Gingivocrevicular transudate for HIV screening

588 Clinical Microbiology and Infection, Volume 3 Number 5, October 1997 that the correctional facilities, which at present have inadequate infection control practices and isolation facilities to prevent transmission of M. tuberculosis, fully comply with the current guidelines to reduce the risk of tuberculosis spread [14,15]. Acknowledgments This work was partly supported by the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT) grant SAF95/0435 by the Ministerio de Educaci6n y Ciencia. Jauier Aznar I, Hassan S&*, Mada C. Conejo’, Jos6 C. Palomares’ ‘Dpto Microbiologia, Apdo 914, Universidad de Sevilla, 41080-Sevilla, Spain; *Department of Microbiology, University of Seville, Seville, Spain Revised version accepted 3 May 1997 References 1. Greifinger RB, Keefus C, Grabau J, et al. Transmission of multidrug-resistant tuberculosis among immunocompromised persons in a correctional system. New York 1991. 2. Sepkowitz KA, R&ah J, Riley L, Kiehn TE, Amstrong D. Tuberculosis in the AIDS era. Clin Microbiol Rev 1995; 8: MMWR 1992; 41: 507-9. 180-99. 3. Valway SE, Richards SB, Kovacovich J, Greifinger RB, Crawford JT, Dooley SW. Outbreak of multi-drug-resistant tuberculosis in a New York State Prison 1991. Am J Epidemiol 1994; 140: 113-22. 4. De March P. Situaci6n actual de la tuberculosis en Espaiia. Med Clin (Barc) 1991; 97: 463-72. 5. Martin V, Gonzalez P, Cayla JA, et al. Case-finding of pulmonary tuberculosis on admission to a penitentiary centre. Tubercle Lung Dis 1994; 75: 49-53. 6. Hermans PWM, van Soolingen D, Dale JW, et al. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol 1990; 28: 2051-8. 7. Small PM, Shafer RW, Hopewell PC, et al. Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection. N Engl J Med 8. van Soolingen D, Hermans PW, de Haas PEW, Soll DR, van Embden JDA. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 1991; 29: 2578-86. 9. Kubica GPW, Dye E, Cohn ML, Middlebrook DH. Sputum digestion and decontamination with N-acetyl-L-cysteine1993; 328: 1137-44. sodium hydroxide for culture of mycobacteria. Am Rev Respir Dis 1963; 87: 775-9. 10. van Embden JDA, Donald Cave M, CrawfordJT, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standarhzed methodology. J Clin Microbiol 1993; 31(2): 406-9. 11. Aznar J, Safi H, Romero J, Alejo A, Gracia MD, Palomares JC. Nosocomial transmission of tuberculosis infection in pediatric wards. Pehatr Infect Dis J 1995; 14: 44-8. 12. Hermans PWM, Messadi F, Guebrexabber H, et al. Analysis of the population structure of Mycobacterium tuberculosis in Ethiopia, Tunisia, and the Netherlands: usefulness of DNA typing for global tuberculosis epidemiology. J Infect Dis 1995; 171: 1504-13. 13. Barnes PE El-Haj H, Preston-Martin S, et al. Transmission oftuberculosis among the urban homeless. JAMA 1996; 275: 14. The role of BCG vaccine in the prevention and control of tuberculosis in the United States. A joint statement by the Advisory Committee for Elimination of Tuberculosis and the Advisory Committee on Immunization Practices. 15. Prevention and control of tuberculosis in correctional facilities. Recommendations for the elimination of tuberculosis. MMWR 1996; 45: 1-27. 305-7. MMWR 1996; 45: 1-14. Gingivocrevicular transudate for HIV screening Clin Microbiol Infect 1997; 3: 588-590 The use of saliva as an alternative biological fluid to serum in diagnosis or screening of infectious diseases by antibody detection has been the main subject of several articles in the last few years [l-61. Sahva samples can be obtained simply, without specialized personnel, and the process is non-traumatic for the patient and economic and poses no contamination risks for health workers. For these reasons, saliva samples may be of great utility in underdeveloped nations, where there is a severe shortage of personnel and specialized equipment. Saliva is a mixture of the secretions produced by the salivary glands and gingival crevicular transudate (GCT). The use of GCT, which has a greater concentration of immunoglobulins (Ig) of the IgG type than does complete saliva [7], seems to improve detection of such Ig in the samples [8]. An oral device without preservatives (Salivette, Starstedt, Leicester, UK) was used to obtain the GCT samples. The device is a cylinder of cotton wool contained in a polystyrene double tube with a snap top (price USA $0.20). The cotton wool was placed in the lateral gingival fold until the individual could perceive that it had become soft because of its absorption of GCT. The samples thus obtained were centrifuged at 4000 g for 15 min, producing a supernatant which was Short Communications and Letters to the Editors 589 aliquoted and frozen at -80°C until processing. A serum sample was simultaneously obtained by venepuncture from each of the individuals included in the study. These samples were then aliquoted and stored at -80°C