Sensitive and specific polymerase chain reaction assays for diagnosis of human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infections in HTLV-I/II-seropositive individuals.

To confirm and differentiate between human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infections, we analyzed by polymerase chain reaction (PCR) samples of peripheral blood lymphocytes from 98 individuals seropositive for HTLV-I/II using pol (SK110/111) and tax (SK43/44) consensus primer pairs. A total of 96 samples (97.9%) were positive by the tax generic probe, while 95 were typed by the HTLV-I and HTLV-II pol probes. The three pol-negative samples were successfully amplified and typed by nested PCR with primers internal to SK110 and SK111. Results of PCR with a lysate of leukocyte nuclei obtained by whole blood lysis were comparable to those obtained with peripheral blood lymphocytes from 16 HTLV-seropositive subjects.     

Comparison of CREB- and NF-kappaB-mediated transactivation by human T lymphotropic virus type II (HTLV-II) and type I (HTLV-I) tax proteins

The function of the transactivator protein Tax from HTLV-II subtype A, subtype B, Brazilian subtype C, and African subtype D isolates was compared to that of Tax from an HTLV-I isolate. HTLV-II subtypes A, B, and C were less active in the transactivation of a NF-kappaB reporter compared to HTLV-I Tax in 293T but not Jurkat T cells. In both cell types there were no significant differences between the functions of HTLV-II B, C, and D and HTLV-I Tax proteins on either a full-length HTLV-I LTR or a 21-bp repeat reporter, suggesting that there is equivalent CREB-mediated transactivation between the viruses and these subtypes. In contrast, Tax of some but not all HTLV-II subtype A isolates, including the prototype Mo, had a greatly decreased ability to transactivate, and this could be directly correlated with a decrease in protein expression. Employment of cDNA clones encoding both Rex and Tax demonstrated that Rex was unable to rescue the expression or activity of the IIA Mo isolate. These studies demonstrate that with the exception of some HTLV-IIA subtypes there are no significant differences in Tax transactivation via the CREB and NF-kappaB pathways between the two viruses and suggest that the HTLV-II Tax may have a pathogenic potential equivalent to that of HTLV-I.     

Isolation, cloning, and complete nucleotide sequence of a phenotypically distinct Brazilian isolate of human T-lymphotropic virus type II (HTLV-II)

Analysis of human T-lymphotropic virus type II (HTLV-II) isolates from North America and Europe have demonstrated the existence of two molecular subtypes of the virus, HTLV-IIa and HTLV-IIb. Recently, studies on HTLV-II infections in Brazil have revealed isolates that are related phylogenetically to the HTLV-IIa subtype but have a HTLV-IIb phenotype with respect to the transactivating protein, tax. To more clearly define this relationship, HTLV-II was isolated from peripheral blood of an IVDA from Sao Paulo, Brazil (SP-WV), and the complete provirus was cloned and sequenced. Comparison of HTLV-II(SP-WV) nucleotide sequences to other available complete HTLV-II proviral sequences revealed that HTLV-II(SP-WV) is most closely related to HTLV-II(Mo), the prototypic HTLV-IIa subtype sequence. Phylogenetic analysis of LTR, env, and tax regions unequivocally demonstrated that HTLV-II(SP-WV) and all other Brazilian sequences examined are members of the IIa subtype. The predicted amino acid sequences of the major coding regions of HTLV-II(SP-WV) are also most closely related to HTLV-II(Mo), with the important exception of tax. The tax protein encoded by HTLV-II(SP-WV) is 96-99% identical to the tax of IIb isolates and is similar in that it has an additional 25 amino acids at the carboxy-terminus compared to the HTLV-II(Mo) tax with which it shares 91% identity. Analysis of tax stop codon usage of a number of HTLV-IIa isolates from North American, Europe, and Brazil demonstrated that isolates from the last region appear to be unique in their extended tax phenotype. It could be demonstrated that the extended tax proteins in the HTLV-IIb and Brazilian isolates had equivalent ability to transactivate the viral LTR, and studies with deletion mutants indicated that the extended C-terminus is not essential for transactivation. In contrast, the HTLV-IIa tax was found to have a greatly diminished ability to transactivate the viral LTR, which appeared to be a consequence of reduced expression of the protein. The studies show that although the Brazilian strains do not represent an entirely new subtype based on nucleotide sequence analysis they are a phenotypically unique molecular variant within the HTLV-IIa subtype.     

