Implementation of SARS-CoV-2 testing at Medical Research Foundation of Trinidad and Tobago

The HIV Clinic at the Medical Reserch Foundation of Trinidad and Tobago (MRFTT) in collaboration with the Ministry of Health (MoH) supports national level diagnostic efforts by conducting HIV viral load testing using the Abbott m2000 real time poymerase chain reation (PCR) platform installed at the MRFTT Laboratory. The MRFTT is strategically poised to scale up diagnostic PCR testing for SARS-CoV-2. In August 2020, the SARS-CoV-2 pandemic in Trinidad and Tobago was categorized as having community spread. To support the government's efforts to rapidly scale up testing and reduce the turn around time whie delivering quality results, an agreement was reached with MoH to expand diagnostic testing for SARS-CoV-2 at the MRFTT laboratory. Goals: 1. Support the efforts of government to rapidly scale up testing for SARS-CoV-2 2. Integrate SARS-CoV-2 testing at a Regional HIV Care and Treatment Site.

Doxycycline treatment of asymptomatic dogs seropositive for Ehrlichia canis

In Grenada, West Indies dogs are at frequent exposure to the rickettsial pathogen, Ehrlichia canis, as demonstrated by high seroprevalence rates. However, many of these seropositive dogs are clinically normal. In this study we identified clinically normal, E. canis seropositive dogs and assigned half to an antibiotic treatment group and half to a no treatment group. All dogs were evaluated for the presence of E. canis DNA by PCR on whole blood before, during and after treatment. Only one seropositive dog was also PCR+ before treatment. Our results suggest that most clinically normal, E. canis seropositive dogs in a highly endemic geographic area are not concurrently infected and thus routine treatment of clinically normal, seropositive dogs is not warranted.

The application of PCR and reverse line blot hybridization to detect arthropod-borne hemopathogens of dogs and cats in Trinidad.

Arthropod-borne diseases are important causes of morbidity and mortality of companion animals in Trinidad. As clinical signs are vague, more sensitive methods to diagnose these diseases based on the polymerase chain reaction (PCR) followed by reverse line blot hybridization (RLB) of amplified products are being developed. An RLB of 14 oligonucleotide probes coupled with polymerase chain-amplified regions of 16S rRNA or 18S rRNA genes of hemoparasites from cats and dogs detected Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, feline mycoplasmas ("Candidatus Mycoplasma haemominutum,"Mycoplasma haemofelis), and some unknown species within the Babesia/Theileria group and the Anaplasma/Ehrlichia tribe. Amplified products were obtained from blood samples collected from 348 dogs and 15 cats. Overall, hemopathogen DNA was detected in 92 (26.4 per cent) dogs and six (40.0 per cent) cats. E. canis (49, 14.1 per cent) and feline mycoplasma (5, 33.3 per cent) DNA were most frequently identified in dogs and cats, respectively. B. canis vogeli (1, 6.7 per cent) and E. canis (1, 6.7 per cent) were also detected in cats. Mixed infections of Anaplasma/Ehrlichia sp. and Babesia sp. were present in five (1.4 per cent) dogs, while mixed infections of the feline mycoplasmas were present in two (13.3 per cent) cats, one of which was also positive for E. canis. Pyrexia was significantly associated with a positive RLB result in dogs (P= 0.00, chi(2), 1 df). This is the first reported application of macro-arraying techniques to detect arthropod-borne hemopathogens of companion animals in the Americas and the first detection of DNA of B. canis vogeli and E. canis in cats in Trinidad.

Antimicrobial drug resistance and genetic diversity of commensal Escherichia coli from caeca of chickens in Grenada.