until processed. A total of 183 paired samples of GCT and serum were analyzed (50 CDC stage 11, 10 CDC stage 111, 33 CDC stage Iv 26 risk factor individuals and 64 blood donors). In both types of sample, the presence of IgG against HIV-I and HIV-2 was detected. With GCT samples, we used a new dot blot (Multispot HIV1 /HIV-2, Sanofi Diagnostics Pasteur, France) capable of discriminating between both IgG HIV-1 and IgG HIV-2. For the screening of serum samples, an indirect enzyme immunoassay (EIA) (VIDAS HIV 1+2, bioMkrieux, France) was used. All reactive samples were confirmed by Western blot (WB) (Bioblot HIVI plus, Genelabs Diagnostics, Switzerland). The WB used combines specific proteins for HIV-1 and a synthetic peptide (gp36) for HIV-2 in the same strip; WHO criteria [9] were used for confirmation of screening reactivity. The results presented in Table 1 show that all GCT samples non-reactive by dot blot belonged to noninfected individuals (EIA negative), except for one sample from a patient of the CDC stage IV group, which was negative by dot blot but positive by GCT WB (reactivity against gp160, gp120 and gp41). The GCT samples reactive by dot blot correspond to infected individuals, with the exception of one sample from a non-infected individual also negative by GCT WB. The characteristics of this test in comparison with serum WB results have been as follows: 99% sensitivity and 99% specificity (positive predictive value (PPV) and negative predictive value (NPV) were 0.9). Several reasons may be suggested in the case of the non-reactive sample from the individual belonging to CDC stage IV The first is the immunologic deterioration presented by individuals in the final stages of the infection, yielding a lower IgG serum concentration and even loss of concrete antibodies (ie. p24 antibodies); second, GCT may therefore not present reactivity when analyzed by insufficiently sensitive techniques. The use of a more sensitive test (WB) leads in this case to the detection of IgG against envelope proteins in the GCT. The confirmed presence of HIV1 envelope antibody in GCT is highly suggestive of HIV-1 infection and also correlates with the reactivities obtained in the serum WB of this individual. The salivary device used presents two main advantages. The first and most important is that it can obtain an adequate volume (= 1 ml) for sample analysis by different techniques and storage. Second, in contrast to the case with other devices, IgG sample concentration does not vary so much, because the sample is not diluted. There is no experimental evidence that the concentration of Ig in GCT declines during storage, but any such changes would be minimal, because the samples were not diluted in any preservative buffer and they were frozen after recovery, thus minimizing the action of saliva enzymes. GCT samples (USA $0.20) are cheaper to obtain than serum samples (USA $0.60 for syringe +needle+ tube). Furthermore, the GCT process permits a single individual to collect samples from several patients at once, instead of the individualized sample collection required with serum. Our results confirm previous work by others [10,11] but using a simple device for sampling and testing. The use of GCT instead of saliva has been proposed in several articles [8,10,11] to facilitate the detection of HIV IgG due to the increased amount of this type of Ig. In conclusion, the use of dot blot together with the oral GCT device could offer an alternative for screening of infection produced by HIV in wide population groups, because of its low cost and easy application. Further studies should be performed in patients with advanced stages of the disease to determine if the lloss in sensitivity could be due to the immunologic condition in this particular stage of the disease. Table 1 Detection of HIV-1 antibodies in the serum and GCT of different population groups Group No. Dotccr EIAscrunl mp" Stage I1 50 50 50 50 Stage I11 10 10 10 10 Stage IV 33 32 33 33 Subjects at risk for HIV infection 26 0 0 0 Blood donors 64 lb 0 0 References Prudencio Martinez, josk Maria Eiros Raul Ortiz dc Lejararu Antonio Rodrkuez Torres Servicio de Microbiologia Clinica, Hospital Universitario, Valladolid, Spain Accepted 25 April 1997 Total 183 93 93 93 1. Parrv IV. Perm KR. Mortimer PP. Sensitive assavs for viral ,_l, , , antibodies in saliva: an alternative to tect 011 cerum. Lancet "Samples with positive Western blot in serum and GCT. "Sample with negative Western blot in GCT. 1987; 2: 72-5. 590 Clinical Microbiology and Infection, Volume 3 Number 5, October 1997 4. 5. 6. 7. 8. 9. 10. 11. 2. Parry JV, Perry KR, Panday S, Mortimer PP. Diagnosis of hepatitis A and B by testing saliva. J Med Virol 1989; 28: 255-60. 3. Thieme T, Yoshihara P, Piacentini S, Beller M: Clinical evaluation oforal fluid samples for diagnosis ofviral hepatitis. J Clin Microbiol 1992; 30: 1076-9. Lucht E, Albert J, Linde A, et al. Human ihmunodeficiency virus type l and cytomegalovirus in saliva. J Med Virol 1993; 39. 