Clinical evaluation of a GP5+/6+-based luminex assay having full high-risk human papillomavirus genotyping capability and an internal control.

The LMNX genotyping kit HPV GP (LMNX) is based on the clinically validated GP5+/6+ PCR, with a genotyping readout as an alternative for the more established enzyme immunoassay (EIA) detection of 14 targeted high-risk human papillomavirus (HPV) types. LMNX is additionally provided with an internal control probe. Here, we present an analysis of the clinical performance of the LMNX using a sample panel and infrastructure provided by the international VALGENT (Validation of Genotyping Tests) project. This panel consisted of cervical specimens from approximately 1,000 women attending routine screening, "enriched" with 300 women with abnormal cytology. Cases were defined as women classified with cervical intraepithelial neoplasia (CIN) grade 2+ (CIN2+) (n = 102) or CIN3+ (n = 55) within the previous 18 months. Controls were women who had normal cytology results over two subsequent screening rounds at a 3-year interval (n = 746). The GP5+/6+-PCR EIA (EIA) was used as a comparator assay and showed sensitivities of 94.1% and 98.2% for CIN2+ and CIN3+, respectively, with a clinical specificity of 92.4% among women aged ≥ 30 years. The LMNX demonstrated clinical sensitivities of 96.1% for CIN2+ and of 98.2% for CIN3+ and a clinical specificity of 92.6% for women aged ≥ 30 years. The LMNX and EIA were in high agreement (Cohen's kappa = 0.969) for the detection of 14 hrHPVs in aggregate, and no significant difference was observed (McNemar's P = 0.629). The LMNX internal control detected 0.6% inadequate specimens. Based on our study results, we consider the LMNX, similarly to the EIA, useful for HPV-based cervical cancer screening.

Universal human papillomavirus genotyping by the digene HPV Genotyping RH and LQ Tests.

BACKGROUND: High-risk (hr)HPV testing plays an important role in primary cervical cancer screening. Subsequent hrHPV genotyping might contribute to better risk stratification. The majority of hrHPV tests do not include identification of individual hrHPV genotypes. OBJECTIVES: The digene HPV Genotyping RH Test (strip-based) and LQ Test (xMAP-based) allow genotyping of GP5+/6+ amplimers, but their probes target a region in the L1 ORF, which is also amplified by other broad-spectrum hrHPV assays, e.g., the Roche Amplicor HPV Test (Amplicor) and the Roche Linear Array. The goal was to test whether the RH Test and LQ Test can be used as an universal hrHPV genotyping test. STUDY DESIGN: Self-collected cervico-vaginal specimens (n=416) from an epidemiologic study were analyzed with Amplicor. The amplimers obtained were also tested with the RH Test and LQ Test for identification of 18 HPV types, including the 13 hrHPVs targeted by Amplicor. RESULTS: 197 specimens were positive by Amplicor, in which the RH Test and LQ Test identified one of the 13 hrHPVs in 94.4% and 98.0%, respectively. In 219 specimens remaining negative by Amplicor, the RH Test and LQ Test, performed on the Amplicor amplification products, still detected one of the 13 hrHPVs in 3.7% and 5.5%, respectively, and include identification of HPV53, 66, and 82. Overall, the RH and LQ Tests demonstrated high concordance with Amplicor for hrHPV detection (κ=0.908 and κ=0.923, respectively). CONCLUSIONS: The digene HPV Genotyping RH and LQ Tests can be directly used for amplimers generated by the Amplicor HPV Test.

Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women.

Chlamydia trachomatis (Ct) comprises 3 serogroups and 19 serovars. Different genotyping methods are available to differentiate between the serovars. The aim of this study was to evaluate the sensitivity and discriminatory power of three genotyping methods, respectively Omp1 sequencing, the Ct Detection and genoTyping (DT) assay and the pmpH real-time PCR discriminating an LGV infection from a non-LGV infection. In total, 50 Aptima Combo 2 (AC2) Ct positive samples were selected and tested with the 3 genotyping methods. The Ct-DT assay detected 3 double Ct infections that caused a non interpretable result by Omp1 sequencing, while Omp1 sequencing has a higher discriminatory power that gave additional information about Ct genovariants. All three methods detected the 6 LGV samples. Although the pmpH real-time PCR detected all LGV infections, a substantial amount (24%) of non-LGV infections were missed. The sensitivity compared to AC2 Ct detection was 80% (95% CI 67-89%) for the Ct-DT assay, 72% (95% CI 58-83%) for Omp1 sequencing and 64% (95% CI 50-76%) for the pmpH real-time PCR. In conclusion, the Ct-DT assay is appropriate for serovar distribution studies, epidemiological studies and differentiation between an LGV and non-LGV Ct infection, while Omp1 sequencing is more appropriate for phylogenetic studies. The pmpH real-time PCR is suitable as second assay to differentiate between an LGV and non-LGV infection, but not as primary detection assay, due to its low sensitivity for non-LGV strains.