The main goal of this study was to generate baseline data on resistance to 11 antimicrobial drugs of global importance among commensal Escherichia coli from healthy chicken in Grenada. For this purpose, a total of 183 commensal Escherichia coli isolates from 197 chickens (147 broilers and 50 layers) originating from 11 poultry farms in Grenada were studies using a standard disk diffusion method. The isolates were further studied for their haemolytic properties using sheep blood agar, and genotypes using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). Sixty-six isolates were positive for alpha haemolysis, and the remaining were non-haemolytic. There was no difference in antimicrobial susceptibility between haemolytic and non-haemolytic isolates. Resistance was highest against tetracycline (58.5%) followed by streptomycin (44.3%) and lowest to chloramphenicol (0.55%). Only three isolates (1.6%) showed resistance to fluoroquinolones. Resistance rates to tetracycline, streptomycin, and gentamicin were significantly lower among isolates from layers, compared with those from broilers. Multiple resistance to three or more classes of drugs was found in 10.4% of total isolates; the most common R-profile was Amp, Str, Tet. Twenty genotypes were identified among 24 randomly selected isolates that originated from 11 unrelated farms and 5 geographical locations. Isolates sharing similar genomic fingerprints by ERIC-PCR had different resistance profiles, whereas isolates with different genotypes shared similar profiles. In conclusion, this study showed the genetic diversity of chicken isolates from Grenada, and their significance and the epidemiological significance of ERIC-PCR genotypes among poultry isolates need further study.

Antimicrobial drug resistance and genetic diversity of commensal Escherichia coli from caeca of chickens in Grenada.

The main goal of this study was to generate baseline data on resistance to 11 antimicrobial drugs of global importance among commensal Escherichia coli from healthy chicken in Grenada. For this purpose, a total of 183 commensal Escherichia coli isolates from 197 chickens (147 broilers and 50 layers) originating from 11 poultry farms in Grenada were studies using a standard disk diffusion method. The isolates were further studied for their haemolytic properties using sheep blood agar, and genotypes using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). Sixty-six isolates were positive for alpha haemolysis, and the remaining were non-haemolytic. There was no difference in antimicrobial susceptibility between haemolytic and non-haemolytic isolates. Resistance was highest against tetracycline (58.5%) followed by streptomycin (44.3%) and lowest to chloramphenicol (0.55%). Only three isolates (1.6%) showed resistance to fluoroquinolones. Resistance rates to tetracycline, streptomycin, and gentamicin were significantly lower among isolates from layers, compared with those from broilers. Multiple resistance to three or more classes of drugs was found in 10.4% of total isolates; the most common R-profile was Amp, Str, Tet. Twenty genotypes were identified among 24 randomly selected isolates that originated from 11 unrelated farms and 5 geographical locations. Isolates sharing similar genomic fingerprints by ERIC-PCR had different resistance profiles, whereas isolates with different genotypes shared similar profiles. In conclusion, this study showed the genetic diversity of chicken isolates from Grenada, and their significance and the epidemiological significance of ERIC-PCR genotypes among poultry isolates need further study.

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago

OBJECTIVES: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS: From March–September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79 percent) gave equivalent PCR results for both enrichment broths—28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago.

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21 per cent) samples, of which 5 (2 per cent) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

A nested-PCR with an Internal Amplification Control for the detection and differentiation of Bartonella henselae and B. clarridgeiae: an examination of cats in Trinidad

BACKGROUND: Bartonella species are bacterial blood parasites of animals capable of causing disease in both animals and man. Cat-Scratch Disease (CSD) in humans is caused mainly by Bartonella henselae and is acquired from the cat, which serves as a reservoir for the bacteria. A second species, B. clarridgeiae is also implicated in the disease. Diagnosis of Bartonellosis by culture requires a week or more of incubation on enriched media containing blood, and recovery is often complicated by faster growing contaminating bacteria and fungi. PCR has been explored as an alternative to culture for both the detection and species identification of Bartonella, however sensitivity problems have been reported and false negative reactions due to blood inhibitors have not generally been addressed in test design. METHODS: A novel, nested-PCR was designed for the detection of Bartonella henselae and B. clarridgeiae based on the strategy of targeting species-specific size differences in the 16S-23S rDNA intergenic regions. An Internal Amplification Control was used for detecting PCR inhibition. The nested-PCR was utilized in a study on 103 blood samples from pet and stray cats in Trinidad. RESULTS: None of the samples were positive by primary PCR, but the Nested-PCR detected Bartonella in 32/103 (31%) cats where 16 were infected with only B. henselae, 13 with only B. clarridgeiae and 3 with both species. Of 22 stray cats housed at an animal shelter, 13 (59%) were positive for either or both species, supporting the reported increased incidence of Bartonella among feral cats. CONCLUSION: The usefulness of a single PCR for the detection of Bartonella henselae and B. clarridgeiae in the blood of cats is questionable. A nested-PCR offers increased sensitivity over a primary PCR and should be evaluated with currently used methods for the routine detection and speciation of Bartonella henselae and B. clarridgeiae. In Trinidad, B. henselae and B. clarridgeiae are the predominant species in cats and infection appears highest with stray cats, however B. clarridgeiae may be present at levels similar to that of B. henselae in the pet population.