15Gh2 The HBe Ag precursor is encoded by the 637-bp precore/core open reading frame starting at nucleotide 1814 of the HBV minus strand genome (nomenclature according to Galibert et al [4]), The frequently described mutation is a G to A substitution at the 3’ end of the pre-C gene (nucleotide 1896), which converts codon 28 for tryptophan (TGG) to a termination codon (TAG) [S], resulting in the failure of HBe - . . . - - .. - . Frerichs R, Htoori M, Eskes N, Soe L. Comparison of saliva and serum for HIV surveillance in developing countries. Lancet 1992; 340: 1496-9. Akker R, Hoek J, Akker W, et al. Detection of HIV antibodies in saliva as a tool for epidemiological studies. Parry JV. A specimen of convenience. Diagn Virol 1991; 3: Martinez P, Ortiz de Lejarazu K, Eiros JM, et al. Comparison of two assays for detection of HIV antibodm in saliva. Eur J Clin Microbiol 1995; 14: 330-6. World Health Organization. AIDS. Proposed WHO criteria for interpreting results fiom Western blot assays for HIV-1, HIV-2 and HTLV-I/HTLV-11. Wkly Epidemiol Rec 1990; G6mez C, Gutitrrez M, Martinez-Acacio P, Soriano V. Evaluation of a new saliva collection device for HIV antibody screening purposes. Vox Sang 1994; 66: 244. Gallo D, George R, Fitchen J, Goldstein A, Hindahl M. Evaluation of a system using oral mucosal transudate for HIV-1 antibody screening and confirmatory testing. JAMA 1997; 277: 254-8. AIDS 1992; 6: 953-7. 13-15. 65: 281-3. More on HBe Ag-negative mutants in chronic HBV infection Clin Microbiol Infect 1997; 3: 590-591 Chronic infection with hepatitis B virus (HBV) leads to a wide spectrum of liver diseases, including asymptomatic carrier state, chronic active hepatitis, cirrhosis and hepatocellular carcinoma. Patients presenting with chronic active hepatitis typically have evidence of HBV replication marked by high levels of HBV DNA, HBV surface antigen (HBs Ag) and the e antigen (HBe Ag) in their serum [l]. In later stages of the disease, seroconversion from HBe Ag to anti-HBe antibody classically indicates a transition to a ‘non-replicative’ phase, in which necro-inflammatory activity ceases. In some cases, however, anti-HBe seroconversion is not followed by such a favorable outcome, and markers for HBV replication remain detectable [2]. Carman et a1 [3] have studied such patients and found that they carried HBV mutants which cannot synthesize the precore precursor protein and therefore HBe Ag. Ag production and the cessation of its secretion into the bloodstream. It is often associated with a G to A silent mutation at nucleotide (nt) position 1899. The frequency of the nt 1896 mutation varies in different studies, ranging from 46% [6] to 80% [3]. This variability led us to investigate the frequency of the nt 1896 mutation and the presence of other putative mutations in a cohort of French patients. We analyzed 32 serum samples from chronically infected patients showing discordant serologic profiles, i.e. positive for HBs Ag and HBV DNA, negative for HBe Ag and positive for anti-HBe antibody. Serum samples were submitted to heat treatment (100 “C) for 30 min and then clarified by centrifugation (12000g) for 30 min. This rapid technique for HBV DNA preparation had been validated in preliminary studies as compared either to classical proteinase K-sodium dodecylsulfate (SDS) lysis followed by phenol extraction or to NaOH treatment of the serum. We applied an amplification-created restriction site method derived from that described by Lindh et a1 [7] for detecting the nt 1896 G to A mutation. According to this procedure, the nt 1865-2058 region of HBV DNA was amplified using P1 primer with a mismatch at position 1893. When the nt 1896 G to A mutation was present, the 194-bp PCR product exhibited a BSU 361 restriction site which could be shown by enzyme digestion and agarose gel electrophoresis. Prior to hgestion, a plasmid containing a unique BSU 361 restriction site was added as a control for the completion of digestion. The specificity of the PCR reaction was checked by hybridization with a digoxigenin-labeled probe specific for the HBV precore region. In parallel, the nt 1728-2160 region of HBV DNA was amplified for the determination of nucleotide sequence using a T7 polymerase sequencing kit (Pharmacia). After digestion with BSU 361, we observed three patterns of migration in an agarose gel among the 32 samples tested. Five cases (16%) showed a digested product of 165 bp characteristic ofthe G to A mutation. Twenty cases (62%) showed an undigested PCR product of 194 bp. In seven (22%) cases, two fragments comigrated in the agarose gel, one of 194 bp, and one of 165 bp, corresponding to a mixed viral population (wild type and mutant). In these cases, the possibility of partial digestion with BSU 361 was ruled out by