Human papillomavirus infection and squamous cell carcinoma of the conjunctiva.

BACKGROUND: Squamous cell carcinoma of the conjunctiva (SCCC) is associated with HIV-related immunosuppression, but human papillomavirus virus (HPV) is also suspected to have a role. We carried out a case-control study to assess the role of cutaneous and mucosal HPV types in SCCC, conjunctival dysplasia, and their combination (SCCC/dysplasia) in Uganda. METHODS: We compared HPV prevalence in frozen biopsies from 94 SCCC cases (79 of whom were found to be HIV-positive), 39 dysplasia cases (34 HIV-positive), and 285 hospital controls (128 HIV-positive) having other eye conditions that required surgery. Highly sensitive PCR assays that detect 75 HPV types were used. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed, adjusting for, or stratifying by age, sex, and HIV status. RESULTS: Cutaneous HPV types were detected in 45% of SCCC cases, 41% of dysplasia cases and 11% of controls. Human papillomavirus virus 5 and 8 were the most common types in SCCC, and most often occurred in combination with other types. Associations were observed between SCCC/dysplasia and detection of both single (OR=2.3; 1.2-4.4) and multiple (OR=18.3; 6.2-54.4) cutaneous HPV types, and were chiefly based on findings in HIV-positive patients. Cutaneous HPV infections were rarely observed among HIV-negative patients and the association with SCCC/dysplasia was not significant (OR=2.4; 0.6-9.6) among them. Squamous cell carcinoma of the conjunctiva/dysplasia risk and mucosal HPV types were not associated in either HIV-positive or HIV-negative patients. CONCLUSIONS: We detected cutaneous HPV types in nearly half of SCCC/dysplasia cases and often multiple types (HPV5 and 8 being most common). The role of HIV (confounder or strong enhancer of cutaneous HPV carcinogenicity) is still uncertain.

High genotyping concordance between the digene HPV Genotyping RH Test and the Reverse Line Blot genotyping assay on GP5+/6+-PCR products.

BACKGROUND: Based on epidemiologic studies, 18 mucosal human papillomavirus (HPV) types have been classified as (probably) high-risk (HR) (i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Recognition of HR HPV at the individual type level may be valuable in clinical management of HR HPV-positive women. OBJECTIVES: The goal of this study was to evaluate the performance of the novel digene HPV Genotyping RH Test (digene RH Test), which uses type-specific probes for the 18 HR HPV genotypes, in comparison to the established in-house Reverse Line Blot (RLB) genotyping assay on PCR products generated with the clinically validated GP5+/6+-PCR method. STUDY DESIGN: GP5+/6+ amplimers, generated from 493 digene High Risk HPV HC2 DNA Test (HC2)-positive and 95 HC2-negative cervical smears, were genotyped by both the digene RH Test and the RLB assay. RESULTS: Both genotyping assays demonstrated high concordance for overall HR HPV detection (ú = 0.886) and type-specific identification of the 18 HR types (overall ú = 0.951, individual ú range 0.777 to 1.000) in 493 HC2-positive samples. The digene RH Test revealed positivity for one or more HR HPV type(s) in 86.6% of the HC2-positive women, and negativity was confirmed in 97.9% of the HC2-negative women. CONCLUSIONS: The digene HPV Genotyping RH Test revealed a high genotyping agreement with the established RLB assay on GP5+/6+ amplimers. Accordingly, this assay following GP5+/6+-PCR could serve as a follow-up test in a clinical setting for women who are HC2-positive to identify the respective HR HPV genotype(s).

High-throughput genotyping of high-risk HPV by the digene HPV Genotyping LQ Test using GP5+/6+-PCR and xMAP technology.