The prevalence of Chlamydia trachomatis in fresh tissue specimens from patients with ectopic pregnancy or tubal factor infertility as determined by PCR and in-situ hybridisation

The prevalence of chlamydial DNA determined by PCR and in-situ hybridisation (ISH) in fresh tissue specimens (endometrium, fallopian tube and ovary) was investigated in 33 women presenting with ectopic pregnancy (EP), 14 women with tubal factor infertility (TFI) and 50 control patients from the UK and the West Indies. In the UK EP group, chlamydial DNA was detected by PCR in 56% of patients; similar results were found in the Trinidad EP group (67%). In the TFI group, chlamydial DNA was detected in (71%) of patients by PCR. The detection of Chlamydia trachomatis DNA by ISH was highest in the TFI group (43%). Women presenting with EP and TFI showed evidence of previous or current genital C. trachomatis infection, underlining the importance of this microorganism in the development of these conditions. Importantly, chlamydial DNA could be detected in DNA preparations from the endometrium, fallopian tube and ovary of EP and TFI patients at the time of surgery.

Chlamydia trachomatis antibody and DNA determination in ectopic pregnancy in Trinidad

OBJECTIVE: To determine the prevalence of C. trachomatis in ectopic pregnancy by serum IgG and IgM antibody and by chlamydia DNA in endometrial, Fallopian tube and ovarian tissues. DESIGN AND METHODS: A cross-sectional study included 32 women presenting with tubal ectopic pregnancy and 94 fertile controls. Methods employed were ELISA for IgG and IgM and Polymerase Chain Reaction (PCR) and in situ hybridization (ISH) for DNA. RESULTS: Chlamydial IgG and IgM antibody detection was higher in the ectopic than the control groups (IgG, p<0.01; IgM, p<0.01). A similar finding was also noted for chlamydia DNA by PCR (p<0.05). DNA detection was also significantly higher at each site in the upper genital tract (endometrium p<0.01, Fallopian tube p<0.05, ovary p<0.05). CONCLUSION: By antibody detection, this study confirms the role played by genital tract C. trachoma infection and subsequently ectopic pregnancy, but more importantly, identifies chlamydial DNA in upper genital tract tissues. These results support allocation of resources towards screening programmes for C. trachomatis.(Au)

Evaluation of two methods for rapid serological diagnosis of acute leptospirosis

OBJECTIVE: To evaluate two rapid assays for serological diagnosis of acute leptospirosis in diagnostic laboratories. DESIGN AND METHODS: 209 specimens were examined from 104 patients admitted to hospital for investigation in a leptospirosis diagnostic protocol. Specimens for serology were taken on days 1 and 4 of the hospital admission. Antibodies were detected using IgM-ELISA, microscopic agglutination (MAT), an IgM-dipstick assay and indirect haemagglutination assay. RESULTS: 51 patients were found to have leptospirosis. The sensitivity of the IgM-dipstick was 98 percent, specificity was 90.6 percent, positive predictive value was 90.9 percent and the negative predictive value was 98 percent. The sensitivity of IHA was 92. percent, specificity was 94.4 percent, positive predictive value was 95.9 percent and negative predictive value was 92.7 percent. The IgM-dipstick assay was positive in 71 percent of the cases in the first sample tested. CONCLUSIONS: Both assays are highly sensitive and specific. Neither requires specialized equipment, and both are suitable for use in diagnostic laboratories.(Au)