Human T-cell lymphotropic virus types I and II infections in patients with leukaemia/lymphoma and in subjects with sexually transmitted diseases in Nigeria

Summary Serological assays that distinguish antibodies to human T-cell lymphotropic virus types I (HTLV-I) and type II (HTLV-II), and polymerase chain reaction (PCR) tests were used to investigate association of these two human retroviruses with several well-defined clinical conditions in Nigeria. We compared the frequency of HTLV-I and HTLV-II infections among patients with lymphoproliferative disorders (n=65), individuals with various sexually transmitted diseases (n=40), patients with genital candidiasis (n=25) and apparently healthy individuals (n=60). Serological analysis of blood samples from all four groups showed that 10 of the 190 (5.3%) individuals tested were confirmed positive for the presence of antibodies to HTLV-I (6) or HTLV-II (4). Using the PCR technique, specific HTLV-I or HTLV-II sequences were amplified from the genomic DNA of 4 of 6 HTLV-I seropositive and 3 of the 4 HTLV-II seropositive individuals respectively. However, sequences of both viruses were amplified from the genomic DNAs of the remaining 3 seropositive individuals. Since one of the 5 sets of primer pairs [(SK110 (II)/SK III (II)], which is used for specific identification of HTLV-II did not amplify the target sequence from the genomic DNAs of any of the 4 HTLV-II-confirmed seropositive individuals in this study, it suggested sequence diversity of these viruses in Nigeria. The virus-infected individuals identified in this study were one (1.5%) of the 65 patients with leukaemia/lymphoma (HTLV-I), 6 of 40 (15.0%) individuals (HTLV-I=1, HTLV-II=3, HTLV-I/II=2) with sexually transmitted diseases (STD), one of 25(4.0%) subjects with genital candidiasis for HTLV-I, and 2 of 60 (33.3%) healthy individuals (one for HTLV-I and one for HTLV-I/II). There was a significant difference (P<0.025) between the prevalence of HTLV-I/II infections among patients with lymphoma/leukaemia and those who attended STD clinic in Ibadan, Nigeria. This study also suggests that while HTLV-I and HTLV-II may be important sexually transmitted viruses, they may not be specific aetiological agents of the common lymphoproliferative disorders in Nigeria.     

Genotypic characteristics of HTLV-II isolates from Amerindian and non-Indian populations

The Amerindian human T-cell lymphotropic virus type II isolate HTLV-II_G12 has been demonstrated to be an HTLV-IIb with several unique features, including several restriction enzyme site changes, a distinctive pre-gag region, a stop codon within thepol gene, and an extended Tax protein. In this study, HTLV-II isolates from Amerindian and non-Indian populations were characterized by restriction enzyme site analysis to determine the prevalent HTLV-II subtype. In addition, DNA amplification by the polymerase chain reaction and Southern blot analyses were used to probe for the HTLV-II_G12 pre-gag region. Our results showed that of 13 Guaymi Indian isolates subtyped, all were HTLV-IIb, and that approximately one third of 17 isolates had the unique pre-gag region. While other HTLV-II-infected groups contained both HTLV-IIa and HTLV-IIb isolates, none of these isolates showed evidence of the distinctive HTLV-II_G12 pre-gag region. Lastly, DNA sequence analysis was used to determine the prevalence of the stop codon within thepol gene open reading frame. These analyses revealed that the occurrence of a stop codon within this sequence appeared to be characteristic of most HTLV-IIb subtypes. These results further our understanding of the genetic variations and evolution of the HTLV-II viruses within the endemically infected Amerindian populations, as well as U.S. intravenous drug users and other non-Indian populations.     

High prevalence of VanB2 vancomycin-resistant Enterococcus faecium in Taiwan

Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bp vanB gene cluster including the vanR(B2), vanS(B2), vanY(B2), vanW(B2), vanH(B2), vanB2, and vanX(B2) genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the vanB1 gene cluster: VanR(B1), 97%; VanS(B1), 97%; VanY(B1), 96%; VanH(B1), 95%; VanB1, 96%; and VanX(B1), 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is a D-Ala-D-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published vanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 microg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.     