BACKGROUND: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) as (probably) high-risk (HR) based on their association with cervical cancer, i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Given the fact that certain HR HPV types confer an increased risk of cervical (pre)cancer, type-specific identification might aid clinical management of women tested positive for HR HPV. Therefore, the development of robust, high-throughput genotyping assays is important. OBJECTIVES: An analytical comparison of the digene HPV Genotyping LQ Test (digene LQ Test), capable of identifying 18 HR types using bead-based xMAP suspension array technology, with the established Reverse Line Blot (RLB) genotyping assay was carried out on amplimers generated with the clinically validated GP5+/6+-PCR method. STUDY DESIGN: GP5+/6+ amplimers, generated from 434 digene High Risk HPV HC2 DNA Test (HC2)-positive and 95 HC2-negative cervical smears, were genotyped by both the digene LQ Test and the RLB genotyping assay. RESULTS: The genotyping assays revealed high agreement for overall HR HPV detection (ú = 0.884) and type-specific identification of the 18 HR HPV types (overall ú = 0.958, individual ú range 0.795 to 1.000). The digene LQ Test demonstrated a very good inter-laboratory reproducibility (ú = 0.987). Among the HC2-positive women, the digene LQ Test revealed positivity for one or more HR HPV type(s) in 85.9%, and negativity was observed in 97.9% of the HC2-negative women. CONCLUSIONS: The digene LQ Test demonstrated a high genotyping agreement with the established RLB genotyping assay on GP5+/6+ amplimers. This novel assay allows for high-throughput genotyping following HR HPV testing by HC2.

Helicobacter species in cancers of the gallbladder and extrahepatic biliary tract.

Helicobacter species have been found in human bile and biliary tract (BT) tissue and are suspected to cause BT diseases, including gallbladder and extrahepatic cancers, collectively referred to in this work as BT cancers. We conducted a literature review of the epidemiological evidence linking the presence of Helicobacter species in bile or BT biopsies to BT cancers and benign diseases. Reports showed great variability with respect to study methods. Nine studies of BT cancers were identified, all with 30 or fewer BT cancers; eight included cancer-free control subjects and used polymerase chain reaction (PCR) as a means of Helicobacter species detection. In four of these studies, Helicobacter species were detected in patients with BT cancer significantly more frequently than in controls, at least when controls without BT diseases were used. In two studies, no Helicobacter species were detected in either cases or controls. Helicobacter species were also often detected in benign BT diseases such as gallstone disease or chronic cholecystitis. As our current knowledge relies on a few small studies that showed substantial differences, larger studies and more standardised protocols for detecting DNA and antibodies against Helicobacter species are needed to investigate a potential association with BT cancer.

Helicobacter genotyping and detection in peroperative lavage fluid in patients with perforated peptic ulcer.

INTRODUCTION AND OBJECTIVES: Certain Helicobacter pylori genotypes are associated with peptic ulcer disease; however, little is known about associations between the H. pylori genotype and perforated peptic ulcer (PPU). The primary aim of this study was to evaluate which genotypes are present in patients with PPU and which genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a way other than by biopsy. MATERIALS AND METHODS: Serum samples, gastric tissue biopsies, lavage fluid, and fluid from the nasogastric tube were collected from patients operated upon for PPU. By means of PCR, DEIA, and LIPA the presence of the "cytotoxin associated gene" (cagA) and the genotype of the "vacuolating cytotoxin gene" were determined. RESULTS: Fluid from the nasogastric tube was obtained from 25 patients, lavage fluid from 26 patients, serum samples from 20 patients and biopsies from 18 patients. Several genotypes were found, of which the vacA s1 cagA positive strains were predominant. Additionally, a correlation was found between the H. pylori presence in biopsy and its presence in lavage fluid (p=0.015), rendering the latter as an alternative for biopsy. Sensitivity and specificity of lavage fluid analysis were 100% and 67%, respectively. CONCLUSION: This study shows the vacA s1 cagA positive strain is predominant in a PPU population. The correlation found between the H. pylori presence in biopsy and its presence in lavage fluid suggests that analysis of the lavage fluid is sufficient to determine the H. pylori presence. Risks associated with biopsy taking may be avoided.

[Vaccination against human papillomavirus for the prevention of cervical cancer]. / Vaccinatie tegen Humaan papillomavirus ter preventie van baarmoederhalskanker.

Persistent infections with high-risk Human papillomavirus (HPV) can lead to cervical cancer. This fact could imply that vaccination against HPV could prevent this disease. Such vaccines could be aimed at the prevention of HPV infections or the treatment of infections, cervical intraepithelial neoplasia (CIN) and cervical cancer. There are two prophylactic candidate vaccines against the high risk HPV types 16 and 18, which appear to be safe and effective in preventing incidental and persistent HPV infections. Phase III studies should reveal whether these vaccines will also have long-term effects by preventing the development of CIN and eventually cervical cancer. The introduction of such an HPV vaccine in the Netherlands, its cost-effectiveness and introduction into the existing national vaccination programmes needs to be studied. The vaccination of girls before they become sexually active seems to be the most effective approach, although older women can also profit from prophylactic vaccination. The current community-based screening, possibly complemented by an HPV test, needs to be continued to identify and treat presently HPV-infected women. In a population with an existing community-based screening program for cervical cancer, a vaccine preventing persisting infection with high-risk HPV will further reduce the incidence of HPV-related cervical cancer.