Estimating the time of HTLV-I infection following mother-to-child transmission in a breast-feeding population in Jamaica

Mother-to-child transmission of human T-cell lymphotrophic virus type 1 (HTLV-I) is primarily due to prolonged breast-feeding (>6 months) in the post-natal period. Most infant infections are not identifiable until 12-18 months of age by available whole virus Western blot serologic tests because of their inability to distinguish passively transferred maternal antibody from infant antibody. We investigated two methods to assess more accurately the time of infant infection. In prospectively collected serial biospecimens, HTLV-I-specific immunoglobulin (Ig) isotypes of IgM and IgA were determined by Western blot and HTLV-I proviral DNA was detected by polymerase chain reaction (PCR). IgA and IgG reactivity was assessed in periodic serum samples from 16 HTLV-I-seropositive children while IgM reactivity was observed in 100 percent of children at 24 months of age and 73 percent of children at 6-12 months of age; however, this could represent maternal and not infant antibody. Both IgA and IgM reactivity were insensitive indicators of infection, with only 50 percent of children showing reactivity at 24 months of age. PCR testing was performed in biospecimens obtained from 11 of these children. An estimated median time of infection of 11.9 months was determined by PCR, which was similar to the median time to infection determined by whole virus Western blot (12.4 months; P=0.72). PCR Tests support a median time to infection that is similar to that estimated by whole virus Western blot. (AU)

Adeno-associated virus and development of cervical neoplasia

Evidence from several sources has suggested that adeno-associated virus (AAV) infection might protect against cervical cancer, in part, by interfering with human papillomavirus (HPV)-induced tumorigenesis. Detection of AAV type 2 (AAV-2) DNA in cervical tissues has been reported. However, there have been few in vivo studies of women with cervical HPV infection or neoplasia, and these have reported inconsistent results. Therefore, we used polymerase chain reaction (PCR) assays targeted to the AAV-2 rep and cap genes to test tissue specimens from women in an epidemiological study of cervical neoplasia in Jamaica. We tested 105 women with low-grade cervical intraepithelial neoplasia (CIN-1), 92 women with CIN-3/carcinoma in situ or invasive cancer (CIN-3/CA), and 94 normal subjects. PCR amplification of human beta-globin DNA was found in almost all cervical specimens, indicating that these materials were adequate for PCR testing. The prevalence of HPV DNA, determined by HPV L1 consensus primer PCR was, as expected, strongly associated with presence and grade of neoplasia. Each of the AAV PCR assays detected as few as 10 copies of the virus genome. However, none of the 291 cervical specimens from Jamaican subjects tested positive for AAV DNA. Negative AAV PCR results were also obtained in tests of cervical samples from 79 university students in the United States. Exposure to AAV was assessed further by serology. Using a whole virus AAV-2 sandwich enzyme-linked immunosorbent assay, we found no relationship between AAV antibodies and presence or grade of neoplasia in either the Jamaican study subjects or women enrolled in a U.S. cervical cancer case (n = 74) - control (n = 77) study. Overall, the data provide no evidence that AAV infection plays a role in cervical tumorigenesis or that AAV commonly infects cervical epithelial cells.(Au)

Molecular diagnosis of dengue by reverse transcriptase-polymerase chain reaction

The techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent PCR were employed in the analysis of serum samples from a range of patients from the Caribbean Epidemiology Centre (CAREC) member countries. Results were compared with those from viral isolation and immuno-fluorescence. In the second part of the study, ten serum samples were stored for one week under four sets of conditions: -20 degrees C, 4 degrees C, 25 degrees C, and thawed (20 degrees C) and frozen (-20 degrees C) daily. After one week of each treatment the samples were analysed by RT-PCR and PCR. 90.4 percent of results from PCR agreed with results from viral isolation (VI) and fluorescent antibody (FA) detection. All PCR positive samples originated from sera collected within five days of the date of onset of fever. Frozen, refrigerated and repeat freeze-thawed samples gave consistent positive results by RT-PCR. The results indicate the sensitivity and reliability of this rapid technique and its applicability in the Caribbean. It provides a preliminary assessment of its advantages and limitations under certain conditions of serum collection and storage (AU)