HIV-1B/C重组型外膜蛋白env基因序列分析及表型预测

目的 克隆并分析HIV-1B/C重组型外膜蛋白env基因序列,根据其氨基酸序列进行表型预测,为疫苗的抗原设计奠定基础.方法 采集北京地区HIV-1 B/C重组型的抗凝全血标本,分离血浆和提取基因组DNA,采用巢式PCR方法扩增rev-env基因,对扩增产物进行序列测定.根据核苷酸序列推导出相应的氨基酸序列,并对重要的Env功能结构域进行深入的分析和比较.结果 从12例B/C重组型HIV-1感染者中成功克隆到7个rev-env基因,序列分析发现其中6个有完整的开放读码框（ORF）,全部为CRF_07B/C重组型.6个Env蛋白氨基酸N糖基化位点和数目没有显著变化;CD4受体结合位点高度保守;根据V3环氨基酸序列及静电荷数目预测全部使用CCR5辅助受体;GP120/GP41剪切位点高度保守,预测所有GP160前体都能有效剪切;对几个已知中和抗体的中和位点分析推测全部的6个序列都对2G12、2F5中和不敏感;对4E10、PG9及PG16中和敏感.结论 有必要进一步阐明env基因型与相关功能的关系,这将为疫苗和药物研究提供依据.     

Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates

Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.     

<Emphasis Type="Italic">Blastocystis</Emphasis> sp. subtype 5: a possibly zoonotic genotype

Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. Previous studies show that Blastocystis sp. subtypes 1–4, 6, and 7 were composed of isolates from humans and animals, while Blastocystis sp. subtype 5 included only pig and cattle isolates. A more recent study on the basis of the SSU rDNA sequence has showed that a single Blastocystis isolate amplified directly from the faeces of a Thai human belongs to Blastocystis sp. subtype 5, but that study failed to cultivate this isolate. We report herein two human isolates from in vitro cultures belonging to Blastocystis sp. subtype 5 and one human isolate from in vitro culture containing two distinct genotypes of Blastocystis sp. subtypes 3 and 5 using PCR amplification with seven kinds of sequence-tagged site (STS) primers. Additionally, 16 Blastocystis isolates from pigs living in the same rural area with the three humans infected Blastocystis sp. subtype 5 were also genotyped by PCR with the STS primers, and all isolates from pigs and humans were compared by small-subunit ribosomal RNA (SSU rRNA) restriction-fragment-length polymorphism (RFLP) analyses using two restriction endonucleases (HinfI and RsaI). The results indicated that all of the isolates from pigs showed Blastocystis sp. subtype 5 and the RFLP patterns of all of the isolates from humans except for the mixed one were identical or quite similar to those of the 16 pig isolates with both HinfI and RsaI enzymes. These findings provide additional molecular-based evidence supporting the zoonotic potential of Blastocystis sp. subtype 5. This study also showed that Blastocystis sp. subtype 3 overgrew Blastocystis sp. subtype 5 in vitro.     

Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of Bartonella henselae Isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Molecular characterization of human T-cell lymphotropic virus type 2 (HTLV-II) from people living in urban areas of Sao Paulo city: evidence of multiple subtypes circulation

BACKGROUND: In Brazil, human T-cell lymphotropic virus type I and type II (HTLV-I and HTLV-II) are co-circulating and possess approximately 65% homology, which results in high cross-reactivity in serological tests. Based on the detection of EIA and Western blot (WB) tests, HTLV serodiagnosis yields indeterminate results in high-risk population, with the true determination of HTLV-II prevalence requiring a combined serological and molecular analysis. Molecular analysis of HTLV-II isolates has shown the existence of four distinct subtypes: IIa, IIb, IIc, and IId. The aim of this study was to evaluate the routine EIA and WB used in Sao Paulo city, as well as molecular methods for confirmation of infection and HTLV-II subtype distribution. Results: Two hundred ninety-three individuals, who were enrolled in the HTLV out-clinic in Sao Paulo city, Brazil, between July 1997 and May 2003, were tested by EIAs, and positive sera 232 (79%) reactive by one of the tests. When these sera were tested by WB revealed 134 were HTLV-I, 28 HTLV-II, 4 HTLV-I/II, and 48 were indeterminate. Polymerase chain reaction (PCR) on the indeterminate group showed that 20 (42%) were HTLV-II and 28 were negative. From a total of 48 HTLV-II subjects with DNA available, restriction fragment length polymorphism (RFLP) of the env region revealed 47 HTLV-IIa and 1 HTLV-IIb. The phylogenetic analysis was performed on 23 samples, which identified 19 as subtype a, Brazilian subcluster, and 4 as subtype b. This is the first time HTLV-II subtype b has been described in Brazil. However, further studies, such as a complete nucleotide DNA sequencing, need to be done to confirm these findings.     