High frequency of gastric colonization with multiple Helicobacter pylori strains in Venezuelan subjects.

Multiple Helicobacter pylori strains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixed H. pylori colonization in 127 subjects from Venezuela, a country of high H. pylori prevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121 H. pylori-positive persons, the prevalence of cagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of the vacA s (mean, 49%) and m (mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization. vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that the H. pylori East Asian vacA genotype has survived in all three populations tested.

Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori.

iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.

Genotyping of Helicobacter pylori strains in formalin-fixed or Formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens.

Different genotypes of Helicobacter pylori can play a role in the development of atrophic gastritis, peptic ulcer disease, and gastric carcinomas. In this study the authors developed polymerase chain reaction assays for the detection and identification of vacA (s- and m-regions), cagA, and iceA genotypes of H. pylori in formalin-fixed or formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens. Polymerase chain reaction products were analyzed by reverse hybridization on a line-probe assay. Complete genotyping was achieved in 26 of 28 samples (93%), and multiple genotypes, indicating the presence of multiple strains, were detected in nine samples (32%). This genotyping method offers the possibility for long-term retrospective studies on H. pylori genotypes and gastric pathology in the same archival gastric tissue specimens.

The importance of vacA, cagA, and iceA genotypes of Helicobacter pylori infection in peptic ulcer disease and gastroesophageal reflux disease.

OBJECTIVE: To study the relationship between the presence of H. pylori virulence factors and clinical outcome in H. pylori infected patients. METHODS: DNA was isolated from an antral biopsy sample and vacA, cagA, and iceA genotype were determined by PCR and a reverse hybridization technique in 183 patients with culture-proven H. pylori infection: 51 with peptic ulcer disease (PUD), 62 with gastroesophageal reflux disease (GERD), and 70 with a normal endoscopy (gastritis only; GO). RESULTS: Forty-four samples (24%) showed more than one allelic variant in the vacA s- or in-region and/or both iceA1 and iceA2 genotypes, indicating multiple strain infection. These were excluded from statistical analysis. vacA s1 and cagA were significantly more common in PUD than in GERD and GO. Logistic regression analysis showed that GERD patients were more often infected with strains lacking both cagA and iceA than GO patients (OR = 0.36; CI = 0.15-0.89). Trend analysis showed that GERD patients were most often infected with less virulent strains (p < 0.002). CONCLUSION: Multiple strain infection is common. H. pylori strains possessing the vacA s1 genotype and/or cagA are associated with PUD. GERD patients, infected with H. pylori, mostly carry less virulent strains possessing neither cagA nor iceA1. Our findings support the hypothesis that virulent strains protect against the development of GERD.

Clarithromycin-susceptible and -resistant Helicobacter pylori isolates with identical randomly amplified polymorphic DNA-PCR genotypes cultured from single gastric biopsy specimens prior to antibiotic therapy.

Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible H. pylori colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous H. pylori populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary H. pylori population is used to test for clarithromycin susceptibility.

Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF(10) PCR and HPV genotyping.

Human papillomavirus (HPV) can be detected by DNA amplification from clinical samples. The aim of the present study was to compare the HPV status in both cervical scrape and biopsy specimens obtained from 174 patients, using the recently developed broad spectrum SPF(10) PCR-LiPA method. The detection rate of HPV in these materials was determined and the spectrum of HPV genotypes was compared. Cervical scrapes and biopsy specimens were obtained, either on the same day (group I), or with an interval of up to almost 2 years (group II, mean interval 97 days, range 1-469 days). HPV DNA was amplified by SPF(10) PCR and detected in a microtitre plate hybridization assay. Of the HPV-positive cases, the genotype was determined by reverse hybridization of the same SPF(10) amplimer on a line probe assay (LiPA), discriminating between HPV genotypes 6, 11, 16, 18, 31, 33-35, 39, 40, 42-45, 51-54, 56, 58, 59, 66, 68, 70, and 74. The results showed that the detection rate and the spectrum of HPV genotypes in cervical scrapes and the corresponding biopsy specimens were highly comparable in both patient groups, even when multiple genotypes were present. In both groups, multiple HPV genotypes were more frequently detected in cervical scrapes than in the corresponding biopsy specimens. In conclusion, HPV infection can be diagnosed in cervical scrapes and biopsy specimens using the SPF(10) PCR-LiPA system. Analysis of cervical scrapes accurately reflects the spectrum of HPV genotypes in the patient's cervical region, even with a sampling interval between the cervical scrape and the biopsy specimen.