Ancestral origin of variation in the triosephosphate isomerase gene promoter

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from Sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which preceeds the separation of African populations.(Au)

Genital ulcers: etiology, clinical diagnosis, and associated human immunodeficiency virus infection in Kingston, Jamaica

Individuals presenting consecutively with genital ulcers in Kingston, Jamaica, underwent serological testing for human immunodeficiency virus (HIV) infection , chlamydial infection, and syphilis. Ulcer material was analyzed by the multiplex polymerase chain reaction (M-PCR) analysis DNA from herpes simplex virus (HSV), Haemophilus ducreyi, and Treponema pallidum was detected in 158 (52.0 percent), 72 (23.7 percent), and 31 (10.2 percent) of 304 ulcer specimens. Of the 304 subjects, 67 (22 percent) were HIV-seropositive and 64 (21 percent) were T. pallidum-seroactive. Granuloma inguinale was clinically diagnosed in nine (13.4 percent) of 67 ulcers negative by M-PCR analysis and in 12 (5.1 percent) of 237 ulcers positive by M-PCR analysis (P = .03). Lymphogranuloma venereum was clinically diagnosed in eight patients. Compared with M-PCR analysis, the sensitivity and specificity of a clinical diagnosis of syphilis, herpes, and chancroid were 67.7 percent, 53.8 percent, and 75 percent and 91.2 percent, 83.6 percent, and 75.4 percent, respectively. Reactive syphilis serology was 74 percent sensitive and 85 percent specific compared with M-PCR analysis. Reported contact with a prostitute in the preceding 3 months was associated with chancroid (P = .009), reactive syphilis serology (P = .011), and HIV infection (P = .007). The relatively poor pleads for syndromic management of genital ulcers in Jamaica. Prevention efforts should be intensified.(Au)

The map1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes

Heartwater is an economically important disease of ruminants caused by the tick-transmitted rickettsia Cowdria ruminantium. The disease is present in Africa and the Caribbean and there is a risk of spread to the Americas, particularly because of a clinically asymptomatic carrier state in infected livestock and imported wild animals. The causative agent is closely related taxonomically to the human and animal pathogens Ehrlichia chaffeensis and Ehrlichia canis. A dominant immune response of infected animals or people is directed against variable outer membrane proteins of these agents known, in E. chaffeensis and E. canis, to be encoded by polymorphic multigene families. We demonstrate, by sequence analysis, the map1 encoding the major outer membrane protein of C. ruminantium is also encoded by a polymorphic multigene family. Two members of the gene family are located in tandem in the genome. The upstream member, orf2, is conserved, encoding only 2 amino acid substitution among six different rickettsial strains from diverse locations in Africa and the Caribbean. In contrast, the downstream member, map1, contains variable and conserved regions between strains. Interestingly, orf2 is more closely related in sequence to omplb of E. chaffeensis than to map1 of C. ruminantium. The regions that differ among orf2, map1, and omp1b correspond to previously identified variable sequences in outer membrane protein genes of E. chaffeensis and E. canis. These data suggest that diversity in these outer membrane proteins may arise by recombination among gene family members and offer a potential mechanism for persistence of infection in carrier animals.(AU)

Clustering of cases of mycobacterium fortuitum infection investigated by molecular typing