Coat Protein Sequence Analysis Reveals Occurrence of New Strains of Sweet Potato Feathery Mottle Virus in Uganda and Tanzania

As subtype C is the most prevalent circulating human immunodeficiency virus type 1 (HIV-1) subtype internationally as well as locally in South Africa, more information on the biological nature and molecular characteristics of these viruses is required. Proviral DNA was isolated from primary cultures of three South African R5 isolates and the near-full length genome amplified by PCR. The resultant PCR product was cloned into the pCR-XL-TOPO vector and a representative clone from each isolate sequenced by primer walking. Phylogenetic analysis showed all three clones clustered within subtype C with a bootstrap value of 100%, and no recombination with other subtypes was identified by distance scan and bootscan analysis. Analysis of the potential coding regions revealed premature truncations of the second rev exon but no other potential structural distortions nor frameshift mutations in the open reading frames. All the clones contained three potential NF-B binding sites, a feature unique to subtype C viruses. The tips of the V3 loops contained the GPGQ sequence motif characteristic of CCR5-utilising subtype C strains, as well as relatively low overall net positive charge characteristic of non-syncytium-inducing isolates. This information contributes to our overall knowledge of circulating strains in South Africa and to the making of effective vaccines and chemotherapeutic agents.     

Human T-lymphotropic virus type II RFLP subtypes a0 and b4/b5 are associated with different demographic and geographic characteristics in the United States

Human T-lymphotropic virus type II (HTLV-II) prevalence is very low among the general U.S. population, intermediate among American Indians, and high among injecting drug users and their sexual partners. However, the transmission dynamics underlying this distribution are not well described. We obtained blood specimens from 493 blood donors found to be seropositive for HTLV-II at blood centers in five U.S. cities. Nested polymerase chain reaction was used to amplify a 672-bp region of the HTLV-II long terminal repeat region, and restriction fragment length polymorphism (RFLP) analysis was performed to classify each virus into subtypes as defined by Switzer et al. (1995, J. Virol. 69, 621-632). Associations between RFLP subtype and other characteristics were analyzed using multivariable logistic regression. HTLV-II subtype a0 was independently associated with age over 30 years (odds ratio (OR) = 2.12, 95% confidence interval (CI) 1.13-3.99) and with Black race/ethnicity (OR = 2.00, 95% CI 1.10-3.65 versus Hispanic race/ethnicity). Conversely, HTLV-II RFLP subtypes b4 and b5 were significantly more common among American Indian (OR = 3.77, 95% CI 1.23-11.57) and Other race/ethnicity (OR = 4.22, 95% CI 1.25-14.27, both versus Black race/ethnicity) and at the Oklahoma City blood center (OR = 3.57, 95% CI 1.08-11.84 compared to Washington, DC/Baltimore). There may have been at least two transmission foci of HTLV-II in the United States: a modest HTLV-II subtype a0 epidemic of unknown source in the 1960s and 1970s spread predominantly among Black persons in several geographic areas and a smaller focus of HTLV-II subtypes b4/b5 among non-Black individuals in Oklahoma and perhaps in other areas not examined by this study.     

Sequence variation of functional HTLV-II tax alleles among isolates from an endemic population: lack of evidence for oncogenic determinant in tax.

Human T-cell leukemia-lymphoma virus type II (HTLV-II) has been isolated from patients with hairy cell leukemia (HCL). We previously described a population with longstanding endemic HTLV-II infection, and showed that there is no increased risk for HCL in the affected groups. We thus have direct evidence that the endemic form(s) of HTLV-II cause HCL infrequently, if at all. By comparison, there is reason to suspect that the viruses isolated from patients with HCL had an etiologic role in the disease in those patients. One way to reconcile these conflicting observations is to consider that isolates of HTLV-II might differ in oncogenic potential. To determine whether the structure of the putative oncogenic determinant of HTLV-II, tax2, might differ in the new isolates compared to the tax of the prototype HCL isolate, MO, four new functional tax cDNAs were cloned from new isolates. Sequence analysis showed only minor (0.9-2.0%) amino acid variation compared to the published sequence of MO tax2. Some codons were consistently different from published sequences of the MO virus, but in most cases, such variations were also found in each of two tax2 clones we isolated from the MO T-cell line. These variations rendered the new clones more similar to the tax1 of the pathogenic virus HTLV-I. Thus we find no evidence that pathologic determinants of HTLV-II can be assigned to the tax gene.     

