.8% +/- 3.5% vs 98.7% +/- 1.51%; P < .01). Pulse oximetry detected 88% and the WHO algorithm 90% of cases of pneumonic ALRI. The WHO algorithm and pulse oximetry detected 72% of radiologic pneumonia. Pulse oximetry misclassified notably fewer well children than did the WHO algorithm (4% vs 35%). Pulse oximetry and the WHO algorithm together (SATWHO) detected 99% and 87% of pneumonic ALRI and radiologic pneumonias, respectively, and both methods detected 94% of all cases of pneumonic and nonpneumonic ALRI diagnosed clinically. CONCLUSIONS: Pulse oximetry and the WHO algorithm are practical, helpful, and appropriate for use in developing countries to identify children with pneumonic and non-pneumonic ALRI who require treatment. The SATWHO is highly sensitive for detecting children with ALRI.

Serodiagnosis of Helicobacter pylori infection by enzyme-linked immunoelectrotransfer blot. Gastrointestinal Physiology Working Group of Cayetano Heredia University.

Immunoreactive bands in sera from 81 Peruvian adults (48 females and 33 males) aged between 18 and 85 years were detected using enzyme-linked immunoelectrotransfer blot with a glycine-extracted Helicobacter pylori antigen. Sensitivity and specificity of bands were determined by comparison with results of H. pylori obtained by culture and silver stain of gastric biopsies. A crude antigen and a commercial enzyme-linked immunosorbent assay (Pylori Stat, Whittaker) were also evaluated for their diagnostic utility. H. pylori was detected in 57/81 (70%) of the subjects. The crude antigen enzyme-linked immunosorbent assay and the commercial enzyme-linked immunosorbent assay kits were, respectively, 84% and 88% sensitive. Specificity of both was 92%. The glycine-extracted antigen produced ten bands. The best single diagnostic band was at 20 kDa (83% sensitive and 96% specific). Three bands (13, 23, 30 kDa) were 100% specific. The combination of two diagnostic bands 20 and 30 kDa was 90% sensitive and 96% specific. Low molecular weight bands could be useful for the diagnosis of H. pylori infection.

Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157:H7 strains by using templates from boiled cultures.

Arbitrarily primed PCR allows genetically different bacterial strains to be distinguished with great sensitivity and efficiency. We report that informative, reproducible arbitrarily primed PCR profiles can be obtained from Escherichia coli O157:H7 strains by using boiled stationary-phase cultures, without the need for time-consuming phenol extraction. This simple template preparation procedure should be especially useful in large epidemiologic studies when many strains must be typed.

Short report: a placebo-controlled study of Lactobacillus GG colonization in one-to-three-year-old Peruvian children.

A pilot, placebo-controlled study conducted in Peruvian toddlers in a periurban shanty-town community demonstrates that 1) a simple fluorescent antibody test performed on bacteria from colonies grown on solid culture media can be used as a presumptive screening tool for Lactobacillus GG, 2) Lactobacillus GG powder sprinkled on flavored gelatin as a nutritional supplement is well-accepted by infants and mothers in this population, and 3) daily doses of Lactobacillus GG result in efficient colonization of the gastrointestinal tract of Peruvian infants. This study demonstrates that Lactobacillus GG should be evaluated as an adjunct for diarrhea control programs at the community level as well as in hospital-based settings.

Community infection ratio as an indicator for tuberculosis control.

The epidemiology of tuberculosis remains poorly understood. We investigated the relative importance of within-household and community transmission of infection among children aged 6 months to 14 years living in a Peruvian shanty-town. The prevalence of Mycobacterium tuberculosis exposure among 175 contact children (sharing a household with a person who had confirmed pulmonary tuberculosis) and 382 control children (living in nearby households free of active tuberculosis) was defined as the proportion of children with a positive purified protein derivative (PPD) skin-test. 97 (55%) contact children and 129 (34%) controls were PPD positive. Living in a contact household (odds ratio 1.74, 95% CI 1.11-2.73) and age (1.11, 1.06-1.18) were significant risk factors for PPD positivity. We calculated the community infection ratio (CIR) as the odds ratio of PPD-positive controls to PPD-positive contacts: CIR = [formula: see text] A low CIR therefore suggests mainly household spread of infection, whereas a high value suggests frequent transmission outside the household. The adjusted odds ratio (for age, sex, within-household correlation, and household size) was 0.40 (95% CI 0.26-0.64), compared with values of 0.18-0.37 in studies elsewhere. Currently recommended tuberculosis control strategies are suitable for areas with low CIRs. Different strategies may be needed for areas, such as that we studied, with high values.

Neonatal tetanus in Peru: risk assessment with modified enzyme-linked immunosorbent assay and toxoid skin test.

We used a modified enzyme-linked immunosorbent assay (ELISA) to investigate tetanus immunity in 232 pregnant Peruvian women. One hundred forty-two (61.2%) had protective antitoxin titers (> or = 0.01 IU/mL). Protective titers correlated positively with the number of toxoid doses reported during the current pregnancy. A majority of women reporting no toxoid doses during the current pregnancy had at least one prenatal health care visit. We evaluated a toxoid skin test in 44 of the subjects, but it correlated poorly with the ELISA. The modified ELISA is a useful in vitro method for studying tetanus immunity in the developing world.