Assessment of Helicobacter pylori vacA and cagA genotypes and host serological response.

Helicobacter pylori strains can be distinguished by genotyping of virulence-associated genes, such as vacA and cagA. Because serological discrimination between strain types would reduce the need for endoscopy, 61 patients carrying H. pylori were studied by vacA and cagA genotyping of H. pylori in gastric biopsy specimens and by detection of specific serum antibodies. Serological responses to H. pylori were determined by Helicoblot (versions 2.0 and 2.1). Antibodies to CagA also were determined by a rapid anti-CagA assay (Pyloriset screen CagA) as well as by two noncommercially developed enzyme immunoassays, each using a recombinant CagA protein. Assessment of performance of the Helicoblot assays indicated substantial interobserver variation, with kappa values between 0.20 and 0.93. There was no relationship between the serological profiles on the Helicoblot and the genotypes from the same patients, except for strong associations between the presence of anti-CagA and the cagA-positive and vacA s1 H. pylori genotypes. Detection of anti-CagA by the five different assays varied considerably, with kappa values ranging from 0.21 to 0.78. Using the cagA genotype as the "gold standard," the sensitivity and specificity of the anti-CagA assays varied from 71.4 to 85.7% and from 54.2 to 100%, respectively. Thus, serological profiles of antibodies to H. pylori are heterogeneous and, with the exception of anti-CagA antibodies, show no relation to the H. pylori vacA and cagA genotypes. Detection of anti-CagA antibodies is strongly dependent on the test used.

Genotypic, clinical, and demographic characteristics of children infected with Helicobacter pylori.

Helicobacter pylori isolates vary between geographic regions. Certain H. pylori genotypes may be associated with disease outcome. Thirty-eight children underwent diagnostic upper endoscopy at four medical centers and were retrospectively analyzed to determine if H. pylori virulence genes were associated with endoscopic disease severity, histologic parameters, and host demographics. The H. pylori virulence genotype was analyzed by a reverse hybridization line probe assay and type-specific PCR. Endoscopic ulcers or erosions were found in 17 (45%) patients, with 13 (34%) of these patients having antral nodularity. Histological gastritis, of varying severity, was present in all children. Four patients harbored more than one H. pylori strain: one subject had both cagA(+) and cagA-negative strains, while three patients harbored either two different cagA-negative strains (two children) or two cagA(+) strains (one child). There were 28 (74%) cagA(+) isolates; 19 were associated with the vacA s1b genotype, 7 were associated with the vacA s1a genotype, 1 was associated with the vacA s1c genotype, and 1 was associated with the s2 genotype. Of 14 cagA-negative isolates, 6 were vacA s2 genotype, 4 were vacA s1b, 3 were vacA s1a, and 1 was vacA s1c. Nine of ten (90%) Hispanics had similar H. pylori strains (vacA s1b,m1), and all Asian-Canadian children were infected by strains with vacA s1c genotype. No correlation between H. pylori strain and endoscopic or histopathologic abnormalities was found. This study provides a baseline framework of North American children and their H. pylori strains, serving as a powerful epidemiological tool for prospective investigations to better understand the transmission and evolution of diverse disease outcomes.

Evaluation of clarithromycin resistance and cagA and vacA genotyping of Helicobacter pylori strains from the west of Ireland using line probe assays.

The prevalence of clarithromycin resistance-associated mutations, the cytotoxin-associated gene (cagA), and the various vacuolating cytotoxin (vacA) genotypes was determined in 50 gastric biopsy specimens from Helicobacter pylori-infected patients, using line probe assays. The clarithromycin resistance-associated mutation A2143G was detected in H. pylori strains from 26% of the specimens, which suggested that the high rate of H. pylori treatment failure in Ireland may be partly attributable to the presence of these mutations. All strains examined carried the vacA s1 genotype, and 76% were cagA positive. Of these 50 specimens, 13 (26%) carried H. pylori strains with vacA midregion genotype m1, 29 (58%) carried strains that were m2, 1 (2%) was infected by a strain that was positive for both m1 and m2, and 7 (14%) carried strains that could not be typed.