Subtype-specific problems with qualitative Amplicor HIV-1 DNA PCR test

BACKGROUND: commercial HIV-1 qualitative DNA PCR tests have the potential to detect virus in patients in whom antibody tests may be ineffective, such as patients with primary HIV infection and infants born to HIV seropositive mothers. However, the genetic diversity of HIV-1 raises concern about the ability of the PCR tests to detect all current subtypes. OBJECTIVES: to asses the sensitivity of the Amplicor HIV-1 test on 126 whole-blood samples representing seven different subtypes and to investigate the sensitivity when the standard assay was modified by including the primer pair SK145 and SKCC1B. RESULTS: of the 126 HIV-1 infected persons, 113 were tested positive and 13 were DNA PCR negative. On the basis of these results, the standard Amplicor HIV-1 test had a sensitivity of 90% in our cohort. In addition, 9% of the positive samples showed a low reactivity but above the cut-off of the assay. The standard assay yielded sensitivities of 100% for subtype B (n=16), D (n=9) and G (n=1), but only 83% for subtype A (n=41), 98% for subtype C (n=43), 79% for subtype E (n=14) and 0% for subtype F (n=2). All samples with low reactivity were non-B subtype. Eight of the DNA PCR negative samples, four subtype A, one C and three E were amplified with the modified Amplicor HIV-1 test with addition of SK145/SKCC1B primers. Using this modified protocol, six samples out of eight became positive. However, two samples (one A and one C) remained DNA PCR negative. CONCLUSION: this study confirms that the Amplicor HIV-1 test does not detect all subtypes with equivalent sensitivity and 10% of the samples, tested negative. Thus, it is preferable to add the SK145/SKCC1B primers to the standard test, where infection with non-B subtype is suspected.     

Helicobacter pylori heat shock protein A: serologic responses and genetic diversity

Helicobacter pylori synthesizes an unusual GroES homolog, heat shock protein A (HspA). The present study was aimed at an assessment of the serological response to HspA in a group of Chinese patients with defined gastroduodenal pathologies and determination of whether diversity is present in the nucleotide sequences encoding HspA in isolates from these patients. Serum samples collected from 154 patients who had an upper gastrointestinal pathology and the presence of H. pylori defined by biopsy were tested for an immunoglobulin G (IgG) serologic response to H. pylori HspA by an enzyme linked immunosorbant assay. HspA-encoding nucleotide sequences in H. pylori isolates from 14 patients (7 seropositive and 7 seronegative for HspA) were analyzed by PCR and direct sequencing of the PCR products. The sequencing results were compared to those of 48 isolates from other parts of the world. Of the 154 known H. pylori-positive patients, 54 (35.1%) were seropositive for HspA. The A domain (GroES homology) of HspA was highly conserved in the 14 isolates tested. Although the B domain (metal-binding site unique to H. pylori) resembled that in the known major variant, particular amino acid substitutions allowed definition of an HspA variant associated with isolates from East Asia. There were no associations between patient characteristics and HspA seropositivity or amino acid sequences. We confirmed in this study that the clinical outcomes of H. pylori infection are not related to HspA antigenicity or to sequence variation. However, B-domain sequence variation may be a marker for the study of the genetic diversity of H. pylori strains of different geographic origins.     

Identification of two cryptic species in the Anopheles (Cellia) annularis complex using ribosomal DNA PCR-RFLP

Anopheles (Cellia) annularis Van der Wulp is a complex of two sibling species provisionally designated as species A and B and can only be differentiated on the basis of the paracentric inversion in the ovarian polytene chromosomes. To analyze the distribution of these two species and to develop a molecular method for the identification of these two cryptic species, we sequenced the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain 3 (D3) of A. annularis specimens collected from Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi (Jharkhand), and Ghaziabad (Uttar Pradesh). We did not find any sequence variation among the specimens collected from Assam, Madhya Pradesh, and Jharkhand states, whereas two types of sequences were obtained from the specimens collected from the state of Uttar Pradesh, which correspond to species A and B of the A. annularis complex. Species A was more prevalent among the all four regions studied. The ITS2 sequence of species A showed unique restriction sites for MvaI and Eco24I, while species B displayed HinfI and NruI sites. Similarly, the D3 sequence of species A showed unique restriction site for Alw26I, while species B showed a unique KpnI site. In this study, we report for the first time the development of ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism methods for identifying these two cryptic species of the Annularis complex.