Organisms of the mycobacterium fortuitum complex are recognised but uncommon causes of pulmonary disease, primary cutaneous disease and a wide spectrum of nosocomial infections. M fortuitum was isolated from 20 patients over a 15 month period, with a apparent clustering of isolates occurring from January to March 1994. The molecular epidemiology of this clustering eas investigated using an arbitrary primer polymerase chain reaction method (AP-PCR). 21 isolates were studied, which yielded 13 distinct profiles. Multiple isolates from a single patient yielded identical profiles. All of seven isolates recovered during the six week period from January to March 1994 shared a common profile which was distinct from all other isolates, suggesting that a single strain was isolated from specimens from all seven patients. The source of this cluster in uncertain. We can find no epidemilogical basis for an episode of cross-infection within the hospital environment, and it is assumed that contamination of the specimens during collection, transport or processing was responsible for the "pseudo-outbreak" of M fortuitum

Population structure of the primary malaria vector in South America, Anopheles darlingi, using isozyme, random amplified polymorphic DNA, internal transcribed spacer 2, and morphologic markers

A genetic and morphologic survey of Anopheles darlingi populations collected from seven countries in Central and South America was performed to clarify the taxonomic status of this major malaria vector species in the Americas. Population genetics was based on three techniques including isozyme, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), and internal transcribed spacer 2 (ITS2) markers. The results of the isozyme analysis indicated moderate differences in the allele frequencies of three putative loci (glutamate oxalaoacetate transaminase-1, isocitrate dehydrogenase-1, and phosphoglucomutase) of the 31 analyzed. No fixed electromorphic differences separated the populations of An. darlingi, which showed little genetic divergence (Nei distances = 0.976-0.995). Fragments produced by RAPD-PCR demonstrated evidence of geographic partitioning and showed that all populations were separated by small genetic distances as measured with the 1 - S distance matrix. The ITS2 sequences for all samples were identical except for four individuals from Belize that differed by a three-base deletion (CCC). The morphologic study demonstrated that the Euclidean distances ranged from 0.02 to 0.14, with the highest value observed between populations from Belize and Bolivia. Based on these analyses, all the An. darlingi populations examined demonstrated a genetic similarity that is consistent with the existence of a single species and suggest that gene flow is occurring throughout the species' geographic range.(Au)

Molecular epidemiology of tetM genes in Neisseria gonorrhoeae

OBJECTIVE: To examine the epidemiology of the tetM gene in Neisseria gonorrhoeae strains with high level resistance to tetracycline (TRNG) using a polymerase chain reaction (PCR) assay. METHODS: A single tube PCR was developed which distinguishes between the American and Dutch variants of the tetM gene. Between 1988 and 1995, 518 strains of TRNG (tetracycline MIC > 8.mg/1) were referred to the Gonococcus Reference Unit by other laboratories or isolated from routine swabs taken at local clinics. The strains were analysed for plasmid content, auxotype, serovar, and the tetM gene type. Travel details of the patients were determined by a questionnaire. RESULTS: A PCR product was obtained from all TRNG examined. 387 TRNG strains produced a 443 by PCR product (American type tetM) and 131 produced a 443 by PCR product (Dutch type tetM). Infections acquired in the United Kingdom contributed 57 percent of the TRNG strains included in this study; 82 percent of these carried the American type of tetM. The number of UK acquired TRNG received by the GRU increased each year except 1993--from four strains received in 1990 to 92 in 1995. After the United Kingdom, Caribbean and African countries contributed most strains, with 56 and 60 TRNG acquired in each area respectively. All strains originating in Africa, except one from South Africa, contained the American type tetM. Infections caught in Nigeria and Kenya contributed most strains (15 and 14 respectively). The TRNG originating from Caribbean countries comprised 36 percent Dutch tetM type. Infections caught in Jamaica accounted for 82 percent of the Caribbean strains. All 35 TNG strains originating in the Far East contained the Dutch type tetM. 25 of the Far East strains were also penicillinase producing (PPNG). Infections originating in Indonesia accounted for 49 percent of the Far East strains but these belonged to 12 different auxotype/serovar combinations. A geographical variation in the type of penicillinase coding plasmids found in PPNG/TRNG was also detected. CONCLUSIONS: These data suggest that the Dutch type tetM may have originated in the Far East and the American type in the African continent. Subsequent spread has resulted in a heterogeneous distribution of TRNG types in other parts of the world.(Au)